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Classification of chemoresponsive tongue units of the cat geniculate ganglion
Brain Research, 54 (1973) 157-175 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
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CLASSIFICATION OF CHEMORESPONSIVE T O N G U E UNITS OF T H E CAT G E N I C U L A T E G A N G L I O N
JAMES C. BOUDREAU* AND NECIP ALEV Leech Farm V. A. Hospital, Pittsburgh, Pa. 15206 and Department o f Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213 (U.S.A.)
(Accepted November 10th, 1972)
SUMMARY Single unit recordings were taken from neurons in the geniculate ganglion of the anesthetized cat. Three groups of chemoresponsive tongue units were identified on the basis of spontaneous activity measures and response to chemical and electrical stimulation of the tongue. Electrical stimulation of the sites of excised fungiform papillae to which tongue units project elicited spikes with latencies of 7-9 msec from group I units, 10-13 msec from group II units and 14 msec or greater from group III units, with little overlap among the groups. The intact tongue was stimulated with different chemical substances. Group I units were preferentially discharged by certain substances such as citric and malic acid, group II units by cysteine and proline. Group III units were affected by fewer substances although some group III units were among those maximally sensitive to mononucleotide salts. Spontaneous activity measures were also utilized to classify the 3 groups, at least in part. The units in the 3 groups innervated fungiform papillae located in different but overlapping regions of the tongue.
INTRODUCTION The geniculate ganglion is a small cranial sensory ganglion located dorsal to the inner ear at the junction of the facial nerve, greater superficial petrosal nerve, and the nervus intermedius. Although the cat geniculate ganglion contains only around 1800 sensory nerve cells, these cells subserve a wide variety of sensory functions, In the first study 1 on single unit recordings from the ganglion, the units were divided into 3 major populations: ear cells that were discharged by moving hairs on the inner surface of the pinna; regular discharge units, most of which were affected by moving * Current address: University of Texas at Houston, Graduate School of Biomedical Sciences, Department of Neural Sciences, Houston, Texas 77005, U.S.A.
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tissues of the soft palate and nasopharynx; and tongue units. Chemoresponsive tongue units were found to comprise a heterogeneous collection of units, but the measures used proved inadequate for classification. In a later study, Kruger and Boudreau s, utilizing response measures to inorganic salt solutions and buffer solutions, identified two major chemoresponsive unit groups which comprised about 7 0 ~ of the chemoresponsive geniculate ganglion units. In this report we present different measures on single unit spike activity which demonstrate that over 90 ~ of chemoresponsive tongue units can be classified into 3 different unit types. The measures used to classify tongue units are spontaneous activity, response to electrical stimulation, and stimulus response measures utilizing a number of chemical stimuli. Similar measures have been found useful for subdividing complex neural populations in other sensory systems. Thus spontaneous activity measures have been found useful for classifying units in peripheral muscle 9 and skin 3 systems and in the cochlear nuclei 7. Latency measurements from peripheral nerve fibers have been used to classify units in muscle ~, joints 2 and skin systemsZ, 6. Stimulus-response measures have been used in all sensory systems to classify neurons and to determine the relationship between input-output variables. The importance of classification in establishing group membership cannot be underestimated. It has been demonstrated in sensory studies that neurons can be grouped into different functional categories. Without adequate criteria for classification, measures from neurons belonging to different groups may be placed together, usually resulting in an impairment in the quantitative stimulus-response relationships measured for the neurons. The measures presented in this report form a solid basis for the classification of tongue units. When group affiliation is known, many response characteristics of a neuron can be predicted and experiments can be performed upon a unit as a representative of a specific group. TECHNIQUES
The preparation Adult cats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg) and, following a tracheotomy, secured in a stereotaxic headholder. The skin and muscles were cleared from the occipital portion of the skull, a portion of the cranium was removed and a partial cerebectomy of the neural matter overhanging the superior aspect of the petrous portion of the temporal bone was performed. The petrous bone overlying the ganglion was planed away with a carbide dental burr until the geniculate ganglion (and portions of the facial and greater superficial petrosal nerves) was exposed. Care was taken to preserve the blood supply to the ganglion. To penetrate the ganglion, either the sheath was nicked with a microprobe or (in most cases) the electrode was inserted through the sheath. The cat's temperature was maintained between 35 and 36 °C. The mean room temperature during recording was 23.2 °C. Relative humidity was kept between 40 ~o and 50 ~o (mean 45.1 ~o) to minimize temperature changes on the surface of the tongue produced by evaporation. Tungsten microelectrodes were advanced into the ganglion with a hydraulic
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mierodrive. The spike activity of a single unit was isolated and fed into an electronic counter or a CAT computer for processing and recorded on analog magnetic tape.
