Comparative collicular tonotopy in two bat species adapted to movement detection, Hipposideros speoris and Megaderma lyra

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1 J Comp Physiol A (1988) 163: Journal of Comparative Physiology A Springer-Verlag 1988 Comparative collicular tonotopy in two bat species adapted to movement detection, Hipposideros speoris and Megaderma lyra R. Rübsamen 1, G. Neuweiler 2 *, and K. Sripathi 3 1 Lehrstuhl fur Allgemeine Zoologie der Ruhr-Universitat, D-4630 Bochum, Federal Republic of Germany 2 Zoologisches Institut der Universität München, Luisenstrasse 14, D-8000 München 2, Federal Republic of Germany 3 Department of Animal Behaviour, Madurai Kamaraj University, Madurai, India Accepted January 11, 1988 Summary. The tonotopic organization of the inferior colliculus (1C) in two echolocating bats, Hipposideros speoris and Megaderma lyra, was studied by multiunit recordings. In Hipposideros speoris frequencies below the range of the echolocation signals (i.e. below 120 khz) are compressed into a dorsolateral cap about μm thick. Within this region, neuronal sheets of about 4-5 μm thickness represent a 1 khz-band. In contrast, the frequencies of the echolocation signals ( khz) are overrepresented and occupy the central and ventral parts of the 1C (Fig. 3). In this region, neuronal sheets of about 80 μm thickness represent a 1 khz-band. The largest 1 khz-slabs ( μm) represent frequencies of the pure tone components of the echolocation signals ( khz). The frequency of the pure tone echolocation component is specific for any given individual and always part of the overrepresented frequency range but did not necessarily coincide with the BF of the thickest isofrequency slab. Thus hipposiderid bats have an auditory fovea (Fig. 10). In the 1C of Megaderma lyra the complete range of audible frequencies, from a few khz to 110 khz, is represented in fairly equal proportions (Fig. 7). On the average, a neuronal sheet of 30 μm thickness is dedicated to a 1 khz-band, however, frequencies below 20 khz, i.e. below the range of the echolocation signals, are overrepresented. Audiograms based on thresholds determined from multiunit recordings demonstrate the specific sensitivities of the two bat species. In Hipposideros Abbreviations: BF Best frequency; CFconstant frequency; FM frequency modulated; 1C inferior colliculus; HS Hipposideros speoris * To whom offprint requests should be sent speoris the audiogram shows two sensitivity peaks, one in the nonecholocating frequency range (10-60 khz) and one within the auditory fovea for echolocation ( khz). Megaderma lyra has extreme sensitivity between khz, with thresholds as low as 24 db SPL, and a second sensitivity peak at 50 khz (Fig. 8). In Megaderma lyra, as in common laboratory mammals, Q 10dB -values of single units do not exceed 30, whereas in Hipposideros speoris units with BFs within the auditory fovea reach Q 10dB -values ofuptol30. In Megaderma lyra, many single units and multiunit clusters with BFs below 30 khz show upper thresholds of db SPL and respond most vigorously to sound intensities below 30 db SPL (Fig. 9). Many of these units respond preferentially or exclusively to noise. These features are interpreted as adaptations to detection of prey-generated noises. The two different tonotopic arrangements (compare Figs. 3 and 7) in the ICs of the two species are correlated with their different foraging behaviours. It is suggested that pure tone echolocation and auditory foveae are primarily adaptations to echo clutter rejection for species foraging on the wing close to vegetation. Introduction The orderly tonotopic arrangement of neuronal best frequencies (BF), is a general principle of organization in the ascending auditory pathway of mammals. It originates from the frequency to place transformation that takes place on the basilar membrane of the inner ear. High frequencies are represented on the basal end of the basilar mem-

2 272 R. Rübsamen et al.: Comparative tonotopy in bats brane, and lower frequencies progressively more apically usually in such a way that octaves cover equal lengths (von Bekesy 1960). In most mammals, this keyboard-like, logarithmic representation of the stimulus spectrum within the inner ear is maintained at every level throughout the hierarchically organized central auditory system (Merzenich et al. 1977). However, in species such as echolocating horseshoe bats, the cochlear frequency representation markedly deviates from a logarithmic relationship. Within the audible frequency range (about 5 to 90 khz), a narrow 3-5 khz band around the frequency of the pure tone component of the echo (ca. 84 khz in Rhinolophus ferrumequinum and khz in R. rouxi) is vastly overrepresented, and occupies over a quarter of the total length of the basilar membrane (Bruns 1976; Vater et al. 1985). This cochlear overrepresentation of about 0.5% of the total audible range displayed on the basilar membrane is also present in auditory brain centers and has been called an 'acoustic fovea' (Pollak and Schuller 1981). We find the term 'auditory fovea' more appropriate and use that terminology in this paper. The center frequency of the auditory fovea coincides with that of a narrowly tuned filter in the audiogram of horseshoe bats (Neuweiler 1970; Schuller 1980). Filter and fovea are tuned to the frequency of the long duration (ca. 50 ms) constant frequency (CF) component in the echo. This CFcomponent serves as a carrier of brief frequency and amplitude modulations superimposed on the echo by reflections from beating insect wings (Schnitzler et al. 1983). Neurons tuned to the foveal frequency band encode these modulations (Neuweiler et al. 1980; Schuller 1984). Such studies suggest that horseshoe bats may only detect wingbeating targets, and a behavioural study showed that indeed Rhinolophus rouxi is specialized to detect such targets, and only attacks insects when they move their wings (Link et al. 1986). Therefore, the long CF echolocation signal and auditory fovea have been interpreted as specializations for fluttering target detection and an adaptation to facilitate prey detection in a cluttered environment such as dense vegetation (Neuweiler et al. 1987). Link et al. (1986) also showed that horseshoe bats are not the only species that detect