Responses of cerebellar neurons of the CF-FM bat,Pteronotus parnellii to acoustic stimuli

Brain Research, 252 (1982) 167-171 Elsevier Biomedical Press

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Responses of cerebellar neurons of the CF-FM bat, Pteronotus parnellii to acoustic stimuli PHILIP H.-S. JEN, XINDE SUN* and TSUTOMU KAMADA**

Division of Biological Sciences, Universityof Missouri, Columbia, MO 65211 (U.S.A.) (Accepted July 20th, 1982)

Key words: CF-FM bats - - cerebellar auditory neurons

Single units (125) which faithfully discharged action potentials to acoustic stimuli (35 ms in duration with 0.5 ms rise and decay times) were recorded in the cerebellar vermis and hemispheres of the CF-FM bat, Pteronotusparnellii. These units had response latencies between 1.5 and 27 ms and minimum thresholds between 2 and 83.5 dB SPL. Best frequencies (BFs) of these units ranged from 30.32 to 79.28 kHz, but more than half (64 units, 51.2~) were between 59.73 and 63.32 kHz. While most tuning curves of these units were either broad or irregular, those curves with BFs tuned at around 61 kHz which is the frequency of the predominant CF component of the bat's echolocation signals were extremely narrow with Q10-dBvalues as high as 153. Those units (29) with BFs tuned near the 61 kHz also showed off-responses. These data indicate that auditory specialization for processing of speciesspecific orientation signals also exists in the cerebellum of this bat. For orientation, the C F - F M bat Pteronotus parnellii emits ultrasonic signals which consist of a long (20-25 ms) constant-frequency (CF) component followed by a short (2-5 ms) downward-sweeping frequency-modulated (FM) component. Each component is composed of 3-4 harmonics in which the second harmonic (around 61 kHz) is the predominant one. During echolocation, the bat adjusts the frequency of its emitted C F component to compensate for the positively Doppler-shifted echoes 19. Neurophysiological investigation has demonstrated that the auditory system of this bat, f r o m peripheral to central, is characterized by a large number of neurons that are specialized for fine frequency analysis of the Doppler-shifted echoeslS, 23-25. Such an overrepresentation of sharply tuned neurons for processing the species-specific C F signals is not found in the F M bats which use frequency-modulated signals for echolocation z2. As is well known, the cerebellum of an animal plays an important role in motor orientation 5 and receives auditory input from the peripherya, 21 and

the cerebrum4, 20. The ability of the cerebellum to process acoustic signals is apparently essential in orienting an animal toward a sound source in space. Recent studies t0-1z have shown that a large area of the cerebellum of the F M bats, Myotis lucifugus and Eptesicus fuscus contains units responding to the bat's ultrasonic signals, but units which are sharply ttmed to an extremely narrow frequency band were not found. In the C F - F M bat, Pteronotusparnellii, a study of evoked potentials for the median lobe of Ingvar of the cerebellum shows both on and off responses sharply tuned to the bat's predominant CF componenO. We report here that our single unit studies show that the cerebellum of this bat also contains units which are specialized for analysis of Doppler-shifted echoes. Four Pteronotus parnellii parnellii (body weight 11.5-13.4 g) from Jamaica and two Pteronotus parnellii rubiginosus (body weight 19.2-20.4 g) from Panama were used for this study. Each animal was lightly anesthetized with Nembutal (25-30 mg/kg body weight) for surgery. Whert necessary, ether was

* Present address: Department of Biology, East China Normal University, Shanghai, People's Republic of China. ** Present address: Department of Oral Physiology, Hokkaido University, Sapporo, Japan. 0006-8993/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

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Fig. 1. Post-stimulus-time (PST) histograms representing responses of 3 single neurons to tone bursts of 63.0 (A), 61.19 (B), and 60.14 (C) BHz. The stimulus intensity is indicated by the figures of the left of the histograms. The duration of the tone burst and its rise-decay time are 35 and 0.5 ms, respectively. Each stimulus was delivered 50 times. The bin width of the histogram is 100 #s. On the bottom, the response of each neuron to a tone burst is shown. used during the initial phase of the surgery. The flat head of a 1.9 cm long nail (3 mm in diameter) was motmted to the anterior portion of the exposed skull with glue and dental cement. The animal was then moved inside a sound-proofed room (temperature 35-38 °C) and tied onto a metal plate with its head immobilized by fixing the shank of the nail into a metal rod with a set screw. A small hole was made in the posterior portion of the bat's skull overlaying the cerebellum. This hole was subsequently enlarged so that 3 M KC1 glass micropipettes (tip resistance 2-8 M f~) electrodes could be inserted into as many places as possible to record neural activity from the same bat. I f the bat awakened and its brain pulsa-

tion interfered with recording, an additional dose of anesthetic (one-third of the original dose) was administered. Recorded action potentials were amplified with conventional techniques and sent to an oscilloscope and audio monitor. The response patterns of single units were studied in post-stimulus-time (PST) histograms with a special purpose computer (Nicolet Med 80). The electronic equipment used to generate acoustic stimuli were similar to those described previously 2z. Acoustic stimuli (35 ms in duration with 0.5 ms rise-decay times) were delivered from a condenser loudspeaker which was placed at 35.3 cm in front of the bat. The loudspeaker was calibrated with a Brfiel and Kjaer 1/4 in. microphone placed at

