Tonotopic organization of rabbit auditory cortex

EXPERIMENTAL

NEUROLOGY

Tonotopic NATHANIEL

75,208-220

Organization T. MCMULLEN

(1982)

of Rabbit Auditory AND EDMUND

Cortex

M. GLASER’

Department of Physiology, University of Maryland School of Medicine, Baltimore, Morylond 21201 Received July IO. 1981; revision received August 25, 1981 The representation of sound frequency within the auditory cortex of New Zealand and Dutch Belted rabbits was investigated using tangential microelectrode penetrations. Both multiunit and evoked potential responses to tone bursts were examined. The auditory cortex of the rabbit was found to lie caudal to the genu of the rhinal sulcus on the lateral surface of the temporal cortex. Two contiguous and tonotopically organized fields were found: a large primary field and a smaller secondary field dorsal and anterior to it. Within the primary field there is a complete and orderly representation of sound frequency. The highest best frequencies (ca. 30.5 kHz) are represented in the dorsal part of the field; the lowest best frequencies (ca 0.4 kHz) are represented in the ventral part of the field. The frequency range of 1 to 18 kHz was represented in greatest detail. The primary field was coextensive with cortex exhibiting cytoarchitectonic features characteristic of sensory cortex: a wide, cell-dense’lamina III/IV, a broad, sparsely cellular lamina V, and a welldeveloped lamina VI. The secondary field also contains an orderly tonotopic organization but it is reverse to that of the primary field. Its highest best frequencies were located ventrally and its lowest best frequencies dorsally. The auditory region in the rabbit is compared with that of two previously mapped, smooth-brain mammals-the squirrel and the guinea pig.

INTRODUCTION The topographic projection from the mammalian cochlea to the cerebral cortex was first demonstrated by recording cortical evoked potentials in response to electrical stimulation of the cochlear nerve fibers (20). Since that pioneering study, microelectrode studies have revealed that there is a systematic representation of cochlear place (the equivalent of sound freAbbreviations: AEP-average evoked potential, BF-best frequency, PSTH-poststimulus time histogram. ’ Supported by National Science Foundation grant BNS 78-05502 to E.M.G. 208 0014-4886/82/010208-13$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form resewed.

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quency) within a functionally and anatomically defined “primary field” in the cerebral cortex. Cortical tonotopic maps of best, or characteristic, frequency have now been obtained for a variety of species including the cat (14), monkey (13), squirrel (15), and guinea pig (9). Although there exist several imprecise mappings of the rabbit auditory cortex obtained by means of surface evoked potentials (4, 19), an extensive mapping with microelectrodes has not yet been made. The absence of a detailed tonotopic map is unfortunate because the rabbit’s relatively large lissencephalic cortex is well suited not only for electrophysiological mapping but also for detailed morphometrical examinations [cf. (5)] which permit correlation of neuronal structure with the functional acoustic map. We are currently engaged in such a study (6). There is also value in comparing the rabbit’s tonotopic organization with that of other mapped species. The differences in (a) the relative area devoted to the different regions of the acoustic spectrum and (b) the number of functionally distinct auditory fields are of interest. The former may provide insights into the animal’s behavior (3), and the latter has relevance to phylogenetic aspects of sensory cortical organization ( 16). In this report we describe the primary auditory cortex of the rabbit based on electrophysiologic and anatomic criteria. We also present evidence for the existence of a second auditory area which lies dorsal and rostra1 to the primary field. Portions of this work were reported elsewhere (6, 11). METHOD Dutch Belted and New Zealand rabbits ( 1.2 to 2.5 kg) were anesthetized with urethane (25%, 1 mg/kg, i.v.). After tracheal cannulation, a headholding pedestal was affixed to the frontal sinus region of the skull and secured to a stereotaxic apparatus (Kopf). A 6-mm-diameter craniotomy was made over the left auditory cortex and an acrylic dam incorporating a small Lucite chamber was installed around the exposed cortex. The dura was carefully removed and the exposed cortex was covered with warm (37’C) mineral oil. A photograph was then taken of the vascular pattern of the exposed cortex. All experiments were carried out in a sound-proof room (Industrial Acoustics Company). Core temperature was maintained at 37°C with a heated water pad. Stimulating earpieces (Brtiel & Kjaer 4034) were inserted into the auditory canal to approximately 6 mm from the tympanic membrane. They were calibrated under experimental conditions with a probe microphone (Brtiel & Kjaer 4033). Acoustical stimulation consisted of clicks, broadband (white) noise, and very narrow-band (~580 Hz between -20-dB frequencies) noise-modulated tone bursts. We have found