fluttering prey. An echolocating hipposiderid species, Hipposideros speoris, does this as well. Hipposiderid bats emit CF/FM echolocation signals. However, the CF-component is only about 5 ms.long (Habersetzer et al. 1984) and therefore should be less suited for coding wingbeats than the long CF of the horseshoe bat. It is not known whether hipposiderids have an auditory fovea. Audiograms derived from evoked potential recordings show only a shallow filter region which is mismatched to the CF-frequency of the echo by about 5 khz (Schuller 1980). In Hipposideros speoris, narrowly tuned units in the frequency range of the CF-echo components are rare and can only be recorded in the most ventral layers of the inferior colliculus (Neuweiler et al. 1984). Therefore the question arises as to whether hipposiderid bats have a poorly developed auditory fovea, even though they selectively detect and attack fluttering insects like horseshoe bats. In horseshoe bats the auditory fovea is a conspicuous feature of the tonotopic organization in the inferior colliculus (Pollak and Schuller 1981), so we decided to answer this question by determining the tonotopy of the 1C in Hipposideros speoris. For comparison we also studied the tonotopic organization in the inferior colliculus of another bat species specialized for the detection of moving prey, the Indian False Vampire Bat, Megaderma lyra. This species also responds to moving prey as do horseshoe bats and hipposiderids, however it does not use echolocation to detect fluttering insects, instead, it listens to the noises made by moving terrestrial prey such as frogs, arthropods, birds, mice, etc. (Marimuthu and Neuweiler 1987). We wanted to see if the very different strategies used by those two species to detect moving prey are reflected in the functional organization of their central auditory systems. Materials and methods The experiments were performed in the Department of Animal Behaviour, Madurai Kamaraj University in Madurai, India. For this study seven Hipposideros speoris and three Megaderma lyra were caught from a cave near the campus. The echolocation pulses of hipposiderid bats were recorded with a Briiel & Kjaer sound level meter (Type 2209) and stored on tape (Lennartz, 78 cm/s). Sound analysis was the same as described by Rubsamen (1987). Each bat was injected i.p. with a muscle relaxant (Rompun 7 μ^g bodyweight) and anesthetized with Nembutal (0.025 mg/g body weight). After surgery a bolt was fixed on the frontal skull with dental cement. When recovery from anesthesia was complete the animals were firmly placed in a holder attached to a stereotaxic device. The wounds were treated with a local anesthetic (Novocaine). Pure tone sound stimuli with a duration of 30 ms and a rise/fall time of 3 ms were delivered at a repetition rate of 4/s by a custom-made ultrasound loudspeaker, calibrated with a Briiel & Kjaer sound level meter Type 2209 and a 1/4" condenser microphone Type The output of the loudspeaker was flat within +5 db from 25 to 130 khz and fell off at a rate

3 R. Rübsamen et al.: Comparative tonotopy in bats 273 of 9 db/10 khz for lower frequencies. The loudspeaker was placed 20 cm in front of the bat and visually adjusted normal to the entrance of the contralateral ear. During the recordings the bat was fully awake and the wound margins were treated by local anesthesia. Thresholds of multiunit clusters were determined audiovisually. When single units were recorded we also calculated dot displays and PST-histograms on a laboratory PC. Through a small hole in the skull a series of electrode penetrations were made with a KC1 filled glass electrode (4 8 Mβ). In order to minimize brain damage at the surface and to guarantee a stable position of the 1C within the skull, we drilled a single small hole (200 μm 0) into the skull of each specimen and changed the angle of penetrations by tilting the preparation stereotaxically by 10 and 20 rostrocaudally, mediolaterally or in combinations of these two planes (Fig. 2). With this fan-like penetration pattern we obtained a rather complete sample of the ventral part of the 1C where the frequencies of the echolocation signals are represented, whereas the dorsal areas of the 1C were scanned less completely. The penetrations mainly sampled the central nucleus, but the external and pericentral regions of the 1C were also included. In multiunit recordings best frequencies (BF) and absolute thresholds were measured in 50 pm steps from the surface to the ventral floor of the 1C until no auditory response could be recorded or the orderly sequence of BFs reversed, indicating that we had penetrated nuclei of the lateral lemniscus. The electrode tracks were marked electrolytically (8 μa for 3 min) at depths of 1000, 2000 or 3000 jim. After the experiments the animals were anesthetized with a lethal dose of Nembutal, and following an intracardial flushing with Ringer solution (100 ml) the brains were fixed with 200 ml formalin (8%). The brains were stored in the same fixative for several weeks, but before histological processing the fixative was washed out with distilled water containing 30% sucrose as a cryoprotective. The brains were embedded in egg yolk fixed by glutaraldehyde in a small chamber with markers to aid in subsequent spatial reconstructions. Alternating frozen sections (48 μm) were collected in two series. One series was stained with cresyl violet, and the other with a silver impregnation (Gallyas 1979) to demonstrate the location of fibers. The electrolytic lesions allowed the calculation of shrinkage due to fixing and histological procedures; they also facilitated reconstruction of electrode tracks so that the recorded BFs could be allotted to the corresponding electrode positions. Data from all experimental animals were pooled to obtain an averaged outline of the 1C, and a composite tonotopy from all recordings for both bat species. For this purpose the outlines of the ICs from four sectioned brains were digitized and averaged to a computer generated three-dimensional standard 1C in which the electrode tracks and the locations of the BF-readings were fitted by the electrolytic marks, known angles of penetrations and the positions of the electrode on the brain surface (Fig. 1). Results Hipposideros speoris This echolocating bat species