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Processing of pure-tone and FM stimuli in the auditory cortex of the FM bat,Myotis lucifugus
Hearing Research, 61 (1992) 179-188
179
© 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00 HEARES 01777
Processing of pure-tone and FM stimuli in the auditory cortex of the FM bat, Myotis lucifugus Sharon Shannon-Hartman, Donald Wong and Masao Maekawa Department of Anatomy, Indiana University School of Medicble, Indianapolis, Indiana, USA (Received 30 September 1991; Revision received 24 March 1992; Accepted 31 March 1992)
FM bats perceive their surroundings during echolocation by analyzing frequency-modulated (FM) acoustic signals. Results from this study indicate a cortical organization in Myotis lucifi~gus which is largely made up of neurons sensitive to FM sounds (FM-sensitive neurons). Three types of neurons were distinguished by their responses to pure-tone and FM stimuli: (1) Type I FM-sensitive units (83%), Type II FM-sensitive units (13%) and pure-tone sensitive units (4%). Type I FM-sensitive units responded to pure tones, but exhibited greater response magnitudes to FM stimuli when the best FM swept through the BF. An orderly frequency representation was found when the frequencies of pure tones essential for response (EPTs) in Type I units were mapped along the cortical surface. The EPTs for Type I neurons were usually found within the last millisecond of a downward FM sweep. As outlined by two neuronal network models, both the responses of Type I and It units could likely result from the convergence of excitatory and inhibitory lower level neurons with slightly differing BFs. Type II units were selective for an FM sweep and showed negligible to no response to pure-tone stimuli. Pure-tone sensitive units exhibited weak or no responses to FM stimuli. These neurons were clustered in a small area located rostrodorsal to the tonotopic zone and had significantly lower best frequencies than adjacent EPT frequencies of Type I FM-sensitive neurons. Spectral processing; Auditory cortex; Frequency organization; FM sweep; Echolocating bats
Introduction
Acoustic stimuli in natural environments commonly consist of temporal shifts in amplitude and spectral composition. The responsiveness of neurons to timevarying stimuli, such as FM sweeps or species-specific vocalizations, has been examined neurophysiologically in the auditory cortex of several mammals, including the cat (Whitfield and Evans, 1965; Mendelson and Cynader, 1985; Phillips et al., 1985), squirrel monkey (Wollberg and Newman, 1972) and bat (Suga, !965b). Cortical neurons have been found that not only were selective to modulation in spectra, but also to sweep direction (Suga, 1965b; Whitfield and Evans, 1965; Phillips et ai., 1985). Moreover, some neurons which exhibit strong responses to FM stimuli have been shown to respond very poorly to pure tones (Suga, 1965b; Whitfield and Evans, 1965). These studies have therefore revealed neuronal populations that are capable of processing different aspects of time-varying stimuli. FM sweeps provide important cues for the recognition of many complex sounds, such as speech. FM sounds are particularly important to bats that use these Correspondence to: Donald Wong, Department of Anatomy, Indiana University School of Medicine, Indianapolis, IN 46202-5120, USA. Fax: (317) 274-3318.
signals for echolocation since they must analyze target-reflected echoes of their emitted sounds to perceive their surroundings (Griffin, 1958). FM sound pairs have been employed in many neurophysiological studies to examine target perception in the auditory cortex of bats (Suga eta!., 1978, 1979, 1983; Feng et al., 1978; O'Neill and Suga, 1979, 1982; Sullivan, 1982; Suga, 1988; Wong and Shannon, 1988; Berkowitz and Suga, 1989). The present study uses Myotis lucifugus, a bat which echolocates primarily with FM sweeps (Griffin, 1958), as a model for the study of basic processing of FM sounds. Suga (1965b) was the first to describe the response properties of cortical neurons stimulated by FM pulses in Myotis. Results from Suga's study indicated that neuronal sensitivity to sweep direction was dependent upon tile temporal sequence in which the stimulus triggered excitatory and inhibitory response areas. A frequency representation based on best frequencies was demonstrated in the tonotopic zone of Myotis cortex usLng pure-tone stimuli (Suga~ 1965b; Wong and Shannon, 1988). Neurons sensitive to FM stimuli were located within this zone and in the overlapping delaysensitive zone (Shannon and Wong, 1989). Within the overlapping zone, neurons that responded to pure-tone and FM stimuli were also found to be sensitive to FM-FM pairs at specific delays and for specific reped-
180
tion rates (Wong et al., 1992). The present study further examined neuronal responsiveness to FM stimuli in Myotis by focusing on whether spectral selectivity existed for a partier'at portion of the FM sweep. The relationship of spectral processing of FM sounds to the tonotopic map was also studied in the present investigation. For auditory-cortical units exhibiting sensitivity to FM stimuli, portions of the FM signal were deleted to determine which FM components were necessary for neuronal response. Cortical organization with regard to neurons exhibiting sensitivity to single stimuli (either a constant frequency tone or FM sweep) was studied by mapping the cortical locations of FM-sensirive neurons. Mapping results were analyzed and related to the known tonotopie organization in Myotis (Suga, 1965b; Wong and Shannon, 1988). Results from these analyses have implications on the basic processing of FM stimuli in the auditory cortex of FM bats.
Materials and Methods
Preparation All experimental protocols and animal care for this study have been approved by the Laboratory Animal Resource Center (LARC) of Indiana University School of Medicine (approval number MD No. 621). A total of 12 tittle brown bats (M. iucij~gus), each with a body wei[ht of 5-10 g, were used. Prior to surgery, the neuroleptanalgesia Innovar-vet (0.04 mg/kg fentanyl and 2 mg/kg droperidol mixture) was injected intramuscularly. A midsagittal incision was made on the scalp and the temporalis muscles were retracted laterally to expose the skull. A small rod (1.5 cm) was mounted onto the dorsal skull with dental cement and eyanoacrylate glue. All surgical wounds were cleaned and treated with antibiotic ointment (Furacin). A local anesthetic (Lidocaine) was topically applied onto exposed tissue to minimize discomfort or pain. Neural recordings commenced after a post-surgical recovery period of 2-7 days. Neural recordings were conducted in an individual animal for up to 8 h on alternate days. The bat was situated in the center of an echo-attenuated, soundproof room thermally maintained at 30-32°C. The bat's body was restrained in a plexiglass holder, which was suspended with an elastic band from a horizontal metal b~.x'. To obtain stable neural recordings, the head was immobilized by securing the skull-mounted rod onto a post with a set screw. All posts suspending the bat and anci~oring the micromanipulators were screwed onto a vibration-isolated experimental table (TMC Micro-g). Previous application of dental cement to the skull surface overlying the auditory cortex usually eliminated the need to retract the scalp for each recording session. However, Lidocaine was topically applied to surgical
wounds if manipulation was necessary. Throughout the r~,cording session, the animal was vigilantly monitored for signs of discomfort. Drinking water was frequently offered. Otherwise, the bat ~ested quietly during recordings~ At the end of each recording session, care was taken to keep the skin around the skull clean and free of infection with topical administration of antibiotic. Typically, five to eight recording sessions were necessary to complete an experiment on one animal. The approximate location of the auditory cortex was determined on the basis of a characteristic bifurcation of a major blood vessel. The vessel was readily visible beneath the thin translucent skull. To provide a general guide for electrode insertion, a sketch of the bat's auditory cortex and its overlying vessels were drawn with the aid of an operating microscope equipped with a grid ocular in one eyepiece. The sharp tip of a scalpel blade was used to make small craniotomies ( ~ 50/.tm in diameter) for electrode insertion into the auditory cortex. During this procedure, the bat did not exhibit excessive movements associated with pain. Electrodes were initially lowered onto the cortical surface with micromanipulators under the visual guidance of an operating microscope and then advanced into the brain with a remote hydraulic microdrive (Narishigc MO-10) outside the soundproof chamber. Columnar organization according to best frequency has been demon: strated in the auditory cortex of FM bats (Shannon, 1988; Jen et. al., 1989) and thus, all electrode penetrations were subsequently charted onto a drawing of the auditory cortex for later reconstruction of the surface location ,of recorded units.