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that such tone bursts enhance the cortical response [cf. (lo)]. The tone bursts had trapezoidal envelopes with 7-ms rise/fall times and lOO-ms duration. The stimulus center frequency was controlled manually with a voltage-controlled oscillator (Wavetek). Glass, and later Parylene-insulated tungsten microelectrodes (Bak) were used to observe both multiunit activity and evoked potentials. Electrode impedances ranged from 0.2 to 2.0 MQ at 1.0 kHz with exposed tips of 15 to 40 pm. A remotely controlled hydraulic microdrive (Kopf) was used to advance the electrode through the cortex parallel to the sagittal plane and normal to a horizontal plane passing through bregma and lambda. The precise location of the electrode’s point of entry into the cortex was determined with a dissecting microscope and marked on a working photograph of the cortical surface. White noise bursts and clicks were used as stimuli until acoustically responsive unit activity was encountered. Tone bursts were then used as stimuli for the remainder of the electrode penetration. In the acoustically responsive cortex, best frequency was determined approximately every 300 pm by simultaneously adjusting stimulus frequency and intensity to find that frequency which evoked multiunit activity at the lowest intensity. The cortical activity was amplified (WP Instruments Model DAM-5A) and fed to a laboratory computer (Digital Equipment Corporation LAB 8/I) to generate poststimulus time histograms (PSTHs) and averaged evoked potentials (AEPs) from the responses to stimuli which were delivered at a l/s rate. Multiunit activity was observed on a stimulus-triggered oscilloscope and monitored by loudspeaker. Electrolytic lesions (10 PA, 5 s) were used to mark the end of an electrode track. During early experiments only the contralateral ear was stimulated. In later experiments the stimuli were delivered in a rotating paradigm: left ear only, right ear only, both ears. There were 50 repetitions for a total of 150 stimuli per mapped site. We report here only on the responses to contralateral (right) ear stimulation. In each of eight rabbits, we were able to make three or four parallel mapping penetrations (0.5-mm separation) through the auditory cortex, obtaining more than 40 best frequency determinations per animal. One or two mapping penetrations through the auditory cortex were obtained in each of nine additional animals. At the conclusion of the mapping phase of the experiment, the animal’s brain was removed after exsanguination, photographed, and processed according to one of two methods: (a) cresyl violet staining after frozen sectioning, and (b) Golgi-Cox Nissl staining after celloidin embedding (7). The Golgi-Cox material was reported in part elsewhere (12). A comprehensive report of this aspect of the work is in preparation (McMullen and Glaser, in preparation).