emits a so called 'short, CF/FM echolocation signal. The CF-component lasts about 3-6 ms and has a frequency between 127 and 140 khz, depending on the individual. The terminal FM-component sweeps down to about 100 khz. When close to obstacles the bats lateral frontal Depth 1640 jjm 500 jum Fig. 1. Position of dorsoventral electrode tracks in four specimens of Hipposideros speoris (HS 2 5) projected onto an averaged horizontal section of the inferior colliculus (1C). The section level is 1640 μm from the surface. Points with crosslines mark the horizontal positions of the penetration hole on the surface of the 1C, that is the point of origin for all penetrations in a given animal. Points outside of 1C mark oblique penetrations which started on the dorsal surface of the 1C, but moved outside of 1C at this level (1640 μm) and more ventrally. o HS2;»HS3; DHS4;AHS5 sometimes emit multiharmonic CF/FM sounds (Habersetzer et al. 1984). The inferior colliculus is an egg-shaped structure which in its largest dimensions extends 2700 μm dorsoventrally, 2100 μm mediolaterally, and 1800 μm rostrocaudally (average from four brains, Fig. 3). In a total of 47 dorsoventral and oblique electrode tracks through the central nucleus of the 1C, best frequencies (BF) and thresholds of multiunit clusters were measured every 50 μm. In addition, tuning curves and PST-histograms of 82 single units were recorded. Tonotopic organization In all penetrations BFs progressed from low to high as the electrode was moved ventrally (Fig. 2). The highest BFs were recorded in the deepest areas, and ranged from 124 khz laterally, to 131 khz rostrally and up to 147 khz in the medial and caudal part. The lowest BF recorded was 11 khz on the surface close to the rostrolateral border of the 1C. This systematic frequency gradient was seen throughout the entire 1C, but the space alloted to equal bands of frequencies of the audible spectrum is not equal since high frequencies occupy far more than their predicted share of space. Representation of frequencies below those of the echolocation signals (up to 120 khz). Frequencies below the range of echolocation signals

4 274 R. Rübsamen et al.: Comparative tonotopy in bats X>0O OOO O OOO 0 caudal 0 H. speoris 3 frontal view 500 Oi H.speoris 3 lateral view «> dorsal distance from midline Cpm] distance from caudal 1C-border Cpm] Fig. 2a-d. Tonotopic arrangement of Best Frequencies (BF) along different electrode penetrations in Hipposideros speoris #3. Figures along the tracks give BFs of multi-unit clusters in khz. a Location of electrode tracks and b tonotopy in a transverse plane, c Location of electrode tracks and d tonotopy in a sagittal plane, ic Inferior colliculus; cer cerebellum; co cortex (<120 khz) were represented in superficial sheets, covering μm of the dorsolateral 1C (20-30% of the entire nucleus). The isofrequency contours are oriented roughly horizontally in the mediocaudal quarter of the 1C, then fall off progressively more steeply in the lateral and rostral directions, so that the isofrequency slabs ran in a more oblique direction in the rostral part of the 1C (Figs. 2 and 3 b), and nearly vertically along the lateral side of the 1C (Fig. 3 c). Since we do not have recordings from the dorsal layers close to the caudal and medial border of the 1C, it is not clear whether the isofrequency sheets tilt downwards along the medial and caudal border as well. It seems more likely that the isofrequency contours remain approximately parallel to the dorsal surface of the 1C since even at depths of 200 μm high BFs were recorded.

5 R. Rübsamen et al.: Comparative tonotopy in bats 275 A 1 khz-band is represented by about 4-6 μm thick neuronal slab for BFs up to 75 khz and only 2-3 μm thick on the average for BFs between E b Distance from caudal border[n ventral -500 and 110 khz. Thus this frequency range is the most sparsely represented part of the hearing range in the 1C of Hipposideros speoris (Figs. 2 and 3). Slabs representing BFs below khz extend to the lateral border of the nucleus, whereas neurons with BFs between 70 and 115 khz were never recorded in the most lateral and frontal penetrations. Isofrequency sheets representing this range of BFs were squeezed into a small space between the dorsal (lower frequency) sheets and the enormous neuronal mass dedicated to frequencies above 120 khz which occupies the central and ventral part of the 1C (Figs. 3 and 4). The frequency range with the smallest spatial representation is 80 to 100 khz. Neurons with these BFs were only found in a thin layer about 50 to 70 μm thick which extended from about 200 μm below the surface at the medial border midway through the 1C to a depth of about 500 to 800 μm in the caudolateral and rostrocentral region. The neuronal layer with BFs between 100 and 120 khz lies just beneath the previously described one and follows the same contours. However, it was on the average 120 μm thick, i.e. as thick as the slab for 60 to 80 khz. The external nucleus of the 1C, located rostrolaterally to the central nucleus, lacks a systematic frequency representation. Units are broadly tuned and lack a well defined BF (Fig. 4). Thus the representation of frequencies below 120 khz, i.e. 4/5 of the entire auditory frequency range in Hipposideros speoris is compressed into the dorsal layers of the 1C, occupying only about 1/4 of the 1C. Representation of frequencies in the range of the echolocation signal ( > 120 khz). The central mass of the 1C is devoted to the main frequency band of the echolocation signals ( khz) and higher frequencies, up to 147 khz. These frequencies are represented in the appropriate sequence within the continuous tonotopic order that begins with low frequencies in the dorsal cap of the 1C. The bulk of the neurons in the high frequency re- ' ' ' I ' ' ' ' I ' ' ' ' I I Distance from midline Fig. 3a-c. Tonotopy of the inferior colliculus in Hipposideros speoris as computed from electrode penetrations in four specimens (see Fig. 1). Figures show isofrequency contours in khz. a Outline of the 1C, rostrocaudal direction is enlarged 1.6 x for clarity. Bold lines indicate the positions of the transverse section T in c and the sagittal section S in b. b Tonotopy in the sagittal plane and c in the transverse plane. Bold lines give isofrequency contours as derived from electrode penetrations, dashed lines as inferred from adjacent recorded BFs of unit clusters. S and T mark the positions of the respective transverse and sagittal sections. Note the huge overrepresentation of frequencies higher than 120 khz. Dotted vertical line in b indicates orderly dorsoventral progression of BFs from 130 to 138 khz