Acoustic stimulation Free-field sounds were presented from a condenser loudspeaker located 75 cm directly in front of the animal. The loudspeaker was calibrated and found to be flat ( + 3 dB) between 20 and 140 kHz. Pure tones and FM sweeps were electronically generated with a Wavetek oscillator and shaped into bursts with a custom-built electronic switch. FM sounds swept linearly 60 kHz in 4 ms with 0.25 ms rise/fall times to simulate biosonar signals emitted by M. lucifugus (Griffin, 19581. To determine whether neurons exhibited a selectivity to a specific part of the FM sound, the entire 4-ms sweep was arbitrarily divided into either (a) four equal segments or quarters (I, II, III, IV), each sweeping 15 kHz in 1 ms (Fig. 1); (b) 2 continuous quarters (I-II, II-IlI, Ill-IV), each sweeping 30 kHz in 2 ms; or (c) 3 coatinuous quarters (I-III, II-IV), each sweeping 45 kHz in 3 ms. The rise/fall time was fixed at 0.25 ms for all FM stimuli used.
Neural recording and data analysis Extracellular unit recordings were obtained in the unanesthetized animal with lacquer-coated tungsten
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electredeg (tip diameter - 5 / t m ) using conventional eiectrophysiological techniques (for details see Wong et al., 1992). In brief, neural activity was amplified, filtered, and displayed on an oscilloscope. Soundevoked unit activity was selected by a window discriminator (Bak Electronics DIS-l) and fed into an audiomonitor and into a 386SX IBM-compatible personal computer (Gateway 2000) for real-time data acquisition. Under software control, the computer both triggered analog sound-generating equipment (ore-set manually during audiovisual measurements) and sampled selected neural activity at a rate of 2 kHz (0.5 ms binwidth) during repetitive sound stimulation for up to 100 trials. Spike counts were represented over time in the form of peri-stimulus-time (PST) and PST cumulative (PSTC) histograms for comparison of response magnitudes. Data acquired in real-time from customwritten software were exported into several commercially-available graphics software for off-line analysis and printouts. Neurons responsive to single FM sweeps were isolated using 60 kHz sweeps in 4 ms as search stimuli. Maximal-response a n d / o r frequency-threshold measurements were then made to determine the best FM for response (BFM). Units were classified as FM-sensitive if either the response magnitude evoked by the BFM at best amplitude (BA) was 50% greater than that for the best pure-tone frequency (BF) at BA, or the threshold was at least 10 dB lower for the BFM than for the BF presented at BA. A neuronal map of
FM-sensitive units was composed on a surface drawing of the auditory cortex with its overlying standar0 vessel pattern. Although variability in vessel pattern was found among individual animals, the major dorsal and ventral branches of cerebral vessels that overlie the auditory. cortex were reliably consistent. Aligning these branches at their point of bifurcation permitted good correspondence among the maps of individual animals. Response magnitudes for the individual quarters were measured using the same stimulus amplitude as the BA for the BFM. The quarter which was essential for response (essential quarter) was determined by comparing the response magnitude elicited by the individual quarters with that evoked by the entire FM sweep. For FM units also showing responsiveness to ~ure tones, the threshold and response magnitude for the BF were measured, and the ]~F was considered to be the pulse component essential for response (essential pure tone; EFT). All stimulus amplitudes were expressed in dB SPL (decibels sound pressure level re 20 #Pa).
Results
Neurons exhibiting responses to both pure-tone and FM stimuli could simply be pure-tone sensitive units responding to a single frequency within the sweep. Hence, units were studied to determine which sweep components were necessary to elicit a response. The BF a n d / o r BFM were measured for each neuron exhibiting responses to pure-tone and/or FM stimuli. For all sampled neurons which responded to both pure-tone and FM stimuli, the BF was found t~o be a frequency within the sweep of the BFM. 'i'wo types of FM-sensitive units were distinguished~ One type (Type I) responded to pure tones, but the response magnitude was at least 50% greater to an FM stimulus when the FM swept through the neuron's BF. Response latencies to the BF and the BFM differed by the time between the onset of the FM sweep and when the sweep passed through the BF. The second type of FM-sensitive unit (Type ll) exhibited responses to pure tones which weie negligible or nonexistent. When a pure-tone response could be determined for Type II units, thresholds were at least 10 dB lower to BFM than to BF. In a sample of 99 neurons, 95 units were classified as FM-sensitive with 82 neurons (83%) meeting the criterion corresponding to Type I and 13 units (13%) corresponding to i~ype II (Fig. 2). The remaining 4 units (4%) were not FM-sensitive but were classified as pure-tone sensitive (PT-sensitive) since (1) thresholds were at least 10 dB lower to pure tones at BF than to FM sweeps and (2) response magnitudes to pure tones at BF were less when ~he BF was presented within an FM sweep.
182 100
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The response properties of Type I FM-sensitive neurons are illustrated for a representative unit in Fig. 3. The BFM sweep (from 111.5 to 51.5 kHz presented at 79 dB SPL) was divided into four quarters (see inset in Fig. 3A) and the essential quarter determined. Fro-
an upward sweep (Fig. 3(2) with the first quarter sweeping through the PT response area. The response latency to a pure tone at the B F was 4.5 ms shorter th~an that to the downward BFM, a time difference due to the time lag between the starting FM frequency and frequencies near the end of the FM sound which swept through the PT response area. A downward FM sound elicited a response four times greater in magnitude than the response elicited by the BF (Fig. 3 D ) but similar thresholds were obtained to both types of stimuli (Fig. 3A). The threshold for the BFM was 6 dB higher for upward than downward sweeps (Fig. 3A), but maximal responses were similar in magnitude regardless of sweep direction (Fig. 3D).
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Fig. 3. Response properties of a representative Type I FM-sensitive neuron. This unit had a best FM sweep from 111.5 to 51.5 kHz at 79 dB SPL and a best frequ~mcyof 57 kHz. Roman numerals indicate sweep quarter. (A) Frequency-tuning curves measured with either a pure tone or an FM sweep (upward and downward) exhibited narrow and closed excitatory response areas. (B) PSTC histogram displaying the downward FM sweep component essential for response (IV quarter) which swept from 66.5 to 51.5 kHz. (C) PSTC histogram showing an essential I quarter to an upward FM swee,r!. (D) PSTC histogram showing responses to both the entire 'upward and downward BFMs~ ;,nd to pure tone presented at best frequency. A downward FM sweep elicited a response four times greater in magnitude than the response evoked by a pure tone. Histograms collected for 50 stimuli.
183
Fig. 4A shows that the FM component essential for response corresponded to the IV quarter of the BFM in 74% ( N = 44) of Type I FM-sensitive units. In 18% of the Type l units ( N = 11), the IIl quarter of the BFM was essential for the response. Interestingly however, the essential III quarter in these units had terminal frequencies that were the same as or slightly higher than the best frequencies (BFs). For three units (5%) the II quarter was essential, and for two units (3%) the I quarter presented alone evoked a response. Onesample t-test indicated that ,his distribution of essential quarters was not random ( P < 0.05). The BF for Type l FM-sensitive neurons varied widely from approximately 25 to 90 kI-Iz. More notably, units that required the IV quarter for response had BFs which were approximately within the same frequency range (from 25 to 85 kHz) (Fig. 413). This finding indicates that the absolute frequen¢,, of the EPT is not as important to Type I neurons, although the timing of the EPT in the entire sweep is likely to be a critical determinant for response. A second experiment was undertaken to further examine Type II FM-sensitive neurons. Analysis of this neuronal class was based on responses recorded from
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42 units. Fig. 5 displays the response magnitudes of a Type II unit evoked from different quarters of the FM sweep. The BFM was downward with an initial frequency of 119 kHz. A weak response was elicited by the III quarter of the FM sweep but no response was evoked by any of the other individual quarters. The response to this essential III quarter was further enhanced with the addition of the IV quarter of the FM sweep. Pure-tone stimuli at BF (81 kl-Iz) evoked a very weak response. For Type II neurons, the III quarter was most often essential for response as indicated by 40% of the sampled neurons (Fig. 6). However, Type II neurons likely do not exhibit selectivity to any particular quarter since one-sample t-test indicated that this distribution of essential quarters was random ( P > 0.05). Two Type II neurons required 2 consecutive quarters for response. Fig. 7 shows PST histograms generated for one of these units. Single quarters and pure tones were not sufficient to elicit a response from this n e u r o n Instead, presentation of both FM quarters I and II was required to evoke a neuronal response. Fig. 8 shows iiiu location ,~f 37 units in the auditory cortex of 3 bats with respect to the characteristic overlying blood vessel pattern. Thirty-three of these units were classified as FM-sensitive. Both types of FM-sensitive neurons were interspersed within a cortical region previously identified as the tonotopic zone in Myotis (Wong and Shannon, 1988). The much smaller
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region containing PT-sensitive units did not overlap with the region containing FM-sensitive neurons and was in the rostrodorsal part of the auditory cortex. Thirty-one units shown in Fig. 8 exhibited responses to pure-tone stimuli. This mapping revealed a region containing Type I FM-sensitive and PT-sensitive units which almost spans both tonotopic and delay-sensitive zones of the auditory cortex. Twenty-seven of these units corresponded to Type I neurons. The EPTs for
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Fig. 8. Spatial distribution of cortical neurons maximally responding to pure-tone or FM stimuli. Overlapping tonotopic and delay-sensitive zones are labelled. The possible borders of a zone containing only PT-sensitive neurons ave drawn with dotted lines. Numbers correspond either to frequencies of EPTs of Type I FM-sens~tive neurons or BFs of PT-sensitive (circled numbers) neurons. Type I FM-sensitive units are arranged tonotopically with the highest frequencies of EPTs represented most rostrally and lowest EPTs being most caudal. A standard blood-vessel pattern overlying the auditory cortex is depicted. ( A = Type I1 units; D = dorsal; R = rostral).
