A SINGLE NEURON ACTIVITY IN THE SECONDARY CORTICAL AUDITORY AREA IN THE CAT

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1 A SINGLE NEURON ACTIVITY IN THE SECONDARY CORTICAL AUDITORY AREA IN THE CAT TAKESHI WATANABE* Department of Physiology, Tokyo Medical and Dental University, Yushima, Tokyo, Japan Several electrophysiological investigations have already been performed on the auditory cortex. However, most of them were concerned with "evoked potentials" produced by sound stimulation and recorded with gross electrodes (Hind, 5; Rose, 13; Tunturi, 14, 15, 16, 17, 18) and only Erulkar et al.(4) reported the results obtained by the method of single unit analysis. In order to obtain further knowledge on the neural mechanism of audition we have been studying the single neuron responses to sound stimulation at various regions along the central auditory pathway in the cat by the use of hyperfine microelectrode method (6, 7, 8) under the common experimental conditions. The present work was carried out along this line and concerned with the function of cortical neurons in the secondary auditory The techniques employed here were almost the same as those reported before. METHODS Experiments were carried out on sixteen adult cats weighing from 2 to 3 kg., under very light general anesthesia. Deep anesthesia was avoided because it hampered seriously the activities of neurons in the brain as described previously (7). The surgical operations for recording the microphonic potentials led off from the round window were performed under ether anesthesia. In this case precaution was taken not to continue the anesthesia for longer than one hour, this is the maximum permissible length of time not to affect the activities of neurons in the brain. Occasionally, thiopental sodium or succynil-choline was given intravenously since these drugs did not much affect the activities of neurons owing to their transient action. Encephale isole method was also used with calm artificial respiration. The epidural application of xylocain in the upper cervical region of the spinal cord was sometimes used supplementarily. The skull was then opened and the ectosylvian gyrus as well as both anterior and posterior sylvian gyri of the cerebrum were exposed. The microelectrodes were similar to those used in the earlier works. The electrode was a glass micropipette with a outside tip-diameter of less than 0.2ƒÊ and filled with 3M-KCl solution, the ohmic resistance of the electrode ranging between 30 and 50Mƒ. The electrode for recording microphonic potentials was a silver wire of about 100ƒÊ in diameter and insulated except the tip was Received for publication January 8, Present adress:department of Physiology, Yokohama University School of Medicine, Yokohama, Japan.

2 246 T. WATANABE placed on the round window of the tested ear. Under a binocular microscope (Zeiss'operation microscope) the microelectrode was inserted very slowly into the brain with a micromanipulator through the pia mater after the incision of the dura mater. Deep precaution was taken against the insertion of electrode perpendicular to the cortical surface in order to measure the depth of the responsive neuron exactly. Since most neurons in the cortical auditory area showed almost no spontaneous discharges, single nueron responses to sound in the secondary area were searched by inserting the electrode into the cortex during the presentation of repetitive sound stimulation to the animal ear. The responses of neuron, the microphonic potential and the 10msec. time mark were led together to each input of three channels of the CRO and in general, photographed on a running film. Details of the specially made oscilloscope were reported previously (6). The amplifiers used in the present experiment were a cathode follower preamplifier with a low grid current (2 ~10-12A) and high gain DC and AC amplifiers. All photographs were taken with Grass Kymograph camera. The upward deflection of the beam presented in figures of the present paper indicates the positive polarity. Continuous pure tones as well as tone bursts were employed as stimuli. Successive 33 tone bursts of about 100msec. duration and with different frequencies in the range from 30 to 20,000cps. were generated by specially designed oscillator, details of which were reported elsewhere (7). The sounds were produced by 3 high-fidelity loudspeakers in combination. A continuous pure tone was generally used as a background tone when simultaneous stimulation of two sounds was desired, the reference zero db of the continuous pure tone being 4 volts at the output of an audio-oscillator as in the previous experiments. The animal was isolated in a sound-proof room in which temperature was kept at about 28 Ž. by air conditioning and the sound stimuli were delivered to the animal in a free field. The main recording and sound producing equipments were set up separately from the sound-proof room, and the photograph of the responses of neuron was thus made outside the room. The experiments, in most cases, were performed on the same day when the operation was made but when the favourable general condition of the animal was maintained they, were often done on the following two days on the same operated cat. RESULTS Prior to the insertion of microelectrode in the secondary cortical auditory area, the thresholds and responsive frequency ranges for tone bursts were examined on the neurons of the primary After confirming the normal activity of the neuron at the primary area recording of the single neuron responses was tried in the secondary area. The size, the shape and other features of recorded spikes were common in the both areas. The extent of the secondary area is ambiguous and at present not defined distinctly. However, in the present work the definition by Rose (11, 13) was followed,