6 276 R. Rübsamen et al.: Comparative tonotopy in bats J o H. speoris 4 frontal view gion have BFs between 130 and 140 khz, the range of the pure tone component of the echolocation signals. In a transverse plane the isofrequency contours resemble the concentric layers of an onion, with the apex for the 120 khz-contour in the central IC-region only about 400 to 500 μm below the surface (Fig. 3). The 120 khz-contour reaches the lateral border about 1800 μm below the surface and extends along the ventral quarter of the lateral side. Neurons with BFs from 140 to 147 khz are restricted to a wedge shaped area along the ventromedial border in the frontal half of the 1C. Medially, the 140 khz-contour starts about 800 to 1000 μm below the surface and thins out laterally along the floor of the 1C (Figs. 2d and 3 c). As best demonstrated in a parasagittal section (Figs. 2b, 3 b) the representation of frequencies above 120 khz fills the ventral part of the rostral 1C and all of the caudal 1C except for the dorsal cap. In the region representing 120 to 140 khz, a 1 khz-band was spread over a layer with an average thickness of 80 μm. This is in sharp contrast to the representation of lower frequencies in the dorsal 1C where a 1 khz-band occupies a sheet only 2-6 μ^i thick. Within the frequency range of the echolocation signals ( khz) the representation is highly differentiated. The largest neuronal layers were dedicated to a 2-3 khz-band which individually varied between 125 and 142 khz (Fig. 5). The thickness of these slabs for 1 khz-bands varied between 250 and 600 μm with an average of 400 μm dorsal medial 2000 distance from midline 1500 Fig. 4 a, b. Tonotopic arrangement in the lateral region of the inferior colliculus in Hipposideros speoris. a Position of electrode tracks in a transverse section and b tonotopic arrangement. Figures along electrode tracks give BFs of unit clusters in khz. In the external nucleus multiunits were broadly tuned with no well defined BF (crosshatched area) Auditory fovea and representation of the individual echolocation pulse frequency. One might assume that in each specimen the most expanded neuronal frequency representation would be that of the individual's CF echolocation signal frequency. We therefore recorded the CF-frequency emitted by each experimental animal and compared it to the BFs represented most extensively in the 1C. If the CF-frequency recorded from the handheld experimental animals would coincide with the BF most extensively represented in the 1C, it should correlate with the frequency of the steepest volume gradient in Fig. 5 b. As the graphs show this occurred only once, in specimen No. 4, and in the other four specimens the CF-frequency was up to 7 khz above or 2 khz below the thickest 1 khz-slab. The total frequency band represented in an expanded fashion was about 13 to 20 khz wide and always included the emitted CF-frequencies. The inconsistent position of the CF-frequency within the expanded tonotopic organization of the

7 R. Rübsamen et al.: Comparative tonotopy in bats 277 Hipposideros speoris No 3 a N = 234 * * -. * ;*^«*. *.* * " *. -v r i 1 1 i r B est Frequency [khz] 120 U'.O Fig. 5. a Individual audiogram in Hipposideros speoris based on thresholds of unit clusters in the 1C (dots). Arrow indicates the individual emitted frequency of the pure tone part of the echolocation signal, b Cumulative percentage of IC-volume dedicated to the frequencies of the echolocation signals. Note that the individual frequency of the emitted pure tone component {CF, vertical line) does not always coincide with the steepest part of the slope, i.e. with the thickest neuronal slab dedicated to a 1 khz-band. CF and dashed line: individual frequency of the tonal part of the echolocation pulses; in No. 1 this frequency varied within the limits of the two dashed lines No. 1 No.4 No.2 No.5 No Best F r e q u e n c y [khz] 1C may be due to incomplete recording scans for each specimen or to the variability of the CF-frequency emitted as demonstrated by No. 1 in Fig. 5 b. From the composite tonotopic organization of the 1C, derived from all penetrations in four specimens (Fig. 3), three conclusions may be drawn: 1. The individual bat's CF-frequency is part of a vastly expanded representation from about 120 to 142 khz. 2. In contrast to horseshoe bats the frequency overrepresentation in Hipposideros speoris is not confined to a few khz around the CF-frequency but is extended to lower frequencies (ca. 20 khz bandwidth). Thus most of the frequencies emitted in the final FM-component of the echolocation signal are also represented in a vastly expanded fashion. 3. A bat's CF-frequency does not occupy a fixed point within the range of most expanded frequency representation (Fig. 5 b). It may be found ventrally at the high frequency border (HS 3 and 2) or more medially (HS 5). This variability is reasonable, since in hipposiderids the frequency emitted by an individual bat may vary by several khz from day to day. H. speoris varied its CF by several khz (unpublished data) and Hipposideros lankadiva varied its CF from 3 khz below to 0.5 khz above the most frequently emitted frequency (Peters 1987). Response properties Audiograms were constructed based on thresholds of the multi-unit recordings (Fig. 5 a). The lowest thresholds demonstrate high sensitivity not only in the frequency range of the echolocation signals but also at much lower frequencies between 10 and 60 khz, and thresholds for these frequencies tended to be several db lower than for those from 120 to 145 khz. Consistently there was a shallow region of relative insensitivity between 70 and 110 khz, with thresholds between 0 and 10 db SPL. We did not systematically investigate the types of neuronal responses. Tuning curves were conven-