Stimulation of the tongue Papillae were electrically stimulated with a small stainless steel p r o b e (anode) with a large cathode clamped to the opposite side of the tongiae. A constant current stimulator was used to deliver 0.1 msec pulses at a rate of 10/sec. Spike latencies were measured from the oscilloscope face and are accurate to within 5 %. In the text there is the occasional remark that the tongue has not been stimulated. This remark means that there has been no prior electrical or chemical stimulation of the tongue. All chemical solutions were mixed in 50 m M concentration in deionized, glass-distilled water and then frozen. Prior to the experiment the chemicals were thawed and brought to room temperature. Chemical substances were applied to the tongue with glass pipettes on to the area known to be innervated by the unit under study. Such an application flooded a large area of the tongue. The change in discharge for a 10 sec period following stimulation was c o m p a r e d t o the spontaneous activity level in one or more 10 sec periods prior to stimulation. Since many chemoreceptive tongue units exhibited high levels of spontaneous activity, the response to chemical stimulation was expressed as the number of spikes during 10 sec o f stimulation minus the spontaneous activity level. Following the stimulus period, the tongue was rinsed with deionized, glass-distilled water. RESULTS
Tongue units can be subdivided into 4 classes. One class of neurons is primarily mechanically sensitive. These mechanically sensitive units display in general little or no spontaneous activity, exhibit latencies to electrical stimulation between 2 and 5 msec, and are maximally sensitive to moving mechanical stimuli. We have not studied mechanically sensitive cells in any detail and will give little further consideration to them other than to acknowledge their existence. The chemosensitive units have been divided into 3 classes of units, labeled group I, group II and group III. Uni~ groups I and II are identical to unit groups I and I1 distinguished by Kruger and Boudreau s primarily on the basis of their response to inorganic salts and physiological buffer solutions. Group III units consist primarily of those units presented in the earlier study as ungrouped. The measures for classifying units presented in this report have not been published before. Each neuron was classified according to a cluster of single unit spike measures. The most useful measures for classification proved to be spontaneous activity measures, response to electrical stimulation and response to chemical stimulation. In addition to displaying differences on these measures, the 3 classes of neurons were found to innervate fungiform papillae located in part on different regions of the tongue.
(I) Spontaneous activity measures All chemosensitive geniculate ganglion tongue neurons display some measurable
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level of spontaneous activity, where spontaneous activity is defined as spike discharge in the absence of experimenter controlled stimulation. The measures taken of tongue unit spontaneous activity were rate measures (counts of spikes/10 sec periods) and interspike interval histograms. These measures were taken under 3 conditions: (1) tongue-in, unstimulated condition; (2) tongue-out, unstimulated condition; (3) tongue-out, stimulated condition. Only rate measures were taken for the third condition. In the tongue-in condition the tongue was in the mouth and at body temperature, whereas the temperature of the tongue tip in the tongue-out condition was close to room temperature. In the unstimulated tongue-in condition, tongue units emit spikes continuously at rates between 5 and 195 spikes/10 sec. Successive 10 sec counts of spontaneous discharge rates from a tongue unit indicate that rate tends to be relatively constant for each cell. The standard deviation tends to increase with the mean rate up to a rate value of about 40 spikes/10 sec, beyond which little increase is seen (Fig. 1). Spontaneous activity rates above about 50 spikes/10 sec are associated with fairly constant standard deviations. Units with rates above 50 spikes/10 sec have proven in our studies almost invariably to be group II neurons (Fig. 1). Rate measures from unstimulated units do not clearly distinguish group I from group III neurons. Interspike interval (ISI) histograms were also measured for units in the tonguein, unstimulated condition. Even when measured from unstimulated units the ISI histograms of tongue units tended to be of bewildering diversity. Examples of ISI histograms from the 3 different groups of tongue units are presented in Fig. 2. The
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modal value of ISI histograms is usually in the extreme short interval end (between 3 and 10 msec). This short interval mode is a result of the presence of bursts o f spikes; the spikes in the burst being separated by relatively constant short intervals. The ISI histograms o f group I units were almost always multipeaked. Group II units often
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exhibited single peaked ISI histograms with the distribution declining slowly from the short interval mode. Group III units exhibited two different types of ISI histograms: one multipeaked and similar to those seen with group I units and the other single peaked and consisting almost entirely of short intervals. This second type of histogram (Fig. 2, cell GG89C1B) results when the spontaneous activity consists almost entirely of multiple spike bursts. All of the ISI histograms in Fig. 2 are asymmetrical with a tail extending toward the long intervals (positively skewed). One measure of the degree of asymmetry of a distribution is to determine the relationship between the mean and median. When the mean and median of the ISI distributions are plotted with respect to one another, it is found that the two values tend to be in a relatively constant relationship to one another (Fig. 3). Regardless of the rate of discharge of the units, the mean interval is always greater than the median interval (with the majority of median to mean ratios between 0.4 and 0.8). Group II units, with lower median and mean interval values, tend to cluster separately on this graph. The ISI histograms of group III units are either skewed in a manner similar to group I ISI histograms or are more markedly skewed with median/mean values below 0.2. It is possible to change the spontaneous activity of a unit simply by changing the condition of the tongue. In our studies this changed condition was accomplished by 40-
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opening the m o u t h and protruding the tongue. Such a procedure usually resulted in decreased spontaneous activity (Fig. 4). The mean percentage decreases in rates for the 3 groups are 29 ~o for g r o u p I, 37 ~ for g r o u p 1I and 68 ~ for g r o u p III. Because o f this
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greater decrease in rate under the new condition, spontaneous activity measures from group III units tended now to separate out from rate measures from the other two groups (Fig. 4). Besides exhibiting a greater proportional decrease in rate with the tongue-out condition, group III units also often showed a major increase in the skewness of their ISI histograms. This change is illustrated by a plot of the median/mean ratio calculated for the two conditions (Fig. 4). The ISI histograms of group I and group II units were less affected by the changing conditions.