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RESULTS Primary Field. We found that the temporal cortex of the rabbit contains two tonotopically organized fields. We designated the largest of the two fields (which was mapped in greatest detail) as the primary auditory field. This designation was based on the short latency and low threshold of the acoustically driven multiunit activity and on the cytoarchitecture of the field. Results of a typical electrode penetration through the auditory cortex are shown in Fig. 1. Averaged evoked potentials to noise bursts were often observed well before acoustically responsive multiunit activity was encountered. In regions where the AEPs were recorded earliest ( 150 pV), and PSTHs showed the shortest latency ( 10 to 12 ms) when the electrode passed through the midcortical laminae. Within the primary auditory field, multiunit clusters were found to respond to tone bursts from 35 to 0.4 kHz, a range which approximates the behavioral hearing range of the rabbit (8). The sites corresponding to these frequencies were located, respectively, at the dorsal and ventral limits of the auditory field. It was common to find that the locus of the auditory cortex varies substantially from animal to animal. The locus of the primary region might shift as much as 1 mm when referred to bregma and the rhinal s&us. Best frequency maps from three rabbits are shown in Figs. 2-4. Note that all three maps show the higher best frequencies to be represented dorsally and the lower best frequencies ventrally. There was a complete and orderly representation of sound frequency (and hence, the cochlear partition) within the primary auditory field. The BF of neuron clusters within this field ranged from 0.4 to 30.5 kHz; BFs between 1 and 18 kHz were represented in greatest detail (i.e., smaller frequency gradient as ex-

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FIG. 1. Reconstruction of a single tangential electrode penetration through the left auditory cortex of rabbit RR 37. The inset indicates the locus of the electrode track. The numbers are the best frequencies (BFs) in kHz of neuron clusters obtained at each recording site. Average evoked potentials to contralateral monaural stimulation at BF (20 dB above threshold) are shown to the right of each recording site. Negativity is upward. In this and the other figures, a star indicates where acoustically responsive multiunit activity was present but no BF could be obtained; nr, no response to acoustical stimulation. The lesion site at the termination of electrode track is indicated by a circle. 212

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pressed by change in BF/mm) (Figs. 2, 4). The spatial distribution of the isofrequency contours was not uniform and was such that the higher frequencies (10 to 30 kHz) had a fuller representation in the anterior region of the primary field (Fig. 2, penetration 3) whereas in the caudal part of the auditory field there was a greater region devoted to lower (1 to 10 kHz) frequencies (Fig. 4). Although the nonuniformity in best frequency representation within the

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primary field made it difficult to fit lines of isofrequency to the best frequency data, they appeared to be oriented approximately 30 to 40 degrees counterclockwise from the rostrocaudal axis (Figs. 2-4). Because the fre-

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quency map differed among animals, there was corresponding variability in the orientation of the lines of isofrequency. Secondary Auditory Field. We mapped portions of a second auditory field which lay dorsal and anterior to the primary field. The existence of the second field was made apparent by a reversal in the sequence of BF measurements as an electrode traversed the cortex (Fig. 5). We took the boundary between the two fields as being where the spatial frequency gradient reversed. Occasionally, two widely different BFs were found at

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FIG. 5. Best frequency representation obtained from two tangential electrode penetrations through the auditory cortex of rabbit RR 38. Details of the enclosed portion of the electrode tracks (left) are shown on the right. Dotted line separates primary field from secondary area. Note the reversal of BF as the electrode traverses the boundary between the two fields.

the border of the two fields (Fig. 3). This was never seen when the electrode had entered the primary field. Although multiunit responses (short latency, low threshold) and AEPs (large amplitude and negative polarity) observed in this field were similar to those observed in the primary field, we found the tonotopic organization of this secondary field to be the reverse of that of the primary field: lower BFs were represented in the dorsal part of the field and higher BFs were represented in the ventral portion. The highfrequency region of both primary and secondary fields abutted one another