Type 1 units form a frequency map with an axis oriented rostrocaudally: high-frequency EPTs located rostrally and low-frequency EPTs caudally. PT-sensitive units have BFs from 31 to 37 kHz which are much lower than the frequencies of EPTs of adjacent Type I units located in the rostrodorsal part of the tonotopic map.
Discussion
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Classification of neurom responding to tonal stimuli Eighty-two of the 95 FM-sensitive units were Type I, with a pure-tone stimu!ius being the frequency .component essential for neural response. The remaining, 13 units, Type II FM-sensitive units, which are characterized by requiring an FM sweep quarter to evoke a response, are interspersed throughout the same cortical zone that is comprised primarily of units belonging to Type I (Fig. 2). Although both Types I and II are classified as FM-sensitive neurons in this study, their neuronal response characteristics differ greatly. Whereas Type I unit.~ (Fig. 3) display frequency selectivity to EPTs, Type II units (Figs. 5 and 7) show negligible to no resp, mse to any single frequency, but respond with low thresholds to FM stimuli. Thus, responses of Type II urdts to pure tones are not exhibited due to inhibitory inputs arising either intracortically or subcorticeJly. These units appear to correspond to 'FM-sensitive' or 'FM-specialized units' which ~ave
185
been previously described in the bat inferior colliculus (Suga, 1968, 1969, 1973) and cortex (Suga, 1965b). However, this neuronal type has not been found at the level of the cochlear nucleus (Suga, 1965a). The percentage of Type II neurons recorded in this study (13%) corresponds to that found in the cortex previously for 'FM-sensitive' units (Suga, 1965b). Four neurons (Fig. 8) were found that were classified as pure-tone sensitive on the basis of threshold measurements. The BFs of these neurons were between 31 and 37 kHz but these units were located rostra! to Type 1 FM-sensitive units with EPTs of similar frequency, range. Hence, PT-sensitive neurons were not among those which made up the orderly frequency map. PT-sensitive neurons have (1) lower thresholds to pure-tone stimuli than to FM sweeps, (2) pure-tone responses which are diminished when the tone is presented within a sweep, and (3) BFs which are noticeably lower compared to nearby Type I FMsensitive units with higher EPTs. These response characteristics are therefore quite distinct from responses of other cortical units and could possibly be a separate functional zone. Why frequencies between 30-40 kHz are re-represented in the cortex is not dear, but may be related ~o the bat's behavioral threshold being lowest between 35-40 kHz (Grinnell, 1963). In addition, behavioral studies of the rTM bat, Eptesicus fuscus, demonstrated that the low-frequency portion of the first harmonic of the FM echo is sufficient for fine distance discriminations (Moss and Schnitzler, 1989). Whether frequencies between 30-40 kHz confer similar information for M. lucifugus has not yet been determined.
Essential components for FM-sensitice neurons The echolocating bat, M. lucifugus, utilizes an FM sound that sweeps downward over an octave (Griffin, 1958). To determine whether the temporal sequence of frequency was an important cue for cortical neurons in the present study, the FM stimulus simulating the bat's biosonar signal was divided arbitrarily into four quarters and the evoked neuronal responses were measured. A single quarter was sufficient for response for most units sampled, although importantly the essential portion of the FM sweep may be shorter in frequency sweep and duration. For those requiring two continuous quarters~ the essential component may simply be a spectral element which requires frequencies found in both quarters. However, if neurons had been found requiring three continuous quarters for response, a wider sequence of frequencies clearly would have been essential. For Type I FM-sensitive units, the fact that the EFT most often was within the IV quarter of the BFM was statistically significant (Fig. 4). Hence, spectral and temporal domains are both important to Type I neu-
rons since the timing of the EPT in an FM sweep appears to be critical. For example, sonograms of Myotis FM pulses indicate that energy is greatest in the last half of the signal (Friend et al., 1966; Sales and Pye, 1974) and that harmonics are present in shorter FM sweeps in later phases of echolocation (Novick and Griffin, 1961). More extensive studies are necessary to determine the salient acoustic cues conveyed in this portion of the FM sound. A neuronal type similar to Type I units in the present study was previously described by Saga (1965b) and referred to as 'high-responsiveness' units. 'Highresponsiveness' neurons were characterized by having greater response magnitudes to FM than to pure tones, but having similar thresholds to both stimulus types. However, the percentage of Type I units in this study was much higher than that reported for 'high-responsiveness' units. The difference in percentage is likely due to 'high-responsiveness' units being classified according to threshold criterion, whereas Type I units in this study were assessed on the basis of maximal response. Fig. 9 shows a neuronal network model which can explain the 1V quarter being essential for response in the majority of Type I units. In Fig. 9E, the Type I unit (Neuron D) receives an inhibitory input from Neuron A, an exotatory input from Neuron B, and an excitatory input from Neuron C which is delayed in time by an interneuron. Neurons A and C differ slightly in their BFs and have an excitatory response area flanked by an inhibitory response area. Such neurons have been extensively characterized in Myotis inferior colliculus (Suga, 1965a, 1968, 1969) and comprise approximately 21% of the neurons at .~hat level (Suga, 1969). The inhibitory response area of Neuron A contains higher frequencies than the excitatory area, whereas the inhibitory response area of Neuron C contains lower frequencies ,h...~,,.,. ~h,..,.. excitatory area. Hence, these neurons would only respond to an FM stimulus which swept through the excitatory area first. Neurons with properties like A and C were previously referred to as "asymmetricar neurons, since they selectively respond to only one direction of an FM sweep (Suga, 1965b, 1968, 1969, 1973). The table in Fig. 9F predicts the effects of stimulation with pure-tone frequencies (fl, f2, f3) and upward and downward FM sweeps on Neurons A through D. Note that Neuron D responds only to pure-tone fl, but not to f2 due to the inhibitory effect of Neuron A (Figs. 9D, 9F). Neuron D maxim',dly responds to a downward FM sweep as a consequence of two key factors. First, the initial sweep through higher frequencies inhibits the response of Neuron A such that Neuron C's excitatory effect on Neuron D is now increased (disinhibition). Second, since Neuron C's response is delayed, Neuron D receives simultaneous excitation from Neurons B and C
186
as the F M sweeps through these n e u r o n s ' excitatory r.esponse areas. N e u r o n D also r e s p o n d s to upward F M sweeps due to the excitatory effect exerted by N e u r o n B, but this response is diminished since the excitatory influence exerted by N e u r o n C is diminished by Neuron A. Type II FM-sensitive neurons described in this study may have a m o r e specialized function in F M bats. T h e essential c o m p o n e n t for these. ,mils is not a single frequency; rather, these units req~ire an FM sweep to elicit a response. T h a t the I I I qttarter most c o m m o n l y (Fig. 6) was the essential F M c o m p o n e n t for T y p e II units lacks statistical significance, but nevertheless supports a neural network model p r o p o s e d previously by Suga (!965b) for 'FM-specialized' units. In this model, shown in Fig. 10, N e u r o n C has the s a m e response
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Fig. 10. Neuronal network model for characteristic responses of Type II FM-sensitive neurons. When lower level neurons A and B have respm~se characteristics as shown respectively in (A) and (B) and converge on Neuron C as shown in (D), Neuron C exhibits response propgrties of a Type II unit (E). Symbols the same as in Fig. 9. Modified from Suga, J. Physiol. (Lend.), 181, (1965).