8 278 R. Rübsamen et al.: Comparative tonotopy in bats Frequency [khz] -110 Fig. 6. Representative tuning curves of single units in the inferior colliculus of Hipposideros speoris. Note the narrow tuning in the frequency range of 130 khz tional and typical examples are shown in Fig. 6. As expected, many of the units with BFs coinciding with the CF-frequency were narrowly tuned. QiodB-vahies above 30 were only found for units with BFs between 120 and 145, and two units with BFs at 132 and 134 had Q 10dB -values of 130. The same relationship between tuning and BF also held for Q 2 odb- va lues, which indicates that sharp tuning was not confined to stimulus intensities close to threshold. Some units had closed tuning curves with upper thresholds between 70 and 90 db SPL. As to the topography of neuron types two trends emerged from the multiunit recordings. a. In rostrolateral penetrations in the 1C units with BFs between 40 and 65 khz frequently were very broadly tuned and responded from 11 to 117 or 15 to 150 khz in some cases. The broadly tuned units responded more vigorously to frequency modulations than to pure tones. In the most rostral penetration in HS 4 (CF-component of the echolocation signal = 140 khz) at a depth of μm a large area was encountered with units tuned to 130 to 138 khz. These units preferentially responded to frequency modulations and also fired in synchrony with spontaneous vocalizations of the bat. b. Tuning curves of neurons medially, caudally and in the ventral half of the 1C frequently were narrow with or without broad low frequency tails. Megaderma lyra In contrast to the narrow band signals of Hipposideros speoris, Megaderma lyra emits a very brief (1 ms duration), broad band echolocation pulse consisting of several harmonics and extending over a frequency range of 20 to 110 khz. In M. lyra the 1C is an egg-shaped structure with its long axis oriented vertically. In its largest dimension it extends 3700 μm in the dorsoventral direction, 2300 μm rostrocaudally, and 3200 μm lateromedially (Fig. 7). Ventrally the 1C is compressed in its rostrocaudal dimension to about 1500 μm (Fig. 7d). In three specimens tonotopic organization was studied by recordings from unit clusters and 74 single units along 16 electrode penetrations (Fig. 7 b). Tonotopic organization In Megaderma lyra the tonotopic organization of the 1C follows the general mammalian pattern. It contains a dorsal-to-ventral sequence of low to high frequencies with equal frequency bands receiving fairly equal spatial representation. In their rostrocaudal dimensions the isofrequency contours coursed in a nearly horizontal plane (Fig. 7d). Only close to the rostral border did they bend slightly ventrally. Medially the 1C contours were also oriented horizontally, but from the center of the 1C towards its lateral border they were steeply inclined in the ventral direction. Consequently the lateral half of the 1C was completely dedicated to frequencies below 20 khz (Fig. 7 b). In the ventral region, isofrequency contours for higher BFs ended progressively more medially so that BFs higher than 60 khz were not represented in the lateral half of the 1C. The lowest BF recorded was 4.9 khz at the surface midway through the rostrocaudal extent of the 1C, and the highest BF (112 khz) was found on the ventral floor 3650 μm from the surface in the central part of the 1C. On the average, neuronal slabs of about 30 μ^i thickness were dedicated to a 1 khz-band. In contrast to the tonotopy in H. speoris, the isofrequency slabs tended to become larger with decreasing BFs in M. lyra. As demonstrated in a transverse section in Fig. 7d frequencies below 20 khz occupied the dorsal and lateral third of the 1C. This slab had an average thickness of 850 μm and reached its maximal extension of 1250 μm at the rostral margin. Thus the frequency range below that of the echolocation signal has the largest representation in the 1C of M. lyra. The thickness of neuronal slabs for frequencies in the range of the echolocation signals decreased with increasing BF: the khz slab was on the average 800 μm, the khz slab 600 μm, the khz slab 590 μm, the khz slab

9 R. Rübsamen et al.: Comparative tonotopy in bats ^ Ml 1000 I5O I 1000 Distance from midline Distance Fig. 7a-d. Tonotopy of the inferior colliculus in Megaderma lyra. a Outline of the inferior colliculus in specimen Ml 6. The rostrocaudal dimension is magnified 1.6 x for clarity. Bold lines indicate position of the sagittal S and transverse T section, shown in d and c respectively, b Position of the electrode tracks (crosses) in a horizontal section (2500 μm from surface of 1C), c Tonotopy in the transverse plane and d in the sagittal plane. Bold lines mark isofrequency contours in khz as derived from electrode penetrations, dashed lines as inferred from BFs of adjacent recorded unit clusters. Dotted line in d shows ventral position of the 100 khz-contour projected into this plane from a more caudal position. S, T and H mark the positions of the sagittal (d), transverse (c) and horizontal (b) sections respectively fro 500 μm and the slab for BFs higher than 100 khz was also 500 μm thick. Isofrequency slabs for BFs above 40 khz were thicker in the mediocaudal area and grew thinner as they sloped ventrally along the lateral side (Fig. 7c). Since we do not have penetrations from the medial border area of the 1C, we do not know whether the slabs also slope ventrally along the medial side. The course of the isofrequency contours in the centre of the 1C suggests that, if they do, it is probably only slightly. BFs with frequencies above 100 khz were confined to a medial band at depths below 2700 μm. This band was largest caudally (550 μm) and became thinner (300 μm) rostrally. Response properties When thresholds of the multiunit recordings are plotted versus their BFs, the lowest thresholds result in an audiogram for Megaderma lyra (Fig. 8) which closely resembles the behavioural one recorded by Schmidt et al. (1984). There is a sensitivity peak outside the frequency range of the echolocation pulses between 10 and 20 khz at thresholds below -20dBSPL. Samples of tuning curves from single units in M. lyra are shown in Fig. 9. The number of units that responded tonically and phasically was about equal; a few units responded phasically on-off to