(2) Latency measures Latencies of discharge to electrical stimulation of the tongue were measured for all chemosensitive tongue units whenever possible. These latencies were measured after chemical stimulation of the tongue. Latencies were measured by electrically stimulating the intact fungiform papillae which a unit innervated (papillae-on condition) and electrical stimulation of these sites after the fungiform papillae had been excised (papillae-off condition). Excision of fungiform papillae was performed because of the great variability in latency measures encountered when electrically stimulating the intact papiUae 1. This variability was believed to be due to the existence of multiple electrically sensitive trigger zones, apparently in the end organ. It was thought that by excising the fungiform papillae the nerve fibers innervating the receptors could be electrically stimulated directly and the variability of latency measures would be reduced. The expected reduction in variability occurred. Latency measurements obtained under the papillae-off condition proved to be less variable than those obtained by stimulating the intact papillae. This reduction in variability is especially noticeable in the threshold latency measurements taken under the two conditions (Fig. 5, top). Threshold latencies measured under the papillae-on condition tended to vary widely for all 3 unit groups. Papillae removal was associated with a sharp reduction in the variability of threshold latency measurements for all 3 unit groups (Fig 5). Latencies were also measured for each unit at suprathreshold intensities (to 3.5 mA). Increasing the intensity of electrical stimulation above threshold often results in large decreases in latencies measured from intact papillae systems1. Only small shifts in latency occur in the papillae-off condition when the intensity of electrical stimulation is raised above threshold. The relationship between suprathreshold latency measurements measured before and after papillae removal are also shown in Fig 5. The latency measures taken after papilla removal proved to be stable and reliable measures for the classification of units. After papillae excision the latencies measured from different papillae innervated by the same tongue unit tended in almost all cases to be quite similar to one another and in practice it proved possible to represent a cell by a single latency measurement (the shortest obtained). Latency measurements for the 3 groups of chemoresponsive tongue units fell into 3 demarcated classes with little overlap. The latencies of group I units ranged from 7 to 9 msec, group II latencies from 10 to 13 msec, and group III latencies were 14 msec or greater. These latency categories proved to be quite reliable and were utilized as one of the chief measures for unit classification. Only two group II units did not exhibit shortest latencies between
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10 and 13 msec, and one of these was 9.5 msec and the other 14 msec, near the lower and upper boundaries of the group II latency range respectively. The validity of the latency measurements for classification is indicated by the relationship between latency and other response measures from tongue units. Group 35"
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II units were those units with the highest level of spontaneous activity and when spontaneous activity rates (tongue-out, stimulated condition) for tongue units are plotted against latency measures for the same units, the group II units are clearly interpolated between the other two low spontaneous activity groups (Fig. 6). Spontaneous activity rates measured under the tongue-out unstimulated condition were similar to tongue-out stimulated rates. It is apparent from Fig. 6 that much of the variability found in the measurements on spontaneous activity was due to the existence of the 3 separate unit groups.
(3) Chemical stimulation In previous work 1,8 on cat geniculate ganglion tongue units involving chemical stimulation with various inorganic salts, it had been found that either the substances did not stimulate units in any reasonable concentration or did not stimulate in a manner that clearly differentiated unit groups. Sodium chloride thresholds for instance were 0.1 M or greater and NaC1 could activate at least some members of all 3 of the unit groups described in this report. Early work had also shown that most cat tongue units could be discharged by weak solutions of various animal components such as chicken breast, cod fillet, pork liver, etc. In an effort to extend our limited list of stimulating substances, to investigate the chemical substrate for the response to foods, and to
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determine if there existed certain chemical substances which more clearly differentiated between units, a great many chemical substances were tested for their stimulatory effectiveness. An initial group of chemical substances was chosen largely from a list by Solms 12 of the nonvolatile compounds important in human perception of food flavors. This group of substances included amino acids, nucleotides, organic acids and various other compounds. The work on the biochemical substrate of the food response is still going on. In this report we wish to present only chemical data relevant to the classification of chemosensitive tongue units. From 17 to 125 different substances were tested on 31 neurons. From these initial substances, 16 different test chemicals were chosen and were tested on 14 additional neurons. These test chemicals were found to clearly distinguish members of the 3 unit groups which could be identified by spontaneous activity or latency measures. The 'response profiles' of 4 units to the test chemicals are illustrated in Fig, 7. The data from one group I and one group II neuron are shown in this figure. Members of either group I or group II were similar to one another in chemical responsiveness although, as can be seen, the two groups varied markedly from each other. Group III units proved less homogeneous on the chemical measures than the other two groups. Two examples of group III unit chemical responsiveness 'are also presented in Fig. 7. The response profiles shown in this figure proved invaluable in the classification of units. The responses of 42 units to 10 of the test stimuli are presented in Fig. 8. In this figure each neuron is arranged with respect to the shortest latency measured for that cell following papilla removal. The chemical selectivity of each of the 3 unit groups in terms of spikes discharged is clearly indicated in this figure. Short latency units (group I) are most responsive to malic acid, citric acid, and O-phosphorylethanolamine, medium latency units (group II) are among those maximally discharged by the amino acids L-proline and L-cysteine, and only long latency units (group III) discharged appreciably to the nucleotide inosine 5'-monophosphate. Two of the chemicals tested, inosine-5'-triphosphate, and inosine-5'-diphosphate (not shown) stimulated members from all 3 groups whereas some other chemicals stimulated members from two of the 3 unit groups. Thus tetrasodium pyrophosphate stimulated group tI and group III units, and butyryl choline chloride stimulated units in groups I and III. It was possible to utilize the results from the chemical stimulation studies to characterize the 3 groups in terms of their chemical responsiveness. Unit groups I and II proved to be more homogeneous in their responsiveness to the test stimuli than group III units. All of the group I units were activated by malic acid, O-phosphoryletha;nolamine, citric acid and butyryl choline. Group I units were also the only units activated by creatinine. Group II units were air discharged by L-proline and L-cysteine and were usually inhibited by L'trypt0Phan and L-isoleucine. Group III units on the other hand seemed to be divided into two subgroups. One subgroup was activated by the nucleotides ITP, IDP and IMP. The other subgroup was in general insensitive to most of the stimuli used and discharged only to phytic acid and butyryl choline chloride. In practice the responses to the test chemicals presented proved adequate for
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classification of almost all tongue units into 3 categories. Classification was done both in terms of the chemicals to which the neurons were sensitive and the chemicals to which the neurons were insensitive. The responsiveness to the chemicals that maximally discharged neurons from the 3 groups also proved to be the responsiveness most resistant to change.