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Fig. 9. Neuronal network model for characteristic responses of Type I FM-sensitive neurons. When lower level neurons A, B and C have response characteristics as shown respecti~velyin Figures (A), (B) and (C), and converge on Neuron D as shown in (E)~ Neuron D exhibits response properties characteristic of a TyI~ 1 unit (D). The table (F) predicts the responses of Neurons A through D to pt, r/e-tone and FM stimuli. Hatched region indicates inhibitory response area. Solid- and dotted-line arrows respectively signify response or no response to FM sweep in the direction of the arrow. Bold arrow signifies dual excitatory response to FM in the direction of the arrow. In (E), ' + ' represents excitatory inputs; '-' represents inhibitory inputs; 'I' represents an interneuron. In (F), stim = sound stimulus; E = excitatory effect; I = inhibitory effect; 0 -- no effecL
properties as a T y p e II ( ' F M - s p e c i a l i z e d ' ) unit and receives inhibitory input from N e u r o n A and excitatory input from N e u r o n B (Fig. 10D). A l t h o u g h N e u r o n s A and B have similar BFs, N e u r o n C d o e s not respond to any p u r e - t o n e stimulus due to the inhibitory effect o f N e u r o n A. Mowever, N' ~. .....,.,.. C d ,v__ "" ~ r e s p o n d at low thresholds to d o w n w a r d F M sweeps since the F M stimulus initially sweeps through the inhibitory response area o f N e u r o n A thereby releasing the excitatory effect exerted by N e u r o n B o n N e u r o n C. H e n c e , for N e u r o n C to be sensitive to d o w n w a r d sweeps only, N e u r o n A m u s t have an inhibitory r e s p o n s e a r e a with frequencies higher than its excitatory response area. T h e present study's finding that the mid-to-low frequency c o m p o n e n t s of the stimulus w e r e essential for ~esponse in m o s t T y p e II neurons lends s u p p o r t to this model.
Cortical organization Ninety-six p e r c e n t o f the n e u r o n s exhibiting sensitive ity to single stimuli could b e classified as F M - and not
187
pure-tone sensitive. These neurons span an area (Fig. 8) which includes the previe,~ly described tonotopic zone (Wong and Shannon, t~88). When the EPTs of Type I FM-sensitive neurons are plotted on a drawing of the cortical surface, they are. systematically arranged along a frequency axis. The resulting map of these units (Fig. 8) closely corresponds with the best frequency representation of neurons arranged tonotopically in Myotis cortex: higher best frequencies are located rostrally, with lower best frequencies located progressively more caudal. Whether previous maps were reconstructed from FM-sensitive neurons rather than pure-tone sensitive is unknown, since previous maps were made based upon BF without regard to whether the units were FM-sensiti,~;e or exclusively PT-sensitive (Suga, 1965b; Wong and Shannon, 1988). The fact that the tonotopic representation in Myotis cortex is based upon units that actually respond better to complex stimuli than to simple tones may be a feature of cortical organization specific to FM bats in general. FM sweeps are integral components of spectra in communication sounds as well as in biosonar pulseg (Suga, 1973). Behavioral studies of FM bats indicate the importance of spectral cues for shape and texture (Simmons et al., 1974; Habersetzer and Vogler, 1983; Simmons et al., 1990) and temporal cues for target feature a n a ! ~ s (Simmons, 1979; Simmons et al., 1990). These findings suggest that nnpott~tt~t c.~e~ for target characterization are derived from a single FM sweep, and a functional organization in which FM-sensitive neurons are found throughout the auditory cortex of M. lucifugus lends support to this type of auditory processing. Neurons exhibiting sensitivity to single pure-tone and FM sweeps could possibly participate in target perception as well as communication under different acoustic conditions. Units responsive to these singlesound stimuli have also been shown to acquire selectivity for FM pairs at particular repetition rates (Wong et el., 1992). Moreover, recognition of targets may not require a comparison ~-.a!ysis between pulse and echo if recognition is based upon wemory. The fact that FM-sensitive neurons are located ill both tonotopic and delay-sensitive zones of the M. iucifugus auditory cortex implicates these neurons in the processing of extensive auditory information that the bat hears, incl,~ding that used during echnlocadon.
Acknowledgements We would like to thank H. Te~g for her valuable comments on the manuscript. This work was supported in part by NIDCD R01 00600 to D. Wong.
References Berkowitz, A. and Suga, N. (1989) Neural mechanisms of ranging are different ~n two species of bats. Hear. Res. 41, 255-264. Feng, A.S., Simmons, J.A. and Kick, S.A. f1978) Echo detection and target-ranging neurons in the auditory system of the bat, Eptestcus fuscus. Science, 202, 645-648. Friend, J.H., Suga, N. and Suthers, R.A. (1966) Neural responses in fhe inferior colliculus of echolocating bats to artificial orientation sounds at~d echoes. J. Cell Physiol./~7, 319-332. Griffin, D.R. (1958) Listening in the dark. Reprinted by Cornell University Press, Ithaca, N.Y. Grinnell, A.D. (1963) The neurophysiology of audition in bats: intensity and freqt~encyparameters. J. Physiol (Lond.) 167, 38-66. Habersetzer, J. and Vogler, B. (1983) Discrimination of surfacestructured targets by the echolocating bat Myotis myotis during flight. J. Comp. Physiol. A 152, 275-282. J~.~n, PH-S, Sun X.D. and Lin PJ-J. (1989) Frequency and space representation in the primary auditory cortex of the FM bat, Eptesicus fuscus. J. Comp. Physiol. A 165, 1~14. Mendelson, J.R. and Cynader, M.S. (1985) Sensitivity of cat primary auditory cortex (At) neurons to the direction and rate of frequency modulation. Brain Res. 327, 331-335. Moss, C.F. and Schnitzler, H-U. (1989) Accuracy of target ranging in echolocating bats: acoustic information processing. J. Comp. Physiol. A 165, 383-393. Novick, A. and Griffin, D.R. (1961) Laryngeal mechanisms in bats for the production of orientation sounds. J. Exp. Zool. 148, 125~146. O'Neill, W.E. and Suga, N. (1979) Target range-sensitive neurons in the auditory cortex of the mustached bat. Science 203: 69-73. O'Neill, W.E. and Suga, N. (1982) Encoding of target-range information and its representation in the auditory cortex of the mustached bat. J. Neurosci. 2, 17-31. Phillips, D.P., Mendelson, J.R., Cynader, M.S. and Douglas, R.M. (1985) Responses of ~ingle neurones in cat auditory cortex to time-varying stimuli: frequency-modulated tones of narrow excursion. Exp. Brain Res. 58, 443-454. Sales, G. and l~e, D. (1974) Ultrasonic Communication by Animals. Chapman and Hall, London. Shannon, S.L. (1988) Encoding of Acoustic Parameters in the Echolocating Bat, Myotis luci]i.~gus.(M.S. Thesis). West Lafayette, IN: Purdue University. ShatQnon, S.L. and Wong, D. (1989) Spectral processing of FM pulses in the audito~ cortex of an FM bat. Soc. Neurosci. Abstr. 15, 111. Simmons, J.A. (1979) Perception of echo phase information in bat sonar. Science 207, 1336-1338. Siminons, J.A., Lavender, B.A., Lavender, C.A., Doroshow, S.W., Keller, R., Livingston and Scallet, A.C. (1974) Target st~mcmre aod echo spectra! disfzimination by echolocating bats. Science 186, ii30-1132. Simmons, J.A., Moss C.F. and Ferragarao, M. (1990) Convergence of temporal and spectral information into acoustic images of complex sonar targets perceived by the echolocating bat, Epwsicus fuscus. J. Comp. Physiol. A 166:449-470. Suga, N. (1965a) Analysis of frequency-modulated sounds by auditory neurones of echo-locating bats. J. Physiol. (Lond.), 179, 26-53. Suga, N. (1965b) Functional properties of auditory neurones in the cortex of echo-locating bats. J. Physiol. (Lond.), 181, 671-700. Suga, N. (1968) Analysis of frequency-modulated and complex sounds ~y single a~ditory neurones of bats. J. Physiol. (Lond.), 19~, 51-80. Suga, N. (1969) Classification of inferior coUicular neurones of bats in terms of responses to pare tones, FM sounds and ~oise bursts. J. Physiol. (Lond.), 200, 555-574.