3 NEURON ACTIVITY IN CORTICAL AUDITORY AREA 247 Evoked potentials Before the start of investigation with hyperfine microelectrodes the potential changes evoked by tone bursts were recorded with a gross silver electrode placed on the surface of the secondary At the area adjacent to the border of the primary area small positive-negative potentials were occasionally observed, while in the remaining large parts of the secondary area evoked potentials were hardly discernible. Such evoked potentials could also be recorded even with a hyperfine microelectrode in a few cases. The latencies of these potentials were almost constant, ranging between 13 and 15msec. These values are longer by a few milliseconds than those of the evoked potentials obtained at the primary Responses in terms of unitary spikes Single neuronal responses to sound stimulation with a hyperfine microelectrode were encountered less frequently at the secondary area than at the primary one. In contrast with the results obtained from the primary area (7) it was noticeable that most neurons encountered in the secondary area responded to strong continuous pure tones and not to brief tones, i.e. tone bursts, alone in spite of their sporadic spontaneous discharges (fig. 1 A). But several neurons were also obtained which responded to tone bursts (fig. 1 B). A B FIG. 1. A. Spontaneous discharges of a single neuron not responding to tone bursts. B. Responses to tone bursts of a single neuron. Responses are shown by upper beam, middle beam shows microphonic potentials led off from round window of contralateral tested ear. Frequencies of tone bursts are 300, 400, 500, 600, 700, 800, ,000, 1,300, 1,700, 2,000cps. from left to right in A and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 13,000, 17,000, 20,000cps. in B. Time:10msec. A. Neurons responding to tone bursts 1. Response pattern. Response patterns of the neurons for tone bursts were divided into two types at the secondary area as in the case of the primary They were responses with a rapid and a slow adaptation respectively. The former was further classified in on, off and on-off responses. Sometimes, either on, off, or on-off responses were recorded from one and the same neuron by changing the frequency of sound. The neurons with a rapid adaptation did not show "sigmoid relation" between the number of spikes per second and

4 T. 248 WATANABE the sound intensity in decibel. In contrast with those brief responses, repetitive spikes appeared for each tone burst in the latter cases with a slow adaptation. However, the cases with a slow adaptation were encountered much less frequently than those with a rapid one. As reported in the preceeding paper (7), a certain tendency was revealed between the depth and the adaptation of each recorded neuron at the primary Responses with a rapid adaptation were recorded from any layer of the cortex, both superficial and deep, but most often they encountered at the superficial layers, while the neurons with long lasting responses were situated at the deep layers. In the present experiment, no single neuronal responses to tone bursts have been recorded from the superficial layers. All neurons responded to tone bursts were obtained at the deep layers, more than 1mm. beneath the cortical surface, showing either a rapid or a slow adaptation. 2. Latency. The latencies of the single neuronal responses at the secondary area were relatively long and not constant, varying from 20 to 100msec. or more. An example of single neuronal response with a relatively long latency is presented in fig. 2. Though the response in this example looks apparently an off-response (left figure), it was confirmed to be an on-response with a relatively long latency by changing the duration of the tone burst. Responses with very short latencies of 9-10msec. were also encountered from the layer 3.5mm. beneath the surface of the anterior sylvian gyrus and the response area of this neuron was very narrow just as those obtained from the medial geniculate body (7). It is, therefore, very likely that such responses are attributable to those recorded from the ascending axons of neurons in the medial geniculate body. FIG. 2. Responses with relatively long latency evoked by a tone burst of 2,000cps. with different duration are shown by upper beam. Middle beam shows the microphonic potentials. Time:10msec. 3. Response Observing the response areas of the neurons responding to tone bursts it was found that their responsive frequency ranges were wide and also that the thresholds for any frequency were very high. Most of those areas differed appreciably from those obtained in the primary The former had no sharply tuned frequency range. In only a few cases response area similar to those at the primary area were found, though the thresholds for tone bursts were much higher compared with those obtained in the primary