10 280 R. Rübsamen et al.: Comparative tonotopy in bats Megaderma Iyra 70-1 Megaderma lyra o Best Frequency [khz] Fig. 8. Audiogram of Megaderma lyra as derived from thresholds of unit clusters recorded in the inferior colliculus (dots). N number of units recorded from all specimens Frequency [khz] a , STIMULUS INTENSITY [dbsptj pure tone stimuli. No systematic arrangement of units with different response patterns was detected. There were, however, three features in the responsiveness of the units which were specific to Megaderma lyra. a. Contrary to the general trend in mammalian audition, many low-frequency units in the 1C of M. lyra were surprisingly narrowly tuned, while those with BFs higher than 50 khz were broadly tuned (Fig. 9). This is also seen in the tuning of multi-unit clusters: as the electrode advanced ventrally, higher BFs and progressively broader tuning curves were encountered. With two exceptions, the QiodB-values of the units were always below 30 and thus tuning was never comparable to the narrow filtering for CF-frequencies in H. speoris. b. Many of the units tuned to lower frequencies had upper thresholds (Fig. 9). These neurons responded most vigorously to pure tone stimuli at rather low sound intensities (e.g. 29 db SPL) and stopped responding at intensities greater than db SPL, which is still a rather moderate intensity level. c. Even though the low frequency units were narrowly tuned, many of them responded preferentially to faint noises. Other units responded only to noise signals and never responded to any pure tones. Such noise-sensitive and noise-specific neurons were encountered over a large extent of the lateral and central 1C from the surface to deeper layers. These units fired vigorously to faint hisses, brushing of the beard or clothing etc, even though the bat was secluded in a sound attenuating chamber. Since we did not have a noise-generator Fig. 9. a Representative tuning curves for single units in the inferior colliculus of Megaderma lyra. Note the upper thresholds in units with BFs from 10 to 55 khz. b Four examples of nonmonotonic spike count functions of single units in the inferior colliculus of Megaderma lyra. The 'best amplitudes' are marked by vertical dashed lines in Madurai this remarkable responsiveness to faint noises could not be systematically studied. Discussion In laboratory mammals the tonotopic map in the inferior colliculus is organized as a stack of approximately horizontal isofrequency sheets in the medial part which tilt downward from caudal to rostral and tilt downward even more steeply from medial to lateral along the sides of the nucleus (Irvine 1986). Low frequencies are represented dorsally and higher ones more ventrally. The tonotopy in the 1C of Megaderma lyra and Hipposideros speoris conforms to this general pattern. However, in H. speoris the representation of frequencies between 120 and 140 khz is greatly expanded towards the surface of the central 1C throughout its rostrocaudal extent. The isofrequency sheets for lower BFs are compressed into the dorsal third of the 1C and give the ventral tonotopic arrangement the shape of a vaulted tunnel running rostrocaudally with frequency sheets for 130 to 140 khz in its center (Fig. 3). This huge overrepresentation of echolocation frequencies comes at the expense of the representation of low frequencies which are densely compressed (Fig. 10).

11 R. Rübsamen et al.: Comparative tonotopy in bats 281 HIPPOSIDEROS SPEORIS Best dorsal ventral Frequencies HORSESHOE BAT MEGADERMA LYRA khz khz > 80 khz HIPPOSIDERID BAT Fig. 10. a Tonotopic arrangement of best frequencies (BF) along electrode tracks in four mammals. Mouse: BFs encountered in a dorsoventral penetration through the inferior colliculus in Mus musculus (Stiebler and Ehret 1985). Horseshoe bat: BFs encountered in a penetration through the anteroventral cochlear nucleus of Rhinolophus rouxi (Feng and Vater 1985). False Vampire and hipposiderid bats: BFs recorded in dorsoventral penetrations through the inferior colliculus of Megaderma lyra and Hipposideros speoris (this study). Overrepresentation and compression of frequency representation is clearly indicated by two distinctly different slopes of the tonotopic gradient in horseshoe and hipposiderid bat. CF marks the frequency range of the pure tone component of the echolocation signals emitted by the rufous horseshoe bat and Hipposideros speoris. b Schematic volume diagram comparing frequency representation in the inferior colliculus of Hipposideros speoris and Megaderma lyra In both bat species the isofrequency contours crossed the cytoarchitectonic subdivisions of the 1C. We could not record any frequency discontinuities when the electrodes were moved from the pericentral nucleus (ICP) into the central nucleus. Tonotopy in the lateral external nucleus (ICX) is unclear since the units in this region were broadly tuned with undefinable BFs (Fig. 4). The continuity of isofrequency stacks across subdivisions conforms to that found in other mammals (monkey: Webster et al. 1984; cat: Serviere et al. 1984; rabbit: Aitkin et al. 1972; rat: Huang and Fex 1986; mice: Stiebler and Ehret 1985) and is in contrast to the subdivided tonotopy reported in the echolocating bat Pteronotus parnellii. In this bat Zook et al. (1985) cytoarchitectonically subdivided the central nucleus into an anterolateral division which contained units with BFs from 10 to 60 khz, a medial division dedicated to BFs from 65 to 115 khz, and a large, dominant dorsoposterior division with many small stellate cells which represented only frequencies from 60 to 65 khz. This narrow band coincides with that of the CF-component of the echolocation signal. The authors suggest that in P. parnellii the ancestral organization of the IC-laminae were horizontal stacks. During evolution the 60 to 65 khz sheet expanded dorsoanteriorly and ventromedially and displaced the sheets for lower frequencies to the lateral and those for higher ones to the medial side. This scenario could be also applied to the 1C of H. speoris with two modifications: 1. The hypertrophied sheets never extend to the surface of the 1C as in P. parnellii, and 2. the three different frequency ranges (below CF, around CF and above CF) do not appear to be represented in morphologically different subdivisions. In both bat species studied here we found the classical subdivision of the central nucleus into a dorsomedial and a larger ventrolateral division. Their