188 Suga, N. (1973) Feature extraction in the auditory system of bats. In: A. M¢ller (Ed.), Basic Mechanisms in Hearing, Academic Press, New York and London, pp. 675-744. Suga, N. (1988) Auditory neuroethology and speech processing: Complex sound processing by combination-sensitive neurons, In: G.M. Edelman, W.E. Gall and W.M. Cowan (Eds.), Functions of the Auditory System, John Wiley and Sons, New York, pp. 679-720. Suga, N., O'Neill, W.E., Kujirai K. and Manabe, T. (1983) Specificity of combination-sensitive neurons for processing of complex biosonar signals in auditory cortex of the mustached bat. J. Neurophysiol. 49, 1573-1626. Suga, N., O'Neill, W,E. and Manabe, T. (1978) Cortical neurons sensitive to combinations of information-bearing elements of biosonar signals in the mustached bat. Science, 200, 778-781. Suga, N., O'Neill, W.E. and Manabe, T. (1979) Harmonic-sensitive neurons in the auditory cortex of the mustached bat. Science, 203, 270-274.
Sullivan, W.E. (1982) Neural representation of target distance in auditory cortex of the echolocating bat Myotis lucifugus. J. Neurophysiol. 48, 1011-1032. Whitfield, I.C. and Evans, E.F. (1965) R,~sponses of auditory cortical neurones to stimuli of changing frequ,~ncy. J. Neurophysiol. 28, 655-672. Wollberg, Z. and Newman, J.D. (1972) Auditory cortex of squirrel monkey: response patterns of single cells to species-specific vocalizations. Science 175, 212-214. Wong, D. and Shannon, S.L. (1988) Functionarl zones in the auditory cortex of the e~holocating bat, Myotis lucifugus. Brain Res. 453, 349-352. Wong, D., Maekawa, M. and Tanaka, H. (1992) The effect of pulse repetition rate on the delay sensitivity of nc,ur6ns in the auditory cortex of the FM bat, Myotis lucifugus. J. C0mp. Physiol. A 170, 393-402.
179
© 1992 Elsevier Science Publishers B.V. All rights reserved 0378-5955/92/$05.00 HEARES 01777
Processing of pure-tone and FM stimuli in the auditory cortex of the FM bat, Myotis lucifugus Sharon Shannon-Hartman, Donald Wong and Masao Maekawa Department of Anatomy, Indiana University School of Medicble, Indianapolis, Indiana, USA (Received 30 September 1991; Revision received 24 March 1992; Accepted 31 March 1992)
FM bats perceive their surroundings during echolocation by analyzing frequency-modulated (FM) acoustic signals. Results from this study indicate a cortical organization in Myotis lucifi~gus which is largely made up of neurons sensitive to FM sounds (FM-sensitive neurons). Three types of neurons were distinguished by their responses to pure-tone and FM stimuli: (1) Type I FM-sensitive units (83%), Type II FM-sensitive units (13%) and pure-tone sensitive units (4%). Type I FM-sensitive units responded to pure tones, but exhibited greater response magnitudes to FM stimuli when the best FM swept through the BF. An orderly frequency representation was found when the frequencies of pure tones essential for response (EPTs) in Type I units were mapped along the cortical surface. The EPTs for Type I neurons were usually found within the last millisecond of a downward FM sweep. As outlined by two neuronal network models, both the responses of Type I and It units could likely result from the convergence of excitatory and inhibitory lower level neurons with slightly differing BFs. Type II units were selective for an FM sweep and showed negligible to no response to pure-tone stimuli. Pure-tone sensitive units exhibited weak or no responses to FM stimuli. These neurons were clustered in a small area located rostrodorsal to the tonotopic zone and had significantly lower best frequencies than adjacent EPT frequencies of Type I FM-sensitive neurons. Spectral processing; Auditory cortex; Frequency organization; FM sweep; Echolocating bats
Introduction
Acoustic stimuli in natural environments commonly consist of temporal shifts in amplitude and spectral composition. The responsiveness of neurons to timevarying stimuli, such as FM sweeps or species-specific vocalizations, has been examined neurophysiologically in the auditory cortex of several mammals, including the cat (Whitfield and Evans, 1965; Mendelson and Cynader, 1985; Phillips et al., 1985), squirrel monkey (Wollberg and Newman, 1972) and bat (Suga, !965b). Cortical neurons have been found that not only were selective to modulation in spectra, but also to sweep direction (Suga, 1965b; Whitfield and Evans, 1965; Phillips et ai., 1985). Moreover, some neurons which exhibit strong responses to FM stimuli have been shown to respond very poorly to pure tones (Suga, 1965b; Whitfield and Evans, 1965). These studies have therefore revealed neuronal populations that are capable of processing different aspects of time-varying stimuli. FM sweeps provide important cues for the recognition of many complex sounds, such as speech. FM sounds are particularly important to bats that use these Correspondence to: Donald Wong, Department of Anatomy, Indiana University School of Medicine, Indianapolis, IN 46202-5120, USA. Fax: (317) 274-3318.
signals for echolocation since they must analyze target-reflected echoes of their emitted sounds to perceive their surroundings (Griffin, 1958). FM sound pairs have been employed in many neurophysiological studies to examine target perception in the auditory cortex of bats (Suga eta!., 1978, 1979, 1983; Feng et al., 1978; O'Neill and Suga, 1979, 1982; Sullivan, 1982; Suga, 1988; Wong and Shannon, 1988; Berkowitz and Suga, 1989). The present study uses Myotis lucifugus, a bat which echolocates primarily with FM sweeps (Griffin, 1958), as a model for the study of basic processing of FM sounds. Suga (1965b) was the first to describe the response properties of cortical neurons stimulated by FM pulses in Myotis. Results from Suga's study indicated that neuronal sensitivity to sweep direction was dependent upon tile temporal sequence in which the stimulus triggered excitatory and inhibitory response areas. A frequency representation based on best frequencies was demonstrated in the tonotopic zone of Myotis cortex usLng pure-tone stimuli (Suga~ 1965b; Wong and Shannon, 1988). Neurons sensitive to FM stimuli were located within this zone and in the overlapping delaysensitive zone (Shannon and Wong, 1989). Within the overlapping zone, neurons that responded to pure-tone and FM stimuli were also found to be sensitive to FM-FM pairs at specific delays and for specific reped-
180
tion rates (Wong et al., 1992). The present study further examined neuronal responsiveness to FM stimuli in Myotis by focusing on whether spectral selectivity existed for a partier'at portion of the FM sweep. The relationship of spectral processing of FM sounds to the tonotopic map was also studied in the present investigation. For auditory-cortical units exhibiting sensitivity to FM stimuli, portions of the FM signal were deleted to determine which FM components were necessary for neuronal response. Cortical organization with regard to neurons exhibiting sensitivity to single stimuli (either a constant frequency tone or FM sweep) was studied by mapping the cortical locations of FM-sensirive neurons. Mapping results were analyzed and related to the known tonotopie organization in Myotis (Suga, 1965b; Wong and Shannon, 1988). Results from these analyses have implications on the basic processing of FM stimuli in the auditory cortex of FM bats.