(4) Location When studying the latency of discharge to electrical stimulation, the receptive field of each unit was also recorded by plotting the location of each unit papilla
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Fig. 9. The approximate locations of the papilla systems of the different unit groups identified in the geniculate ganglion. Each unit is represented by one point - - the locus of the papilla site from which the shortest latency to electrical stimulation was measured. No. of units: 37, group I; 39, group II; 26, group III.
system on a standardized schematic diagram of the tongue. It was found that each of the unit groups projected preferentially to different regions of the tongue although there was much overlap. Group I units innervated the posterior center and side of the tongue; group H units innervated the side and tip of the tongue; and group III units innervated fungiform papillae on the sides of the tongue. This differential innervation of the tongue by the different unit groups is illustrated in Fig. 9. These graphs include the units classified according to the criteria discussed in this report and units classifiable on spontaneous activity measures, latency measurements without papilla removal, and the response to NaC1, citric acid, quinine and often other chemical stimuli. The basis for classifying units on these chemical measures is presented in the discussion of this report. The latency measurements for the 3 unit groups were found to be independent of the cat studied and the locations of the papilla systems innervated. Latency measurements did not vary as a function of distance from the tip of the tongue, rather latency was correlated with group membership (Fig. 10).
(5) Unclass~ed neurons Of the 42 neurons for which spontaneous activity, latency and adequate chemical measures were available, only 3 units could not be readily classified into the 3 unit groups described. These 3 units did not straddle the 3 established categories in such a way as-to render ~ classification invalid; rather they seemed to represent somewhat anomalous units. All 3 of the unclassified units were similar to one another. Two of the latency measurements from those 3 cells were in the group II range, both being 12.5 msec. The latency measurement for the third cell was 14.0 msec. Their unstimulated, tongue-in, spontaneous activity rates were low (6.8, 8.6 and 18.3) and their ISI histograms were of the multimodal group I variety. They were in general not highly responsive to the test chemicals, and what response existed did not clearly indicate a classification.
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DISCUSSION
The measures presented in this report definitely establish the fact that the chemoresponsive geniculate ganglion units constitute at least 3 separate sensory sub-systems. These 3 sub-systems are composed of neurons that innervate fungiform papillae on different parts of the tongue. The latency measures indicate that the peripheral fibers of the 3 systems are also probably of different fiber diameters. Spontaneous activity measures suggest the possibility that different neural pulse codes are utilized by the 3 separate systems. It is extremely unlikely that there are fewer than 3 unit groups, although the possibility exists that there may be more than 3 groups. Our present measures are inadequate for further subdivisions. Each of the 3 neural groups delimited is not homogeneous however; rather, there are often differences in spontaneous activity and chemical responsiveness among the neurons grouped together. To what extent these differences may represent functional subdivisions within the group is yet to be determined. The measures used to demarcate these 3 separate peripheral neur~tl chemoreceptor groups are at least as valid as measures that have been utilized by other workers in analyzing other sensory systems. In other sensory systems when neurons are functionally grouped with measures similar to those reported here, the peripheral
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sensory systems have often proven to have certain structural correlates. In most cases the different unit groups have been shown to have anatomically distinct peripheral connections, usually different receptor types. Thus muscle afferents may be broken down into fiber groups innervating Golgi tendon organs, primary spindle afferents, secondary spindle afferents and others 13. The fibers present in a nerve innervating the skin innervate anatomically distinct structures such as hair follicles, Vater-Pacini corpuscles, tactile pads, etc. 6. In most cases neural recordings from fibers innervating distinct structures can be distinguished functionally.