borders did not coincide with any specific frequency contour. Although there might be further parcellations in the ICs, their possible demarcations are too poor and equivocal for further subdivision. In any case we could not find in H. speoris a specific morphological correlate to the hypertrophied slabs in the 1C representing the frequencies of the echolocation signals. Except for two bat species, overrepresentation of specific frequency bands within the range of audition has been never systematically studied. Huang and Fex (1986) mention that in the 1C of rats significantly more tissue was devoted to frequencies above 8 khz than to lower frequencies. No further details are given. In the 1C of mice, the volume of tissue devoted to each frequency band increases continuously with increasing BFs to reach a maximum for frequencies from 20 to 26 khz, then decreases again at higher BFs. The frequencies that are maximally represented coincide with those emitted by lost pups to initiate retrieval by their mothers (Stiebler and Ehret 1985). This indicates that overrepresentations might identify frequency bands of special importance for behaviour. The term ' overrepresentation' itself has never been clearly defined. As shown in Fig. 10 the best way to determine whether a frequency band is overrepresented is to examine a plot of best frequency versus place. A tonotopy containing an

12 282 R. Rübsamen et al.: Comparative tonotopy in bats overrepresentation is characterized by two different slopes, the inflection point demarcating the border frequency when the electrode track was perpendicular to the course of isofrequency sheets. We suspect that overrepresentations of behaviourally relevant frequency bands are more common than so far reported. For instance, a plot of BF versus recording depth in the 1C of Tadarida brasiliensis was shown with a straight slope suggesting that frequencies are represented in an orderly tonotopy (Bodenhamer and Pollak 1981). The data, however, are best fitted by two lines with different slopes which intersect at 26 khz, a steep one for lower frequencies and a shallow one for higher frequencies. This would indicate that in this echolocating bat frequencies below 26 khz are overrepresented compared to higher frequencies. Auditory foveae There is a discussion among us what should be called an auditory fovea. The term may designate either overrepresentation of a frequency band, or a narrow frequency band with very sharp tuning characterized by unusually high Q 10dB -values. The term was coined by Schuller and Pollak (1979) because of the following analogies between a visual fovea and the frequency overrepresentation associated with Doppler shift compensation in horseshoe bats: 1) The foveal part of of the visual field is overrepresented in the neuronal centers of the visual pathway. Similarly, a frequency band within the auditory range is overrepresented on the basilar membrane and at all subsequent stages of the ascending auditory pathway. 2) A moving object is kept centered on the fovea by eye movements. Similarly, a horseshoe bat maintains an insonified target within the 'auditory fovea' by Doppler shift compensation. Therefore an auditory fovea requires a frequency overrepresentation and a control of the emitted frequency in the echolocation signal in order to actively maintain the echo within the fovea. A broader definition would include any overrepresentations of frequency bands that occur on the basilar membrane whether or not a focustracking mechanism is present. This would be a more suitable definition for comparative auditory physiology. So far, however, only very few frequency maps of basilar membranes exist, so we will use the first definition which restricts the possible occurrence of auditory foveae to echolocating species. According to this definition not only horseshoe bats and mustached bats, but also, hipposiderid bats have an auditory fovea: as this study shows, the frequencies of their echolocation signals are greatly overrepresented in the auditory pathway and a corresponding expanded frequency representation on the basilar membrane has been found in Hipposideros lankadiva (Peters 1987); they also compensate for Doppler shifts of echoes (Gustafson and Schnitzler 1979; Habersetzer et al. 1984). In the 1C of horseshoe bats the representation of the auditory fovea seems to be restricted to a frequency band from 7 khz below the emitted CFfrequency to 3 khz above (Schuller and Pollak 1979). In the auditory cortex this band extends from 10 khz below the CF-frequency to 3 khz above (Ostwald 1984). Apparently, in horseshoe bats the fovea includes major frequencies of the final FM-part of the echo. In the 1C of H. speoris overrepresentation includes 2/3 of the final FMsweep in the echoes. Therefore the auditory foveae of both rhinolophid and hipposiderid bats incorporate at least some frequencies of the FM-component, and the difference between the two is only a matter of degree: the foveae of horseshoe bats are relatively narrowly tuned and the foveae of hipposiderid bats are more widely tuned. The auditory fovea in H. speoris is about 20 khz wide; similarly the expanded frequency representation on the basilar membrane in Hipposideros lankadiva ranges from 63 to 80 khz (Peters 1987). Such wide foveae might also explain why H. speoris, H. bicolor and H. lankadiva can afford to vary the frequency of the CF component by 500 to 3000 Hz from day to day and on the average compensate for Doppler shifts of the echoes with an accuracy of only 55% (Habersetzer et al. 1984; Peters 1987). Behavioural tests show that hipposiderid bats with a wide fovea detect wing beating prey as well as do horseshoe bats (Link et al. 1986). In discriminating wingbeat rates, hipposiderid bats with brief CF-components, and even FM-bats without a CFcomponent in their echolocation signals, perform surprisingly well compared to horseshoe bats with their long CF-signals (R. Roverud, personal communication). Why then a narrowly tuned fovea in horseshoe bats? We propose that overcoming echo clutter and not wingbeat detection per se was the main evolutionary driving force for perfecting pure tone echolocation. A pure tone signal is more noise resistant than any other type of a signal if the receiver is tuned to the pure tone frequency. Theoretically, the narrower the receiving filter, the longer the duration of the signal must be. This may explain why horseshoe bats and mustached bats, with their sharply tuned cochleae, use long pure tone components and perform close to 100% Doppler shift