5 NEURON ACTIVITY IN CORTICAL AUDITORY 249 AREA Fig. 3 shows responses of a neuron to tone bursts obtained from the layer 2.2mm. beneath the surface of the middle sylvian gyrus. As seen in the figure, the present neuron has a threshold of-40db. for a 20,000db. burst, which is the characteristic frequency of the neuron and an unusual elevation of the threshold is seen for a 13,000db. burst. Such a dip on the threshold-frequency curve for a single neuron response was seen, though rarely, at the deep layer of the primary area, too. It is conceivable that such an elevation of threshold may be due to some kind of interaction among adjacent neurons at the medial geniculate body. FIG. 3. Response area obtained from secondary Each gives a series of responses to tone bursts in equal intensity. beams show the response (upper), the microphonic potentials and time in 10msec.(bottom) respectively. B. Neurons responding to continuous pure column Three (middle) tones Among neurons which were activated by sound stimulation, about 60% of them responded only to strong continuous pure tones and not to short tone bursts. In those cases the intensities of sound used were much stronger than those used in the previous experiments on the primary In these cases the responses were provided with wide responsive frequency ranges and the frequency of spikes per unity interval very slightly increased with increasing intensity for the respective sound frequency. Neurons showing prolonged afterdischarges after cessation of sound stimulation were also rarely observed. The

6 250 T. WATANABE feature of responses of the neuron which responded only to strong continuous pure tones resembled to those obtained from the ascending reticular system in the thalamus, details of which will be discussed later. The depth and the responsive frequency ranges of the neurons responding to strong continuous pure tones obtained in the whole series of the present experiments are summarized in table 1. As seen in this table, responses of those neurons were encountered at any layer of the cortex, either at a superficial or at a deep layer. The spacial distribution of the neurons in this area is also illustrated in fig. 4. At the anterior part of the secondary area, no responses have so far been recorded by the present exploration. But small negative evoked potentials due to tone bursts were rarely observed. Probably the density of the active units in this area is quite low. On the contrary responses were frequently recorded at the posterior part. This situation is just opposite to that in the primary TABLE 1. Locations and Responsive Ranges of Neurons Responding to Continuous Pure Tones in the Secondary Area *SCC:Succynilcholine FIG. 4. Tonotopic localisation in secondary auditory Left hemisphere: S.S.; Suprasylvian sulcus, S.E.A.; Anterior ectosylvian sulcus, S.E.P., Posterior ectosylvian sulcus, S.Sy.; Sylvian sulcus. Solid circles indicate the locations of recorded neurons the numbers correspond those in table 1. Open circles indicate the locations of responding to tone bursts and with characteristic frequency.

7 NEURON Responses ACTIVITY to two sounds IN CORTICAL delivered AUDITORY AREA 251 simultaneously When a tone burst was applied during the background stimulation continuous pure tone, responses to tone bursts were often facilitated background tone of a moderate intensity. Similar phenomenon has also observed at the primary Fig. 5 shows a typical example obtained the layer 1.9mm. beneath the surface of the middle sylvian gyrus. by by a been from FIG. 5. Facilitatory and suppressive effects of second tones on the responses to tone bursts obtained from the depth of 1.9mm. Frequencies of tone bursts are marked at left side of figure. Left column shows responses as a control. Other columns shows change of response pattern by simultaneous presentation of a second tone, frequency of which is marked at bottom. Responses of neuron are shown by upper beam, middle beam shows the microphonic potentials. Time at bottom 10msec. As seen in the control records at the left column, the present neuron responded to tone bursts of from 300 to 2,000cps. The response to a tone burst of 400cps. was especially facilitated by a 2,000cps. background tone. Occasionally, neurons were observed which did not respond to tone bursts alone but responded to them when accompanied with a certain background tone. The effect of a background tone with different frequency on the tone burst response was examined. Fig. 5 also shows such an example. The frequencies of tone bursts are shown at the left side of the first column and those of back-