The measures utilized (1) Spontaneous activity. It is often possible to classify a geniculate ganglion unit simply on the basis of its spontaneous activity. Thus most group II units and some group III units could be clearly identified on the basis of rate and ISI measures. In other cases spontaneous activity measures for neurons from different groups did not clearly indicate group membership. Spontaneous activity patterns of geniculate ganglion tongue units are extremely complex and it is possible that more precise measures of these spike trains would more clearly differentiate units. (2) Latency measures. The latency measures proved to be among the most reliable measures for grouping cells. The fact that the threshold latencies measured for stimulating the intact papillae system often differ markedly from those measured by stimulating the site of the excised papilla indicates that there exists more than one electrically excitable component in the peripheral system. Since latencies almost invariably shorten when the fungiform papillae are removed, it is likely that the end organs for the neural units are in most cases located in the fungiform papillae. Latency measures for members of each neural group do not vary as a function of position along the tongue, suggesting that greater distances are compensated for by larger diameter fibers. This adjustment for distance insures that within a group information from different parts of the tongue arrives centrally at the same time. (3) Chemical measures. The results from the studies on chemical responsiveness of geniculate ganglion tongue units suggest that each neural group has a different chemical stimulus dimension. That members of all 3 groups discharge to substances normally present in animal tissues the cat is likely to ingest (at least in part) suggests that the 3 systems are concerned with different aspects of feeding and nutrition. Studies in the neurophysiology of oral chemoreception have been dominated by the so-called '4 basic tastes' derived from human psychophysical studies. Certain chemical substances are blandly assumed to elicit these 4 basic sensations. The substances used are normally NaCI, HCI, sucrose and quinine. Neurophysiological work has been dominated by these stimuli, even though any relationship between human psychophysics and first order neural discharge from another species must be highly speculative. We have investigated in some detail the response of geniculate ganglion units to NaCI, quinine, acids and sugars 1,s. We have found that there is little or no response to sugars from any neural group. The other 3 types of stimuli have been limited in their effectiveness in delimiting the different groups. The thresholds for NaCI are in general
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TABLE I SUMMARY OF THE RESPONSIVENESS OF GENICOLATE GANGLION TONGUE UNITS TO N a C I ,
ACIDS AND
QUININE
NaCI
Acids
Quinine
Group I
Variable, some units relatively insensitive
All units highly responsive
Variable, units either insensitive or excited
Group II
Most units highly responsive
Variable, many units excited
Most units inhibited
Group III
Variable,some units excited
Variable,some units excited
Variable,many units excited
above 0.1 M and although group II units are more responsive to NaC1 than other units, NaC1 will activate members of the other two groups also. Acids, both organic and inorganic (HCI), will excite members of all 3 groups although in general group I units are preferentially activated. Quinine normally inhibits group II neurons but will excite some members of group I and group III. The response of units from the 3 groups to NaC1, acid and quinine is summarized in Table I. The data summarized in this table will be used to compare our cat geniculate ganglion with published work on cat chorda tympani units. In this comparison, it must be realized that the experimental conditions differed markedly among investigators. Often, for instance, only the responses to a single concentration of a few chemicals were used in the different studies and the concentration of any chemical substance often differed from experimenter to experimenter.
Comparison with chorda tympani studies The peripheral processes of geniculate ganglion tongue neurons leave the ganglion with the facial nerve, depart from the facial nerve in the chorda tympani, and join the lingual nerve to innervate the tongue. There have been several neurophysiological studies on cat chorda tympani fibers. Unfortunately these studies were in most cases not quantitative and did not include many of the measures presented in this report. In almost all cases the measures taken were spike discharge to solutions of NaC1, quinine and HC1. As we have seen, these measures by themselves are often inadequate for distinguishing in the geniculate ganglion the unit groups presented here. The one attempt to determine whether chorda tympani fibers can be quantitatively grouped on the basis of their responses to NaCI, quinine and HC1 found a great deal of overlap among the response measures 10. These results were essentially in agreement with our conclusions 1: the responses of units to NaCl, quinine and acids are often inadequate criteria for distinguishing neural groups. Investigators who have extensively studied chorda tympani fibers in the cat have tended to parcel them out into groups, even though these groups were often only qualitatively described. The fiber groups distinguished by 3 different teams of investi-
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TABLE 1I COMPARISONOF CAT CHORDATYMPANIFIBERGROUPSWITHCAT GENICULATEGANGLIONGROUPS Investigators
Characteristics o f groups
Probably included in geniculate ganglion group
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1. Acid sensitive Water response labile 2. Acid and NaCI sensitive 3. Acid and quinine sensitive
1
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I
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2. Cohen et al. 4 A. 4 groups
3. Nagaki et al. x°
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I, I1, 11I I, Ill
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1. NaCI sensitive, no water response II, lIl, I 2. Water sensitive, not NaC1 1 sensitive 3. Quinine sensitive 111
gators are presented in T a b l e II. The fiber groups o f Pfaffmann 11 a n d C o h e n et al. 4 are the g r o u p s distinguished by these investigators. The groups of N a g a k i et al. ~° are our i n t e r p r e t a t i o n o f their data. I n every case we have indicated the p r o b a b l e c o r r e s p o n d ing unit g r o u p or groups in the geniculate ganglion based on o u r experience with the response o f the different groups to stimuli as indicated in Table 1. The first g r o u p i n g o f cat c h o r d a t y m p a n i fibers was a t t e m p t e d by Pfaffmann ~ , who g r o u p e d t h e m a l m o s t exclusively on the basis o f their responses to a few chemical stimuli. As can be seen, his g r o u p i n g w o u l d in m o s t cases include m o r e t h a n one g r o u p as d e t e r m i n e d by geniculate ganglion classification. O u r g r o u p I units could be classified in all 3 o f Pfaffmann's groups. The tentative groupings derived f r o m the d a t a o f N a g a k i et al. 1° include all 3 o f o u r geniculate ganglion groups. The closest c o r r e s p o n dence between c h o r d a t y m p a n i fiber groups a n d geniculate ganglion g r o u p s can be seen in the c o m p a r i s o n with the g r o u p s o f C o h e n et al. 4. Three o f their 4 groups corres p o n d to o u r 3 groups. Even the s p o n t a n e o u s activity rates o f the first two groups closely c o r r e s p o n d . Their f o u r t h g r o u p m a y consist o f g r o u p I units that are relatively insensitive to NaC1 a n d quinine or have lost sensitivity to these chemicals. The response o f units to distilled water is frequently used as a criterion for grouping. In o u r experience only g r o u p I units will discharge to distilled water. As o t h e r investigators 4,~1 have observed, however, this response is extremely labile. W e find the response o f g r o u p I units to deionized, glass-distilled water to be extremely weak a n d occasionally absent.