Materials and Methods
Preparation All experimental protocols and animal care for this study have been approved by the Laboratory Animal Resource Center (LARC) of Indiana University School of Medicine (approval number MD No. 621). A total of 12 tittle brown bats (M. iucij~gus), each with a body wei[ht of 5-10 g, were used. Prior to surgery, the neuroleptanalgesia Innovar-vet (0.04 mg/kg fentanyl and 2 mg/kg droperidol mixture) was injected intramuscularly. A midsagittal incision was made on the scalp and the temporalis muscles were retracted laterally to expose the skull. A small rod (1.5 cm) was mounted onto the dorsal skull with dental cement and eyanoacrylate glue. All surgical wounds were cleaned and treated with antibiotic ointment (Furacin). A local anesthetic (Lidocaine) was topically applied onto exposed tissue to minimize discomfort or pain. Neural recordings commenced after a post-surgical recovery period of 2-7 days. Neural recordings were conducted in an individual animal for up to 8 h on alternate days. The bat was situated in the center of an echo-attenuated, soundproof room thermally maintained at 30-32°C. The bat's body was restrained in a plexiglass holder, which was suspended with an elastic band from a horizontal metal b~.x'. To obtain stable neural recordings, the head was immobilized by securing the skull-mounted rod onto a post with a set screw. All posts suspending the bat and anci~oring the micromanipulators were screwed onto a vibration-isolated experimental table (TMC Micro-g). Previous application of dental cement to the skull surface overlying the auditory cortex usually eliminated the need to retract the scalp for each recording session. However, Lidocaine was topically applied to surgical
wounds if manipulation was necessary. Throughout the r~,cording session, the animal was vigilantly monitored for signs of discomfort. Drinking water was frequently offered. Otherwise, the bat ~ested quietly during recordings~ At the end of each recording session, care was taken to keep the skin around the skull clean and free of infection with topical administration of antibiotic. Typically, five to eight recording sessions were necessary to complete an experiment on one animal. The approximate location of the auditory cortex was determined on the basis of a characteristic bifurcation of a major blood vessel. The vessel was readily visible beneath the thin translucent skull. To provide a general guide for electrode insertion, a sketch of the bat's auditory cortex and its overlying vessels were drawn with the aid of an operating microscope equipped with a grid ocular in one eyepiece. The sharp tip of a scalpel blade was used to make small craniotomies ( ~ 50/.tm in diameter) for electrode insertion into the auditory cortex. During this procedure, the bat did not exhibit excessive movements associated with pain. Electrodes were initially lowered onto the cortical surface with micromanipulators under the visual guidance of an operating microscope and then advanced into the brain with a remote hydraulic microdrive (Narishigc MO-10) outside the soundproof chamber. Columnar organization according to best frequency has been demon: strated in the auditory cortex of FM bats (Shannon, 1988; Jen et. al., 1989) and thus, all electrode penetrations were subsequently charted onto a drawing of the auditory cortex for later reconstruction of the surface location ,of recorded units.
Acoustic stimulation Free-field sounds were presented from a condenser loudspeaker located 75 cm directly in front of the animal. The loudspeaker was calibrated and found to be flat ( + 3 dB) between 20 and 140 kHz. Pure tones and FM sweeps were electronically generated with a Wavetek oscillator and shaped into bursts with a custom-built electronic switch. FM sounds swept linearly 60 kHz in 4 ms with 0.25 ms rise/fall times to simulate biosonar signals emitted by M. lucifugus (Griffin, 19581. To determine whether neurons exhibited a selectivity to a specific part of the FM sound, the entire 4-ms sweep was arbitrarily divided into either (a) four equal segments or quarters (I, II, III, IV), each sweeping 15 kHz in 1 ms (Fig. 1); (b) 2 continuous quarters (I-II, II-IlI, Ill-IV), each sweeping 30 kHz in 2 ms; or (c) 3 coatinuous quarters (I-III, II-IV), each sweeping 45 kHz in 3 ms. The rise/fall time was fixed at 0.25 ms for all FM stimuli used.
Neural recording and data analysis Extracellular unit recordings were obtained in the unanesthetized animal with lacquer-coated tungsten
18!
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Fig. 1. Schematic of stimulus paradigm. Ultrasonic FM sounds with linear downward sweeps of 60 kHz in 4 ms were electronically generated and presented free-field to the bat inside a soundproof room. For each identified FM-sensitive neuron, the best FM (entire 60 kHz sweep) was determined. The sweep was then arbitrarily divided into four spectral quarters (I, lI, III and IV), each qt~arter sweeping 15 kHz in 1 ms.
electredeg (tip diameter - 5 / t m ) using conventional eiectrophysiological techniques (for details see Wong et al., 1992). In brief, neural activity was amplified, filtered, and displayed on an oscilloscope. Soundevoked unit activity was selected by a window discriminator (Bak Electronics DIS-l) and fed into an audiomonitor and into a 386SX IBM-compatible personal computer (Gateway 2000) for real-time data acquisition. Under software control, the computer both triggered analog sound-generating equipment (ore-set manually during audiovisual measurements) and sampled selected neural activity at a rate of 2 kHz (0.5 ms binwidth) during repetitive sound stimulation for up to 100 trials. Spike counts were represented over time in the form of peri-stimulus-time (PST) and PST cumulative (PSTC) histograms for comparison of response magnitudes. Data acquired in real-time from customwritten software were exported into several commercially-available graphics software for off-line analysis and printouts. Neurons responsive to single FM sweeps were isolated using 60 kHz sweeps in 4 ms as search stimuli. Maximal-response a n d / o r frequency-threshold measurements were then made to determine the best FM for response (BFM). Units were classified as FM-sensitive if either the response magnitude evoked by the BFM at best amplitude (BA) was 50% greater than that for the best pure-tone frequency (BF) at BA, or the threshold was at least 10 dB lower for the BFM than for the BF presented at BA. A neuronal map of
FM-sensitive units was composed on a surface drawing of the auditory cortex with its overlying standar0 vessel pattern. Although variability in vessel pattern was found among individual animals, the major dorsal and ventral branches of cerebral vessels that overlie the auditory. cortex were reliably consistent. Aligning these branches at their point of bifurcation permitted good correspondence among the maps of individual animals. Response magnitudes for the individual quarters were measured using the same stimulus amplitude as the BA for the BFM. The quarter which was essential for response (essential quarter) was determined by comparing the response magnitude elicited by the individual quarters with that evoked by the entire FM sweep. For FM units also showing responsiveness to ~ure tones, the threshold and response magnitude for the BF were measured, and the ]~F was considered to be the pulse component essential for response (essential pure tone; EFT). All stimulus amplitudes were expressed in dB SPL (decibels sound pressure level re 20 #Pa).
Results
Neurons exhibiting responses to both pure-tone and FM stimuli could simply be pure-tone sensitive units responding to a single frequency within the sweep. Hence, units were studied to determine which sweep components were necessary to elicit a response. The BF a n d / o r BFM were measured for each neuron exhibiting responses to pure-tone and/or FM stimuli. For all sampled neurons which responded to both pure-tone and FM stimuli, the BF was found t~o be a frequency within the sweep of the BFM. 'i'wo types of FM-sensitive units were distinguished~ One type (Type I) responded to pure tones, but the response magnitude was at least 50% greater to an FM stimulus when the FM swept through the neuron's BF. Response latencies to the BF and the BFM differed by the time between the onset of the FM sweep and when the sweep passed through the BF. The second type of FM-sensitive unit (Type ll) exhibited responses to pure tones which weie negligible or nonexistent. When a pure-tone response could be determined for Type II units, thresholds were at least 10 dB lower to BFM than to BF. In a sample of 99 neurons, 95 units were classified as FM-sensitive with 82 neurons (83%) meeting the criterion corresponding to Type I and 13 units (13%) corresponding to i~ype II (Fig. 2). The remaining 4 units (4%) were not FM-sensitive but were classified as pure-tone sensitive (PT-sensitive) since (1) thresholds were at least 10 dB lower to pure tones at BF than to FM sweeps and (2) response magnitudes to pure tones at BF were less when ~he BF was presented within an FM sweep.
182 100
quency-tuning curves measured with either a pure tone
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or an FM sweep (upward and downward) typically N=89
exhibited narrow and closed excitatory response areas
83~.
(Fig. 3A). Tuning curves obtained with presentation of
80
9 F M were plotted according to the initial frequency o f the sweep. The IV quarter of the B F M which swept downward from 66.5 kHz to 51.5 kHz (Fig. 3B) was essential for response° This quarter contained frequencies found in the PT response area (BF = 57 kHz) (Fig.
60 z
3A). This unit responded either to a downward sweep (Fig. 3B) terminating within the PT response area or to
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Fig. 2. Distribution of types of neurons sensitive to single stimuli.
The response properties of Type I FM-sensitive neurons are illustrated for a representative unit in Fig. 3. The BFM sweep (from 111.5 to 51.5 kHz presented at 79 dB SPL) was divided into four quarters (see inset in Fig. 3A) and the essential quarter determined. Fro-
an upward sweep (Fig. 3(2) with the first quarter sweeping through the PT response area. The response latency to a pure tone at the B F was 4.5 ms shorter th~an that to the downward BFM, a time difference due to the time lag between the starting FM frequency and frequencies near the end of the FM sound which swept through the PT response area. A downward FM sound elicited a response four times greater in magnitude than the response elicited by the BF (Fig. 3 D ) but similar thresholds were obtained to both types of stimuli (Fig. 3A). The threshold for the BFM was 6 dB higher for upward than downward sweeps (Fig. 3A), but maximal responses were similar in magnitude regardless of sweep direction (Fig. 3D).