8 252 T. WATANABE ground tones are indicated at the bottom of each column. The range and the pattern of response to the tone burst were changed by changing the frequency of the background tone. When the frequency of the second tone is 3,000cps., the facilitatory effects appear for tone bursts with 300 and 400cps. In the cases of 2,000cps. and 1,000cps. background tones, similar effects are seen at 400 and 500cps. bursts, and at 400, 500, 600 and 1,700cps. bursts respectively. However, in the latter case a suppressive effect occurs for a 300cps. burst. We have already found at the primary area that there is usually a simple ratio between the frequencies of tone burst and of background tone when they produce marked facilitation. In the present case, as shown by fig. 5, the ratios between the frequencies of these two sounds are 1:10, 1:5 and 2:5, when the frequency of the background tone is 3,000, 2,000 and 1,000cps. respectively. Tone bursts with other frequencies were either facilitated or suppressed somewhat. The threshold change associated by facilitation or suppression of responses were examined for different frequencies. In one case, the threshold for tone burst of 500cps. alone was-27db. When a background tone of either 1,000, 2,000, 3,000 or 5,00cps. was delivered the threshold of the neuron varied to 5,000cps. elevated the threshold appreciably, but a 1,000 or 2,000cps. tone gave no noticeable change of the threshold. In another case, the threshold was delivery of background tone of either 500, 1,000, 2,000, 4,000, 8,000, 10,000, 16,000 or 20,000cps. brought the thresholds to-25,-22,-26,-24,-34,-27,-24 and-24db. respectively, i.e. a 8,000cps. background tone lowered the threshold, but the background tones of other frequencies afforded almost no effect on the threshold of the neuron. These situations in the secondary area is almost completely coincident with that in the primary Tonotopic localisation The tonotopic localisation is defined by the systematic spacial distribution of the neurons for their characteristic frequencies. At the secondary area, however, such neurons as having distinct characteristic frequencies were not frequently encountered. The locations of those neurons were presented schematically in fig. 4 together with those of the neurons which responded to continuous pure tones without any characteristic frequency. As seen in the figure, it can be said that the tonotopic localisation in the secondary area is at least very complicated just as in the primary area, though the number of neurons explored was not satisfactorily large enough. DISCUSSION Determination of the auditory areas has so far been made electrophysiologically and neurohistologically. In the latter case the method of retrograde cell-degeneration were used for that purpose after ablation of the cortical areas and the electrocoagulation at the medial geniculate body. Some different

9 NEURON ACTIVITY IN CORTICAL AUDITORY AREA 253 characteristic features have thus been found in the primary and the secondary areas. These two areas, however, has not been as yet delimitated distinctly in the cat (1, 2, 13, 19). Therefore, the present experiment was performed according to Rose's description. Woolsey and Walzl (3) suggested by their histological findings that fibers from the small-cell part (pars principalis) of the medial geniculate body in the thalamus apparently go to the primary auditory area and those from the large-cell portion (pars magnocellularis) reach the secondary On the other hand, appreciable differences of the difficulty in recording the responses of single neuron were found in the present experiment between the primary and the secondary areas. It is highly probable that the population of active neurons responding to sound stimulation is much less at the secondary area than at the primary one. Rose and Galambos reported previously (12) on the responses to sound stimulation in the medial geniculate body that they were encountered mainly at the tars principalis but almost not at the pars magnocellularis which remained silent or at best gave rise to responses of small magnitude to clicks. Histological evidences show that two parts of the medial geniculate body project their fibers to the primary and the secondary areas respectively as already mentioned. Although no direct physiological evidence has so far been found to prove such connections, if the impulses conduct through the magnocellular portion to reach the secondary area, the small-number of the active neurons at this area observed in the present experiment corresponds to the results at the medial geniculate body obtained by Rose and Galambos. On the contrary, it was remarkable that the recorded neurons responding to tone bursts in this area were located only at the relatively deeper layers than 1mm. from the cortical surface but not at the superficial layers. They might represent the responses from the neurons projecting from the medial geniculate body to the secondary At the primary area as reported previously, about 63% of 109 recorded neurons responding to tone bursts were located at the superficial layers which are not deeper than 1mm. from the cortical surface. Deep care was paid for the effect of anesthesia on the activities of neurons but the responses of neurons to tone bursts were still not found at the superficial layers in the secondary area, though neurons were frequently encountered which showed spontaneous discharges and were not influenced by sound stimulation. The reason for this fact is at present unknown. As already described the evoked potentials due to tone burst stimulation were hardly discernible in the most parts of the secondary area while at the primary area they were easily observed under the same experimental condition. The obscurity of the evoked potentials in the secondary area well explains the difficulty in recording of the responses of neurons to sounds. In anyway response impulses due to the sound stimulation are forwarded into this region through a relatively small number of neurons at the medial geniculate body or elsewhere and only very small portion of neurons in the secondary area might be activated. The response areas of the single neurons examined by a tone burst stimulation in the secondary area were very wide, Many of them did not show any