169 the bat's ear and its output was expressed in dB SPL referred to a 0.0002 dyne/cmz root mean square. As in previous studies~0-~2, penetration of the electrode through the cerebellar cortex encountered marry spontaneously active cells (10-100 impulses/ s). With one exception, the firing (amplitude 0.5-5 mV) of these cells was not affected by the acoustic stimuli. Judging from their waveforms and injury discharges, they likely originated from Purkinje cells1,5. In addition to such cells, recordings were made from 125 units whose firing activities were influenced by the presented acoustic stimuli. Among them, 54 units (43.2 ~) were isolated from cerebellar vermis and the remaining 71 units (56.8~o) from cerebellar hemispheres. Only about two-thirds of these cerebellar auditory units were spontaneously active and their action potentials were small (300-500 #V). Most (100 units, 80 ~) of these auditory units were isolated at depths less than 990 #m. In the remaining 25 units, 16 were recorded between 1000 and 1400 #m and 9 were between 1500 and 2700/~m. Response patterns of 96 units were studied. Phasic responses were found in 90 units; 18 units generally fired only one impulse per stimulus (Fig. 1A) and 72 units fired 3-5 impulses during the acoustic stimulus (Fig. 1B). Tonic responses were found in only 5 units which fired action potentials throughout acoustic stimulus. The response pattern of the remaining unit was rather interesting. It fired phasically when the stimulus intensity was up to 20 dB above its minimum threshold (MT). It became a tonic responder when the stimulus intensity increased from 20 to 40 dB above its MT. Then, it changed back to a phasic responder with further increases in stimulus intensity. The complex response pattern of such a unit has been found in other CF-FM batslZ, 15. Latencies of response of 122 units were examined. They ranged between 1.5 and 27 ms but most (100 units, 829/00)were below 10 ms. Those 9 units whose latencies were shorter than 3 ms, may receive their inputs directly from the dorsal cochlear nucleus 17. It is interesting to note that the latencies of the 18 oneimpulse phasic units always fluctuated from stimulus to stimulus regardless of stimulus intensity so that their PST histograms are similar to those of the 72 multi-.impulse phasic units (Fig. 1A and B). There were 29 units which were tuned near the bat's second

harmonic CF frequency and which also discharged impulses upon the cessation of the acoustic stimulus (Fig. 1C), similar to neurons in the auditory nuclei7, 13,16,1s,24,25. Latencies of these off responses were between 3.5 and 27 ms upon cessation of the stimulus but most (20 units, 69 ~) were below 9 ms. The minimum thresholds of these 125 units were between 2 and 83.5 dB SPL but most (84 units, 67.2 ~) were below 52 dB SPL. Although the best frequencies (BFs) of these units ranged from 30.32 to 79.28 kHz, 64 units (51.2~) had their BFs between 59.73 and 63.32 kHz which corresponds approximately to the predominant CF component in the bat's orientation sounds and Doppler-shifted echoes. As all these units were sampled across the vermis and hemispheres, it is tempting to speculate that there is also a disproportionate representation for processing the biologically significant CF component of the bat's orientation signals irt the bat's cerebellum, similar to the situation in the auditory cortexz3 and other auditory nuclepS. 25. However, a careful mapping of the auditory area such as that performed in the cerebellum of the FM bat, Myotis lucifugus 12 is essential in order to confirm such a speculation. Coding of the stimulus frequency by these cerebellar auditory units was studied by measuring their tuning curves. Among the 80 tuning curves measured, their shapes cart be described as either broad, narrow or irregular. All of the 20 (25~) broad tuning curves have simple triangular shapes (Fig. 2A). They were tuned to a rather wide band of frequency with gradually increasing slopes on both sides of their BFs. Their Q10-dB values (a value obtained by dividing the BF by the bandwidth at 10 dB above a unit's MT 14) are between 1.18 and 11.81. The shapes of 11 (13.8~) irregular tuning curves consist of at least two peaks (Fig. 2B). These 11 units fired action potentials to stimuli over an extremely broad frequency spectrum similar to previous studies in the cerebellum of cats1, 2. The Q10dB values calculated for the peaks having the lowest MTs ranged between 3.26 and 62.4. However, these Q10-dB values do not faithfully reflect the sharpness of these turfing curves because of the irregular shapes of the curves. The tuning curves of the remaining 49 (61.2 ~) units were extremely narrow "solid curves of Fig. 2C, D, E). They had very sharp

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Fig. 2. Representative tuning curves of 8 cerebellar auditory neurons that are either broad (A), irregular (B) or nar row (C-E) Respectively, the ordinates and abscissae represent the stimulus amplitude in dB SPL and stimulus frequency in kHz. The upper solid lines represent the maximum available sound stimulus level. C-E: the solid curves are the tuning curves of on-responses and the dashed curves are the tuning curves of off-responses (see text for details).