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I n no case has a c h o r d a t y m p a n i unit g r o u p been identified t h a t c a n n o t be e q u a t e d with at least one o f the 3 geniculate g a n g l i o n groups. The criteria established in o u r study for g r o u p i n g the m a j o r i t y o f g e n i c u l a t e ganglion c h e m o r e s p o n s i v e tongue units should also be a p p l i c a b l e for classifying c h o r d a t y m p a n i fibers in the cat. ACKNOWLEDGEMENTS This study was s u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t NB06547. W e t h a n k S a n d r a M c C a r t n e y , Jackie Truxell a n d J o s e p h Oravec for technical assistance, a n d M a r y P e t r u s k a a n d Lillian G w y n n for secretarial assistance.
REFERENCES 1 BOUDREAU,J. C., BRADLEY,B. E., BIERER,P. R., KRUGER,S., AND TSUCHITANI,C., Single unit recordings from the geniculate ganglion of the facial nerve of the cat, Exp. Brain Res., 13 (1971) 461-488. 2 BURGESS,P. R., AND CLARK, F. J., Characteristics of knee joint receptors in the cat, J. Physiol. (Lond.), 203 (1969) 317-335. 3 BURGESS,P. R., PETIT, D., AND WARREN,R. M., Receptor types in cat hairy skin supplied by myelinated fibers, J. NeurophysioL, 31 (1968) 833-848. 4 COHEN, M. J., HAGIWARA,S., AND ZOTTERMAN,Y., The response spectrum of taste fibers in the cat: A single fiber analysis, Actaphysiol. scand., 33 (1955) 316-332. 5 HUNT, C. C., Relation of function to diameter in afferent fibers of muscle nerves, J. gen. Physiol., 38 (1954) 117-131. 6 IGGO, A., Somesthetic sensory mechanisms. In M. J. SWENSON (Ed.), Dukes' Physiology of Domestic Animals, Cornell University Press, Ithaca, N.Y., 1970, pp. 947-990. 7 KIANG, N. Y.-S., PFEIFFER,R. R., WARR,W. B., AND BACKUS,A. S. N., Stimulus coding in the cochlear nucleus, Ann. Otol. (St. Louis), 74 (1965) 463-485. 8 KRUGER,S., ANDBOUDREAU,J. C., Responses of cat geniculate ganglion tongue units to some salts and physiological buffer solutions, Brain Research, 47 (1972) 127-145. 9 MATTHEWS,P. B. C., AND STEIN,R. B., The regularity of primary and secondary muscle spindle afferent discharges, J. Physiol. (Lond.), 202 (1969) 59-82. 10 NAGAKI,J., YAMASHITA,S., AND SATO, M., Neural response of cat to taste stimuli of varying temperatures, Jap. J. Physiol., 14 (1964) 67-89. 11 PFAFFMANN,C., Gustatory afferent impulses, J. cell. comp. Physiol., 17 (1941) 243-258. 12 SOLMS,J., Nonvolatile compounds and the flavor of foods. In G. OHLOFFAND A. F. THOMAS (Eds.), Gustation and Olfaction, an International Symposium, Academic Press, New York, 1971, pp. 92-110. 13 STACEY,M. J., Free nerve endings in skeletal muscle of the cat, J. Anat. (Lond.), 105 (1969) 231-254.
157
CLASSIFICATION OF CHEMORESPONSIVE T O N G U E UNITS OF T H E CAT G E N I C U L A T E G A N G L I O N
JAMES C. BOUDREAU* AND NECIP ALEV Leech Farm V. A. Hospital, Pittsburgh, Pa. 15206 and Department o f Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, Pa. 15213 (U.S.A.)
(Accepted November 10th, 1972)
SUMMARY Single unit recordings were taken from neurons in the geniculate ganglion of the anesthetized cat. Three groups of chemoresponsive tongue units were identified on the basis of spontaneous activity measures and response to chemical and electrical stimulation of the tongue. Electrical stimulation of the sites of excised fungiform papillae to which tongue units project elicited spikes with latencies of 7-9 msec from group I units, 10-13 msec from group II units and 14 msec or greater from group III units, with little overlap among the groups. The intact tongue was stimulated with different chemical substances. Group I units were preferentially discharged by certain substances such as citric and malic acid, group II units by cysteine and proline. Group III units were affected by fewer substances although some group III units were among those maximally sensitive to mononucleotide salts. Spontaneous activity measures were also utilized to classify the 3 groups, at least in part. The units in the 3 groups innervated fungiform papillae located in different but overlapping regions of the tongue.
INTRODUCTION The geniculate ganglion is a small cranial sensory ganglion located dorsal to the inner ear at the junction of the facial nerve, greater superficial petrosal nerve, and the nervus intermedius. Although the cat geniculate ganglion contains only around 1800 sensory nerve cells, these cells subserve a wide variety of sensory functions, In the first study 1 on single unit recordings from the ganglion, the units were divided into 3 major populations: ear cells that were discharged by moving hairs on the inner surface of the pinna; regular discharge units, most of which were affected by moving * Current address: University of Texas at Houston, Graduate School of Biomedical Sciences, Department of Neural Sciences, Houston, Texas 77005, U.S.A.