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Fig. 3. Response properties of a representative Type I FM-sensitive neuron. This unit had a best FM sweep from 111.5 to 51.5 kHz at 79 dB SPL and a best frequ~mcyof 57 kHz. Roman numerals indicate sweep quarter. (A) Frequency-tuning curves measured with either a pure tone or an FM sweep (upward and downward) exhibited narrow and closed excitatory response areas. (B) PSTC histogram displaying the downward FM sweep component essential for response (IV quarter) which swept from 66.5 to 51.5 kHz. (C) PSTC histogram showing an essential I quarter to an upward FM swee,r!. (D) PSTC histogram showing responses to both the entire 'upward and downward BFMs~ ;,nd to pure tone presented at best frequency. A downward FM sweep elicited a response four times greater in magnitude than the response evoked by a pure tone. Histograms collected for 50 stimuli.
183
Fig. 4A shows that the FM component essential for response corresponded to the IV quarter of the BFM in 74% ( N = 44) of Type I FM-sensitive units. In 18% of the Type l units ( N = 11), the IIl quarter of the BFM was essential for the response. Interestingly however, the essential III quarter in these units had terminal frequencies that were the same as or slightly higher than the best frequencies (BFs). For three units (5%) the II quarter was essential, and for two units (3%) the I quarter presented alone evoked a response. Onesample t-test indicated that ,his distribution of essential quarters was not random ( P < 0.05). The BF for Type l FM-sensitive neurons varied widely from approximately 25 to 90 kI-Iz. More notably, units that required the IV quarter for response had BFs which were approximately within the same frequency range (from 25 to 85 kHz) (Fig. 413). This finding indicates that the absolute frequen¢,, of the EPT is not as important to Type I neurons, although the timing of the EPT in the entire sweep is likely to be a critical determinant for response. A second experiment was undertaken to further examine Type II FM-sensitive neurons. Analysis of this neuronal class was based on responses recorded from
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T i m e in m s e c Fig. 5. PST histograms obtained from a representative Type II FM-sensitive neuron, Roman numerals designate FM sweep quarter(s). The best FM for this unit was a downward FM sweep from 119 kHz to 59 kHz presented at 70 dB SPL. The Ill quarter of the FM was essential for response, but addition of the IV quarter clearly enhanced the response. Histograms collected for 50 stimuli.
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42 units. Fig. 5 displays the response magnitudes of a Type II unit evoked from different quarters of the FM sweep. The BFM was downward with an initial frequency of 119 kHz. A weak response was elicited by the III quarter of the FM sweep but no response was evoked by any of the other individual quarters. The response to this essential III quarter was further enhanced with the addition of the IV quarter of the FM sweep. Pure-tone stimuli at BF (81 kl-Iz) evoked a very weak response. For Type II neurons, the III quarter was most often essential for response as indicated by 40% of the sampled neurons (Fig. 6). However, Type II neurons likely do not exhibit selectivity to any particular quarter since one-sample t-test indicated that this distribution of essential quarters was random ( P > 0.05). Two Type II neurons required 2 consecutive quarters for response. Fig. 7 shows PST histograms generated for one of these units. Single quarters and pure tones were not sufficient to elicit a response from this n e u r o n Instead, presentation of both FM quarters I and II was required to evoke a neuronal response. Fig. 8 shows iiiu location ,~f 37 units in the auditory cortex of 3 bats with respect to the characteristic overlying blood vessel pattern. Thirty-three of these units were classified as FM-sensitive. Both types of FM-sensitive neurons were interspersed within a cortical region previously identified as the tonotopic zone in Myotis (Wong and Shannon, 1988). The much smaller
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region containing PT-sensitive units did not overlap with the region containing FM-sensitive neurons and was in the rostrodorsal part of the auditory cortex. Thirty-one units shown in Fig. 8 exhibited responses to pure-tone stimuli. This mapping revealed a region containing Type I FM-sensitive and PT-sensitive units which almost spans both tonotopic and delay-sensitive zones of the auditory cortex. Twenty-seven of these units corresponded to Type I neurons. The EPTs for
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Type 1 units form a frequency map with an axis oriented rostrocaudally: high-frequency EPTs located rostrally and low-frequency EPTs caudally. PT-sensitive units have BFs from 31 to 37 kHz which are much lower than the frequencies of EPTs of adjacent Type I units located in the rostrodorsal part of the tonotopic map.
Discussion
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Classification of neurom responding to tonal stimuli Eighty-two of the 95 FM-sensitive units were Type I, with a pure-tone stimu!ius being the frequency .component essential for neural response. The remaining, 13 units, Type II FM-sensitive units, which are characterized by requiring an FM sweep quarter to evoke a response, are interspersed throughout the same cortical zone that is comprised primarily of units belonging to Type I (Fig. 2). Although both Types I and II are classified as FM-sensitive neurons in this study, their neuronal response characteristics differ greatly. Whereas Type I unit.~ (Fig. 3) display frequency selectivity to EPTs, Type II units (Figs. 5 and 7) show negligible to no resp, mse to any single frequency, but respond with low thresholds to FM stimuli. Thus, responses of Type II urdts to pure tones are not exhibited due to inhibitory inputs arising either intracortically or subcorticeJly. These units appear to correspond to 'FM-sensitive' or 'FM-specialized units' which ~ave
185
been previously described in the bat inferior colliculus (Suga, 1968, 1969, 1973) and cortex (Suga, 1965b). However, this neuronal type has not been found at the level of the cochlear nucleus (Suga, 1965a). The percentage of Type II neurons recorded in this study (13%) corresponds to that found in the cortex previously for 'FM-sensitive' units (Suga, 1965b). Four neurons (Fig. 8) were found that were classified as pure-tone sensitive on the basis of threshold measurements. The BFs of these neurons were between 31 and 37 kHz but these units were located rostra! to Type 1 FM-sensitive units with EPTs of similar frequency, range. Hence, PT-sensitive neurons were not among those which made up the orderly frequency map. PT-sensitive neurons have (1) lower thresholds to pure-tone stimuli than to FM sweeps, (2) pure-tone responses which are diminished when the tone is presented within a sweep, and (3) BFs which are noticeably lower compared to nearby Type I FMsensitive units with higher EPTs. These response characteristics are therefore quite distinct from responses of other cortical units and could possibly be a separate functional zone. Why frequencies between 30-40 kHz are re-represented in the cortex is not dear, but may be related ~o the bat's behavioral threshold being lowest between 35-40 kHz (Grinnell, 1963). In addition, behavioral studies of the rTM bat, Eptesicus fuscus, demonstrated that the low-frequency portion of the first harmonic of the FM echo is sufficient for fine distance discriminations (Moss and Schnitzler, 1989). Whether frequencies between 30-40 kHz confer similar information for M. lucifugus has not yet been determined.