slopes on both limbs of the BF. Their Q10-dB values are between 14.10 and 153.05 with most (41 units, 83.7~) above 25. One unit had a BF of 51.54 kHz. The remaining 48 units had BFs between 61.17 and 64.67 kHz. Threshold curves of off responses could be measured for 24 of the 49 units and they are also extremely narrow with Q10-dB values ranging between 8.94 and 208.7 (e.g. dashed curves of Fig. 2C-E). From an evolutionary perspective, the auditory sensitivity of a species evolves under the selective pressure of its acoustic environment. Thus, the auditory system of an animal is most effective in detection of its biologically significant acoustic signals. Such an auditory specialization for processing

of species-specific orientation sounds has been demonstrated in several species of echolocating bats (ref. 8). Previous studies on the cerebellum of FM bats 10-12 and the present study on a CF-FM bat indicate that there exists such an auditory specialization in the bat's cerebellum. Thus it is not surprising to find out that while auditory cerebellar units of the F M bat and C F - F M bat are generally tuned to a wide spectrum of frequencies, those cerebellar traits of the CF-FM bat with BFs tuned at around the bat's predominant CF component are as sharply tuned as those units in the bat's auditory nucleilS, z4, zs. In view of the role of the cerebellum in motor orientation 5 and the fact that echolocating bats rely upon sound processing for survival 6, it should be

171 interesting to study h o w these cerebellar a u d i t o r y units c o d e the i n f o r m a t i o n o n the source o f the a u d i t o r y stimulus in space. This w o r k was s u p p o r t e d b y a g r a n t f r o m N a t i o n al Science F o u n d a t i o n (BNS 80-07348), a R e s e a r c h C a r e e r D e v e l o p m e n t A w a r d f r o m N a t i o n a l Institutes o f H e a l t h ( U S P H 1-K04-NS-00433-02) a n d a

1 Aitkin, L. M. and Boyd, J., Responses of single units in cerebellar vermis of the cat to monaural and binaural stimuli, J. NeurophysioL, 38 (1975) 418--429. 2 Altman, J. A., Bechterev, N. N., Radionova, E. A., Shmigidina, G. N. and Syka, J., Electrical responses of the auditory area of the cerebellar cortex to acoustic stimulation, Exp. Brain Res., 26 (1976) 285-298. 3 Dow, R. S., Cerebellar action potentials in response to stimulation of various afferent connections, J. NeurophysioL, 2 (1939) 543-555. 4 Dow, R. S., Cerebellar action potentials in response to stimulation of the cerebral cortex in monkeys and cats, J. NeurophysioL, 5 (1942) 121-136. 5 Eccles, J. C., Ito, M. and Szentagothai, J., The Cerebellum as a Neuronal Machine, Springer, Berlin, 1967. 6 Griffin, D. R., Listening in the Dark, Yale University Press, 1958, reprinted Dover, 1974. 7 Grinnell, A. D., Rebound excitation (off responses) following non-neural suppression in the cochlea of echolocating bats, J. comp. PhysioL, 82 (1973) 179-194. 8 Grinnell, A. D., Neural processing mechanisms in echolocating bats, correlated with differences in emitted sounds, J. Acoust. Soc. A, 54 (1973) 42-48. 9 Guterman, L. R. B., Adaptations of the Posterior Colliculus and Cerebellum of the Bat, Pteronotus parnellii for Biosonar, Ph.D. thesis, Dept. Zoology, Yale Univ. 1977. 10 Jen, P. H.-S., Echolocation in the bat" obstacle avoidance by the bat and signal coding in the bat's cerebellum, Proc. nat. Sci. Counc. (ROC), 6 (1982) 71-80. 11 Jen, P. H.-S. and Schlegel, P. A., Neurons in the cerebellum of echolocating bats respond to acoustic signals, Brain Research, 196 (1980) 502-507. 12 Jen, P. H.-S., Vater, M., Harnischfeger, G. and Rubsamen, R., Mapping of the auditory area in the cerebellar vermis and hemispheres of the little brown bats, Myotis lucifugus, Brain Research, 219 (1981) 156-161. 13 Jen, P. H.-S. and Suthers, R. A., Responses of inferior collicular neurons to acoustic stimuli in certain FM and CF-FM paleotropical bats, J. comp. PhysioL, 146 (1982) 423-434. 14 Kiang, N. Y.-S., Discharge Patterns of Single Nerve Fibers

g r a n t f r o m the R e s e a r c h C o u n c i l o f U n i v e r s i t y o f M i s s o u r i at C o l u m b i a ( D H H S B i o - M e d 2491) to P. H.-S. Jen. W e t h a n k Drs. G. A u d e s i r k a n d T. A u d e s i r k for c o r r e c t i n g the English o f this m a n u script a n d Drs, G. P o l l a k a n d A. G r i n n e l l f o r s u p p l y i n g the a n i m a l s used in this research. W e also t h a n k S. A l l i s o n a n d G. H. G u for their technical assistance.

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