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tissues of the soft palate and nasopharynx; and tongue units. Chemoresponsive tongue units were found to comprise a heterogeneous collection of units, but the measures used proved inadequate for classification. In a later study, Kruger and Boudreau s, utilizing response measures to inorganic salt solutions and buffer solutions, identified two major chemoresponsive unit groups which comprised about 7 0 ~ of the chemoresponsive geniculate ganglion units. In this report we present different measures on single unit spike activity which demonstrate that over 90 ~ of chemoresponsive tongue units can be classified into 3 different unit types. The measures used to classify tongue units are spontaneous activity, response to electrical stimulation, and stimulus response measures utilizing a number of chemical stimuli. Similar measures have been found useful for subdividing complex neural populations in other sensory systems. Thus spontaneous activity measures have been found useful for classifying units in peripheral muscle 9 and skin 3 systems and in the cochlear nuclei 7. Latency measurements from peripheral nerve fibers have been used to classify units in muscle ~, joints 2 and skin systemsZ, 6. Stimulus-response measures have been used in all sensory systems to classify neurons and to determine the relationship between input-output variables. The importance of classification in establishing group membership cannot be underestimated. It has been demonstrated in sensory studies that neurons can be grouped into different functional categories. Without adequate criteria for classification, measures from neurons belonging to different groups may be placed together, usually resulting in an impairment in the quantitative stimulus-response relationships measured for the neurons. The measures presented in this report form a solid basis for the classification of tongue units. When group affiliation is known, many response characteristics of a neuron can be predicted and experiments can be performed upon a unit as a representative of a specific group. TECHNIQUES
The preparation Adult cats were anesthetized with an intraperitoneal injection of sodium pentobarbital (60 mg/kg) and, following a tracheotomy, secured in a stereotaxic headholder. The skin and muscles were cleared from the occipital portion of the skull, a portion of the cranium was removed and a partial cerebectomy of the neural matter overhanging the superior aspect of the petrous portion of the temporal bone was performed. The petrous bone overlying the ganglion was planed away with a carbide dental burr until the geniculate ganglion (and portions of the facial and greater superficial petrosal nerves) was exposed. Care was taken to preserve the blood supply to the ganglion. To penetrate the ganglion, either the sheath was nicked with a microprobe or (in most cases) the electrode was inserted through the sheath. The cat's temperature was maintained between 35 and 36 °C. The mean room temperature during recording was 23.2 °C. Relative humidity was kept between 40 ~o and 50 ~o (mean 45.1 ~o) to minimize temperature changes on the surface of the tongue produced by evaporation. Tungsten microelectrodes were advanced into the ganglion with a hydraulic
CAT TONGUE UNITS
159
mierodrive. The spike activity of a single unit was isolated and fed into an electronic counter or a CAT computer for processing and recorded on analog magnetic tape.
Stimulation of the tongue Papillae were electrically stimulated with a small stainless steel p r o b e (anode) with a large cathode clamped to the opposite side of the tongiae. A constant current stimulator was used to deliver 0.1 msec pulses at a rate of 10/sec. Spike latencies were measured from the oscilloscope face and are accurate to within 5 %. In the text there is the occasional remark that the tongue has not been stimulated. This remark means that there has been no prior electrical or chemical stimulation of the tongue. All chemical solutions were mixed in 50 m M concentration in deionized, glass-distilled water and then frozen. Prior to the experiment the chemicals were thawed and brought to room temperature. Chemical substances were applied to the tongue with glass pipettes on to the area known to be innervated by the unit under study. Such an application flooded a large area of the tongue. The change in discharge for a 10 sec period following stimulation was c o m p a r e d t o the spontaneous activity level in one or more 10 sec periods prior to stimulation. Since many chemoreceptive tongue units exhibited high levels of spontaneous activity, the response to chemical stimulation was expressed as the number of spikes during 10 sec o f stimulation minus the spontaneous activity level. Following the stimulus period, the tongue was rinsed with deionized, glass-distilled water. RESULTS
Tongue units can be subdivided into 4 classes. One class of neurons is primarily mechanically sensitive. These mechanically sensitive units display in general little or no spontaneous activity, exhibit latencies to electrical stimulation between 2 and 5 msec, and are maximally sensitive to moving mechanical stimuli. We have not studied mechanically sensitive cells in any detail and will give little further consideration to them other than to acknowledge their existence. The chemosensitive units have been divided into 3 classes of units, labeled group I, group II and group III. Uni~ groups I and II are identical to unit groups I and I1 distinguished by Kruger and Boudreau s primarily on the basis of their response to inorganic salts and physiological buffer solutions. Group III units consist primarily of those units presented in the earlier study as ungrouped. The measures for classifying units presented in this report have not been published before. Each neuron was classified according to a cluster of single unit spike measures. The most useful measures for classification proved to be spontaneous activity measures, response to electrical stimulation and response to chemical stimulation. In addition to displaying differences on these measures, the 3 classes of neurons were found to innervate fungiform papillae located in part on different regions of the tongue.