Essential components for FM-sensitice neurons The echolocating bat, M. lucifugus, utilizes an FM sound that sweeps downward over an octave (Griffin, 1958). To determine whether the temporal sequence of frequency was an important cue for cortical neurons in the present study, the FM stimulus simulating the bat's biosonar signal was divided arbitrarily into four quarters and the evoked neuronal responses were measured. A single quarter was sufficient for response for most units sampled, although importantly the essential portion of the FM sweep may be shorter in frequency sweep and duration. For those requiring two continuous quarters~ the essential component may simply be a spectral element which requires frequencies found in both quarters. However, if neurons had been found requiring three continuous quarters for response, a wider sequence of frequencies clearly would have been essential. For Type I FM-sensitive units, the fact that the EFT most often was within the IV quarter of the BFM was statistically significant (Fig. 4). Hence, spectral and temporal domains are both important to Type I neu-
rons since the timing of the EPT in an FM sweep appears to be critical. For example, sonograms of Myotis FM pulses indicate that energy is greatest in the last half of the signal (Friend et al., 1966; Sales and Pye, 1974) and that harmonics are present in shorter FM sweeps in later phases of echolocation (Novick and Griffin, 1961). More extensive studies are necessary to determine the salient acoustic cues conveyed in this portion of the FM sound. A neuronal type similar to Type I units in the present study was previously described by Saga (1965b) and referred to as 'high-responsiveness' units. 'Highresponsiveness' neurons were characterized by having greater response magnitudes to FM than to pure tones, but having similar thresholds to both stimulus types. However, the percentage of Type I units in this study was much higher than that reported for 'high-responsiveness' units. The difference in percentage is likely due to 'high-responsiveness' units being classified according to threshold criterion, whereas Type I units in this study were assessed on the basis of maximal response. Fig. 9 shows a neuronal network model which can explain the 1V quarter being essential for response in the majority of Type I units. In Fig. 9E, the Type I unit (Neuron D) receives an inhibitory input from Neuron A, an exotatory input from Neuron B, and an excitatory input from Neuron C which is delayed in time by an interneuron. Neurons A and C differ slightly in their BFs and have an excitatory response area flanked by an inhibitory response area. Such neurons have been extensively characterized in Myotis inferior colliculus (Suga, 1965a, 1968, 1969) and comprise approximately 21% of the neurons at .~hat level (Suga, 1969). The inhibitory response area of Neuron A contains higher frequencies than the excitatory area, whereas the inhibitory response area of Neuron C contains lower frequencies ,h...~,,.,. ~h,..,.. excitatory area. Hence, these neurons would only respond to an FM stimulus which swept through the excitatory area first. Neurons with properties like A and C were previously referred to as "asymmetricar neurons, since they selectively respond to only one direction of an FM sweep (Suga, 1965b, 1968, 1969, 1973). The table in Fig. 9F predicts the effects of stimulation with pure-tone frequencies (fl, f2, f3) and upward and downward FM sweeps on Neurons A through D. Note that Neuron D responds only to pure-tone fl, but not to f2 due to the inhibitory effect of Neuron A (Figs. 9D, 9F). Neuron D maxim',dly responds to a downward FM sweep as a consequence of two key factors. First, the initial sweep through higher frequencies inhibits the response of Neuron A such that Neuron C's excitatory effect on Neuron D is now increased (disinhibition). Second, since Neuron C's response is delayed, Neuron D receives simultaneous excitation from Neurons B and C
186
as the F M sweeps through these n e u r o n s ' excitatory r.esponse areas. N e u r o n D also r e s p o n d s to upward F M sweeps due to the excitatory effect exerted by N e u r o n B, but this response is diminished since the excitatory influence exerted by N e u r o n C is diminished by Neuron A. Type II FM-sensitive neurons described in this study may have a m o r e specialized function in F M bats. T h e essential c o m p o n e n t for these. ,mils is not a single frequency; rather, these units req~ire an FM sweep to elicit a response. T h a t the I I I qttarter most c o m m o n l y (Fig. 6) was the essential F M c o m p o n e n t for T y p e II units lacks statistical significance, but nevertheless supports a neural network model p r o p o s e d previously by Suga (!965b) for 'FM-specialized' units. In this model, shown in Fig. 10, N e u r o n C has the s a m e response
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properties as a T y p e II ( ' F M - s p e c i a l i z e d ' ) unit and receives inhibitory input from N e u r o n A and excitatory input from N e u r o n B (Fig. 10D). A l t h o u g h N e u r o n s A and B have similar BFs, N e u r o n C d o e s not respond to any p u r e - t o n e stimulus due to the inhibitory effect o f N e u r o n A. Mowever, N' ~. .....,.,.. C d ,v__ "" ~ r e s p o n d at low thresholds to d o w n w a r d F M sweeps since the F M stimulus initially sweeps through the inhibitory response area o f N e u r o n A thereby releasing the excitatory effect exerted by N e u r o n B o n N e u r o n C. H e n c e , for N e u r o n C to be sensitive to d o w n w a r d sweeps only, N e u r o n A m u s t have an inhibitory r e s p o n s e a r e a with frequencies higher than its excitatory response area. T h e present study's finding that the mid-to-low frequency c o m p o n e n t s of the stimulus w e r e essential for ~esponse in m o s t T y p e II neurons lends s u p p o r t to this model.
Cortical organization Ninety-six p e r c e n t o f the n e u r o n s exhibiting sensitive ity to single stimuli could b e classified as F M - and not
187
pure-tone sensitive. These neurons span an area (Fig. 8) which includes the previe,~ly described tonotopic zone (Wong and Shannon, t~88). When the EPTs of Type I FM-sensitive neurons are plotted on a drawing of the cortical surface, they are. systematically arranged along a frequency axis. The resulting map of these units (Fig. 8) closely corresponds with the best frequency representation of neurons arranged tonotopically in Myotis cortex: higher best frequencies are located rostrally, with lower best frequencies located progressively more caudal. Whether previous maps were reconstructed from FM-sensitive neurons rather than pure-tone sensitive is unknown, since previous maps were made based upon BF without regard to whether the units were FM-sensiti,~;e or exclusively PT-sensitive (Suga, 1965b; Wong and Shannon, 1988). The fact that the tonotopic representation in Myotis cortex is based upon units that actually respond better to complex stimuli than to simple tones may be a feature of cortical organization specific to FM bats in general. FM sweeps are integral components of spectra in communication sounds as well as in biosonar pulseg (Suga, 1973). Behavioral studies of FM bats indicate the importance of spectral cues for shape and texture (Simmons et al., 1974; Habersetzer and Vogler, 1983; Simmons et al., 1990) and temporal cues for target feature a n a ! ~ s (Simmons, 1979; Simmons et al., 1990). These findings suggest that nnpott~tt~t c.~e~ for target characterization are derived from a single FM sweep, and a functional organization in which FM-sensitive neurons are found throughout the auditory cortex of M. lucifugus lends support to this type of auditory processing. Neurons exhibiting sensitivity to single pure-tone and FM sweeps could possibly participate in target perception as well as communication under different acoustic conditions. Units responsive to these singlesound stimuli have also been shown to acquire selectivity for FM pairs at particular repetition rates (Wong et el., 1992). Moreover, recognition of targets may not require a comparison ~-.a!ysis between pulse and echo if recognition is based upon wemory. The fact that FM-sensitive neurons are located ill both tonotopic and delay-sensitive zones of the M. iucifugus auditory cortex implicates these neurons in the processing of extensive auditory information that the bat hears, incl,~ding that used during echnlocadon.
Acknowledgements We would like to thank H. Te~g for her valuable comments on the manuscript. This work was supported in part by NIDCD R01 00600 to D. Wong.
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188 Suga, N. (1973) Feature extraction in the auditory system of bats. In: A. M¢ller (Ed.), Basic Mechanisms in Hearing, Academic Press, New York and London, pp. 675-744. Suga, N. (1988) Auditory neuroethology and speech processing: Complex sound processing by combination-sensitive neurons, In: G.M. Edelman, W.E. Gall and W.M. Cowan (Eds.), Functions of the Auditory System, John Wiley and Sons, New York, pp. 679-720. Suga, N., O'Neill, W.E., Kujirai K. and Manabe, T. (1983) Specificity of combination-sensitive neurons for processing of complex biosonar signals in auditory cortex of the mustached bat. J. Neurophysiol. 49, 1573-1626. Suga, N., O'Neill, W,E. and Manabe, T. (1978) Cortical neurons sensitive to combinations of information-bearing elements of biosonar signals in the mustached bat. Science, 200, 778-781. Suga, N., O'Neill, W.E. and Manabe, T. (1979) Harmonic-sensitive neurons in the auditory cortex of the mustached bat. Science, 203, 270-274.
Sullivan, W.E. (1982) Neural representation of target distance in auditory cortex of the echolocating bat Myotis lucifugus. J. Neurophysiol. 48, 1011-1032. Whitfield, I.C. and Evans, E.F. (1965) R,~sponses of auditory cortical neurones to stimuli of changing frequ,~ncy. J. Neurophysiol. 28, 655-672. Wollberg, Z. and Newman, J.D. (1972) Auditory cortex of squirrel monkey: response patterns of single cells to species-specific vocalizations. Science 175, 212-214. Wong, D. and Shannon, S.L. (1988) Functionarl zones in the auditory cortex of the e~holocating bat, Myotis lucifugus. Brain Res. 453, 349-352. Wong, D., Maekawa, M. and Tanaka, H. (1992) The effect of pulse repetition rate on the delay sensitivity of nc,ur6ns in the auditory cortex of the FM bat, Myotis lucifugus. J. C0mp. Physiol. A 170, 393-402.