10 254 T. WATANABE sharply tuned characteristic frequency, and only in a few cases such sharply tuned one was seen as that of observed at the primary The thresholds for each tone bursts were nevertheless very high in this area in either of two cases. It was suggested in our previous report that the analysis of sounds in reference to frequency and intensity is completed at the level of the medial geniculate body, because of the narrowest response area of single neuron at this level and of the maintenance of the "sigmoid relation" between the frequency of discharges in a unit time interval and the intensity of sound in decibel up to this level. On the other hand the role of the primary auditory area seemed to be the integration of component sounds by mutual facilitation of neurons. Similar facilitatory effects were also observed in the secondary From these present experimental results the function of the neurons in the secondary area does not seem to be directly concerned with the analysis of sounds but with the integration. However, it is likely to be of a different order from that at the primary area, because of high thresholds, longer latencies, after-discharges after cessation of sound stimulation and also the more complicated patterns in the interaction among neurons. Only from those results, however, nothing further can be deduced about the auditory sensation of the cat. In order to discuss more about the function of neurons in this area which may contribute something to the auditory sensation further studies are needed on the functional relation of neurons in this area to those in other regions. The latencies of responses to tone bursts in this area were generally found to be much longer than any of those in the primary This fact agreed well with the results obtained by the evoked potentials. In the primary and also the secondary areas responses with unusually long latencies of 50msec., even over 100msec. were often encountered besides those with short latencies. Such responses should have passed through complicated synaptic pathways before reaching the cortical neurons. They might come from extralemniscal pathways, e.g. the ascending reticular activating system proposed by Magoun (10). Attention was drawn by Magoun (9) and others to the fact that several kinds of sensory stimuli including auditory one fired the neurons in the ascending reticular activating system involving the medial reticular formation, the subthalamus and hypothalamus and also the ventromedial thalamus. The comparison between the responses obtained from the secondary area and from the reticular system were tried. Those experimental results showed that many neurons were provided with quite similar responses, e.g. the wide response areas without sharply tuned characteristic frequencies on. Details of them will be reported elsewhere., long latencies and so On the tonotopic localisation, Tunturi (14) figured out the beautiful map at the middle ectosylvian gyrus of the dog by means of the surface evoked potentials by the use of the strychnine techniques. However, as already reported (7), the present results in the cat, in terms of single neuron activity explored with a hyperfine microelectrode, were not completely coincident with

11 NEURON ACTIVITY IN CORTICAL AUDITORY AREA 255 them. General tendency of the tonal localisation in the primary area, however, were found to be similar in the dog and the cat, i.e. low frequency tones activate neurons in the more caudal portion of the gyrus, while progressively higher tones activate those more rostrally located. Contrary to the primary area, Woolsey and Walzl (19) revealed the tonal localisation at the secondary area to be arranged reversely in direction, i.e. the high frequency tones activate the posterior neurons, while low tones anterior ones. However, such tonal localisations in the secondary area were not clearly found, similarly as in the case of the primary area, as far as the measurements described above were concerned. SUMMARY Discharges of a single neuron in response to tone bursts or continuous pure tones were studied in the secondary cortical auditory area of the cats under very light general anesthesia by means of hyperfine microelectrode technique. Difference of responses of a single neuron to sound stimulation was found between the primary and the secondary areas. (1) Small evoked potentials by tone burst stimulation were recorded from the regions adjacent to the primary area while in the remaining regions they were hardly found. (2) The thresholds of all neurons examined for any stimulating sounds were much higher in the secondary area than in the primary (3) The latencies of responses to tone bursts were generally much longer, though variable, in the secondary area than in the primary (4) Most neurons in the secondary area responded only to strong continuous pure tones and not to tone bursts alone. Their responsive frequency ranges were very wide. Sometimes, the prolonged after-discharges were observed. (5) Neurons were also encountered responding to tone burst alone. Their responses had the same feature as those of the primary (6) Measurements were done on the response areas of neurons. Some of them showed a characteristic properties similar to those in the primary area, while the others were different, the areas being wider and not having the distinct characteristic frequency. (7) By two sound stimulation facilitatory and suppressive interactions were observed on the single neuronal responses just as in the case of the primary (8) Tonotopic localisation was not clearly found in this area, because many neurons explored had no distinct characteristic frequencies. Even on the neurons having a narrow response area the situation was very complicated. In contrast to the primary area neurons were easy to be recorded from the lower half of the posterior ectosylvian gyrus. (9) From the results described above it may be concluded that neurons of the secondary area do not play an important role for the analysis of sound, i.e for the discrimination of pitch and intensity of sound, but rather have integrative functions for analyzed component sounds.