13 R. Rübsamen et al.: Comparative tonotopy in bats 283 compensation, while hipposiderids with their less precisely tuned auditory foveae can afford to use a brief CF-signal of a more variable frequency and less complete Doppler shift compensation. Thus, the need for clutter rejection might have driven species that use pure tone echolocation signals to develop narrow auditory foveae which necessarily demand long duration signals. The information that can be obtained from such long pure tones is largely limited to detection of glints from fluttering insects. Therefore, horseshoe bats should depend more on wingbeat detection than do hipposiderids which may also detect non-wingbeating insects moving on the ground (Habersetzer et al. 1984; Link et al. 1986). We suggest that in evolution the development of narrower auditory foveae is an adaptation for clutter resistance and the increasing restriction to wingbeat detection is its inadvertent byproduct. The wider auditory foveae of hipposiderids invite speculation on the phylogeny of pure tone echolocation systems. Long narrow band signals are used as a searching signal in many bat species that forage in open spaces above the canopy (Pye 1980; Neuweiler 1984). Use of long narrow-band signals is thus interpreted as an adaptation to long range echolocation, and the signal will necessarily be sensitive to glint-reflecting targets, i.e. wingbeating insects. Narrow cochlear tuning for the frequency of the search signal, coinciding with the best frequency of hearing, would allow echolocation to become more clutter resistant (Neuweiler 1984), and such a bat species could effectively forage close to vegetation. Rhinopoma hardwickei may be a species in the process of evolving clutter resistance through narrower filtering (Neuweiler et al. 1984). When such auditory filters are complemented by Doppler shift compensation and by focusing the filter precisely to the echo frequency, the resulting clutter resistant echolocation system allows detection of small wingbeating prey even within dense foliage. This advantage is offset by the restriction of prey detection to wingbeating insects. Comparison between Megaderma and Hipposideros It is rewarding to compare the tonotopy of Megaderma lyra and Hipposideros speoris and its implication for prey detection. Both species belong to the same superfamily Rhinolophoidea. The two species have to detect moving prey in a complex, echo-cluttered environment. Each has solved this problem in a very different way (Marimuthu and Neuweiler 1987; Habersetzer et al. 1984). The smaller species, H. speoris, hunts only flying insects, close to but never within the foliage, while the larger species, Megaderma lyra, uses a sit-and-wait strategy and often catches larger prey from the ground. H. speoris detects its prey exclusively by echolocation and specializes in wingbeat detection by means of an auditory fovea as described above. In contrast, Megaderma lyra has given up echolocation for prey detection and instead passively listens for and locates noises generated by ground-dwelling prey. Consequently the tonotopy in Megaderma lyra is systematically organized over a wide range of frequencies with a moderate overrepresentation for frequencies between 10 and 20 khz (Fig. 7). This frequency band is a prominent part of the power spectrum of rustling noises (Marimuthu and Neuweiler 1987). Thus, as exemplified for the two species described here, the tonotopy faithfully reflects the specific acoustical behaviour of a species. In addition, the huge pinnae of Megaderma lyra not only result in a precise sound lateralization which is best at 20 khz (Witzke 1987), but also contribute to the low thresholds at lower frequencies (-24 db SPL at 18 khz, Fig. 8). This incredible sensitivity to even the faintest noise may be attributed to the large proportion of IC-units which have their best amplitude at sound intensities as low as 19 db SPL, their upper thresholds at db SPL, and that respond preferentially or exclusively to noise signals. This specificity to faint noises was in sharp contrast to recordings from IC-units in Hipposideros speoris where units with upper thresholds or noise sensitivity were rare. However, noise sensitive neurons may be a prominent, though not exclusive, feature of the auditory system of Megaderma lyra. Such neurons have occasionally been reported in other echolocating bat species as well (Suga 1969; Metzner, personal communication). Among 125 units recorded from the 1C of Myotis yumanensis and Plecotus townsendi Suga (1969) found two 'pure tone deaf units which responded only to noise and FMsignals, and three 'noise-specialized' units which only responded to noise. Thus it is conceivable that many bat species have the appropriate neuronal mechanisms to passively detect prey generated noise, as well as elaborate auditory neuronal areas reserved for echolocation. An analysis of the afferent input and of the synaptic connectivity in such faint noise sensitive neurons provides some insight into how feature detectors may be created in the auditory system. In contrast to these features of auditory neu-

14 284 R. Rübsamen et al.: Comparative tonotopy in bats rons in Megaderma lyra, we found many neurons with BFs within the acoustical fovea in H. speoris which were especially sensitive to linear frequency modulations around the BF. As in horseshoe bats (Vater 1982; Schuller 1984), this specific feature is probably correlated with the narrow tuning of these units. Their Q 10d B-values are far above those of any units in Megaderma lyra. This modulation sensitivity should greatly enhance glint detection in time smeared narrow band echo sequences reflected from vegetation. The comparison of tonotopy and responsiveness of auditory units in Megaderma lyra and Hipposideros speoris demonstrates the wide capacity for diverse adaptations in the mammalian auditory system and the precision with which these adaptations match the species-specific acoustical behaviour. 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