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(Figs. 3-5), making it difficult to draw a precise boundary between the two fields (Fig. 5). The two auditory fields appeared to be surrounded by a belt of acoustically responsive cortex containing high-threshold, broadly tuned elements for which BFs could not be determined. Cytoarchitecture of the Primary Field. A Nissl-stained coronal section through the primary auditory field is shown in Fig. 6. We found the primary auditory field, as defined by its responsive properties, to be coextensive with cortex exhibiting koniocortical characteristics ( 15). The primary field was characterized by a broad, cell-dense lamina III/IV, a wide, sparsely cellular lamina V, and a well-developed lamina VI (Fig. 6). As seen in coronal sections, the cortex immediately dorsal to the primary field was lacking in these koniocortical features. Most noticable was the loss of the cell-dense lamina III/IV and a reduction in the width and sparseness of lamina V. It appeared, therefore, that the secondary auditory field could be distinguished from the primary field based on these criteria. DISCUSSION The present study showed that the acoustically responsive cortex of the rabbit consists of two, tonotopically organized fields: a large primary field and a smaller secondary one. Both fields are caudal to the genu of the rhinal sulcus on the lateral surface of the temporal cortex. The location of the auditory fields varies among animals, as was reported in the cat and squirrel (14, 15). Within the primary field, BFs in the range of 1 to 18 kHz were represented in greatest detail indicating that most ,of the neurons in the auditory cortex are devoted to this spectral region. This is also the spectral range to which rabbits are maximally sensitive as shown by behavioral methods (8). The electrophysiologically defined primary region appears to be coextensive with cortex having koniocortical characteristics. These sensory cortical features include a broad, dense lamina III/IV and a wide, cell-sparse lamina V. The location of the primary auditory cortex delineated in the present study conforms closely to the Te 1 region of Fleischhauer et al. (2), which they assume to be the primary auditory region. Their identification was derived purely from cytoarchitectonic criteria. Although we lack data on the thalamocortical connections of the rabbit’s primary field, its complete and orderly representation of the cochlear partition and its koniocortical cytoarchitecture are strong evidence that this primary auditory field is the homolog to AI of the squirrel, cat, dog, guinea pig, and monkey (9, 13-15, 18). A secondary auditory field was located dorsal and anterior to the primary

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field. Although we have not completely mapped this field, the response characteristics of neurons within this field were similar to those in the primary field. This secondary field also appears to contain a complete rep resentation of the cochlear partition. However, its tonotopic organization is spatially the reverse of that in the primary field. The secondary field lies in the Te 2.1 region of Fleischhauer et al. (2) immediately dorsal to Te 1. The tonotopic organization of the rabbit auditory cortex described in the present study is in agreement with the surface evoked response study of Galli et al. (4) and the cochlear stimulation study of Woolsey (19). A similar BF representation was also described for the rabbit inferior colliculus (1). The studies of Galli et al. (4) and Woolsey (19) also provide evidence for a second auditory field situated similarly to the one which we partially mapped. The rabbit auditory cortex appears to contain only two auditory fields. The evidence for the existence of only two auditory fields was derived from more than 40 successful mapping penetrations through the auditory cortex. Although the failure to find additional auditory fields in this species may reflect experimental difficulties in isolating small cortical regions, we consider this unlikely. Two rodents -the squirrel and guinea pig-also appear to have only two tonotopically organized auditory fields (9, 15). This is in contrast to the cat, whose auditory cortex contains at least four tonotopically organized fields (17). The existence of two auditory cortical fields may be a generalized “ancestral characteristic” of lissencephalic mammals (16). It is interesting that although the temporal cortical locus of the primary field is nearly identical in the rabbit, squirrel, and guinea pig, the organization of BFs, lines of isofrequency, and relative position of the secondary auditory fields are not. Thus, the rabbit’s primary field is rotated counterclockwise on the temporal cortex in comparison with the cortices of the guinea pig and squirrel. There, the high frequencies are represented posteriorly and low frequencies anteriorly (9, 15). In both the rabbit and squirrel, the secondary auditory field lies rostra1 and dorsal to the primary field whereas in the guinea pig, it lies posterior to the primary field. Furthermore, the primary and secondary cortices of the rabbit and guinea pig interface at the high-frequency region of the acoustic spectrum. The squirrel’s auditory fields are joined at the low-frequency region (9, 15). In all three species lines of isofrequency take an erratic course and their orientation varies among animals. REFERENCES 1. AITKIN, L. M., S. FRYMAN, D. W. BLAKE, AND W. R. WEBSTER. 1972. Responses of neurons in the rabbit inferior colliculus. I. Frequency specificity and topographic arrangement. Brain Res. 42: 77-90.

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2. FLEISCHHAUER,

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71: 315-344.