12 256 T. WATANABE I wish to express my thanks to Professor Y. Katsuki for suggesting this investigation as well as for his constant guidance in the course of this work. My thanks are also due to Dr. S. Hagiwara for some valuable suggestions and to Mr. M. Ootori and Mr. N. Suga for their technical assistance. This work is supported by a grant from the Ministry of Education of Japan. REFERENCES 1. ADES, H. W. A secondary acoustic area in the cerebral cortex of the cat. J. Neurophysiol. 6: 59-63, BREMER, F. AND DOW, S. The acoustic area of the cerebral cortex in the cat. A combined oscillographic and cytoarchitectonic study. J. Neurophysiol. 2: , DAVIS, H. Psychophysiology of hearing and deafness. Handbook of experimental psychology, pp New York: John Wiley and Sons, ERULKAR, S. D., ROSE, J. E. AND DAVIES, P. W. Single unit activity in the auditory cortex of the cat. Bull. Johns. Hopk. Hosp. 99: 55-86, HIND, J. E. An electrophysiological determination of tonotopic organisation in auditory cortex of cat. J. Neurophysiol. 16: , KATSUKI, Y., Sumi, T., UCHIYAMA, H. AND WATANABE, T. Electric responses of auditory neurons in cat to sound stimulation. J. Neurophysiol. in press KATSUKI, Y., WATANABE, T. AND MARUYAMA, N. Functional analysis of the auditory neurons in the upper brain of cat. J. Neurophysiol. in press KATSUKI, Y. AND WATANABE, T. Electric responses of auditory neurons in cat to sound stimulation, 111. Proc. Jap. Acad. 34: 64-69, MAGOUN, H. W. An ascending reticular activating system in the brain stern. Arch. Neurol. and psychiat. 67: , MAGOUN, H. W. Brain stem and higher centers. pp.11-93, Nerve impulse. Transactions of the fifth conference. New York: Josiah Macy Jr. Foundation, ROSE, J. E. The cellular structure of the auditory region of the cat. J. Comp. Neurol. 91: , ROSE, J. E. AND GALAMBOS, R. Microelectrode studies on medial geniculate body of cat. I. Thalamic region activated by click stimuli. J. Neurophysiol. 15: , ROSE, J. E. AND WOOLSEY, C. N. The relations of thalamic connections cellular structure and evokable electrical activity in the auditory regions of the cat. J. comp. Neurol. 91: , Tunturi, A. R. Audio-frequency localisation in the acoustic cortex of the dog. Am. J. Physiol. 141: , TUNTURI, A. R. Physiological determination of the boundary of the acoustic area in the cerebral cortex of the dog. Am. J. Physiol. 160: , TUNTURI, A. R. Physiological determination of the arrangement of the afferent connections to the middle ectosylvian auditory area in the dog. Am. J. Physiol. 162: , TUNTURI, A. R. A difference in the respresentation of auditory signals for the left and right ears in the iso-frequency contour of the right middle ectosylvian auditory cortex of the dog. Am. J. Physiol. 168: , TUNTURI, A. R. Analysis of cortical auditory responses with the probability pulse. Am. J. Physiol. 181: , WEVER, E. G. Theory of hearing, pp New York: John Wiley and Sons, 1949.