(I) Spontaneous activity measures All chemosensitive geniculate ganglion tongue neurons display some measurable
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level of spontaneous activity, where spontaneous activity is defined as spike discharge in the absence of experimenter controlled stimulation. The measures taken of tongue unit spontaneous activity were rate measures (counts of spikes/10 sec periods) and interspike interval histograms. These measures were taken under 3 conditions: (1) tongue-in, unstimulated condition; (2) tongue-out, unstimulated condition; (3) tongue-out, stimulated condition. Only rate measures were taken for the third condition. In the tongue-in condition the tongue was in the mouth and at body temperature, whereas the temperature of the tongue tip in the tongue-out condition was close to room temperature. In the unstimulated tongue-in condition, tongue units emit spikes continuously at rates between 5 and 195 spikes/10 sec. Successive 10 sec counts of spontaneous discharge rates from a tongue unit indicate that rate tends to be relatively constant for each cell. The standard deviation tends to increase with the mean rate up to a rate value of about 40 spikes/10 sec, beyond which little increase is seen (Fig. 1). Spontaneous activity rates above about 50 spikes/10 sec are associated with fairly constant standard deviations. Units with rates above 50 spikes/10 sec have proven in our studies almost invariably to be group II neurons (Fig. 1). Rate measures from unstimulated units do not clearly distinguish group I from group III neurons. Interspike interval (ISI) histograms were also measured for units in the tonguein, unstimulated condition. Even when measured from unstimulated units the ISI histograms of tongue units tended to be of bewildering diversity. Examples of ISI histograms from the 3 different groups of tongue units are presented in Fig. 2. The
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modal value of ISI histograms is usually in the extreme short interval end (between 3 and 10 msec). This short interval mode is a result of the presence of bursts o f spikes; the spikes in the burst being separated by relatively constant short intervals. The ISI histograms o f group I units were almost always multipeaked. Group II units often
162
J. C. BOUDREAU AND N. ALEV
exhibited single peaked ISI histograms with the distribution declining slowly from the short interval mode. Group III units exhibited two different types of ISI histograms: one multipeaked and similar to those seen with group I units and the other single peaked and consisting almost entirely of short intervals. This second type of histogram (Fig. 2, cell GG89C1B) results when the spontaneous activity consists almost entirely of multiple spike bursts. All of the ISI histograms in Fig. 2 are asymmetrical with a tail extending toward the long intervals (positively skewed). One measure of the degree of asymmetry of a distribution is to determine the relationship between the mean and median. When the mean and median of the ISI distributions are plotted with respect to one another, it is found that the two values tend to be in a relatively constant relationship to one another (Fig. 3). Regardless of the rate of discharge of the units, the mean interval is always greater than the median interval (with the majority of median to mean ratios between 0.4 and 0.8). Group II units, with lower median and mean interval values, tend to cluster separately on this graph. The ISI histograms of group III units are either skewed in a manner similar to group I ISI histograms or are more markedly skewed with median/mean values below 0.2. It is possible to change the spontaneous activity of a unit simply by changing the condition of the tongue. In our studies this changed condition was accomplished by 40-
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opening the m o u t h and protruding the tongue. Such a procedure usually resulted in decreased spontaneous activity (Fig. 4). The mean percentage decreases in rates for the 3 groups are 29 ~o for g r o u p I, 37 ~ for g r o u p 1I and 68 ~ for g r o u p III. Because o f this
164
J. C. BOUDREAU AND N. ALEV
greater decrease in rate under the new condition, spontaneous activity measures from group III units tended now to separate out from rate measures from the other two groups (Fig. 4). Besides exhibiting a greater proportional decrease in rate with the tongue-out condition, group III units also often showed a major increase in the skewness of their ISI histograms. This change is illustrated by a plot of the median/mean ratio calculated for the two conditions (Fig. 4). The ISI histograms of group I and group II units were less affected by the changing conditions.
(2) Latency measures Latencies of discharge to electrical stimulation of the tongue were measured for all chemosensitive tongue units whenever possible. These latencies were measured after chemical stimulation of the tongue. Latencies were measured by electrically stimulating the intact fungiform papillae which a unit innervated (papillae-on condition) and electrical stimulation of these sites after the fungiform papillae had been excised (papillae-off condition). Excision of fungiform papillae was performed because of the great variability in latency measures encountered when electrically stimulating the intact papiUae 1. This variability was believed to be due to the existence of multiple electrically sensitive trigger zones, apparently in the end organ. It was thought that by excising the fungiform papillae the nerve fibers innervating the receptors could be electrically stimulated directly and the variability of latency measures would be reduced. The expected reduction in variability occurred. Latency measurements obtained under the papillae-off condition proved to be less variable than those obtained by stimulating the intact papillae. This reduction in variability is especially noticeable in the threshold latency measurements taken under the two conditions (Fig. 5, top). Threshold latencies measured under the papillae-on condition tended to vary widely for all 3 unit groups. Papillae removal was associated with a sharp reduction in the variability of threshold latency measurements for all 3 unit groups (Fig 5). Latencies were also measured for each unit at suprathreshold intensities (to 3.5 mA). Increasing the intensity of electrical stimulation above threshold often results in large decreases in latencies measured from intact papillae systems1. Only small shifts in latency occur in the papillae-off condition when the intensity of electrical stimulation is raised above threshold. The relationship between suprathreshold latency measurements measured before and after papillae removal are also shown in Fig 5. The latency measures taken after papilla removal proved to be stable and reliable measures for the classification of units. After papillae excision the latencies measured from different papillae innervated by the same tongue unit tended in almost all cases to be quite similar to one another and in practice it proved possible to represent a cell by a single latency measurement (the shortest obtained). Latency measurements for the 3 groups of chemoresponsive tongue units fell into 3 demarcated classes with little overlap. The latencies of group I units ranged from 7 to 9 msec, group II latencies from 10 to 13 msec, and group III latencies were 14 msec or greater. These latency categories proved to be quite reliable and were utilized as one of the chief measures for unit classification. Only two group II units did not exhibit shortest latencies between
165
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10 and 13 msec, and one of these was 9.5 msec and the other 14 msec, near the lower and upper boundaries of the group II latency range respectively. The validity of the latency measurements for classification is indicated by the relationship between latency and other response measures from tongue units. Group 35"
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Fig. 6. Relationship of latency measures and spontaneous activity measures for 51 geniculate ganglion tongue units. Each unit is represented by a single latency value: the shortest latency found in papillaoff condition. Spontaneous activity rates measured during beginning of chemical stimulation (tongueout, stimulated condition).
II units were those units with the highest level of spontaneous activity and when spontaneous activity rates (tongue-out, stimulated condition) for tongue units are plotted against latency measures for the same units, the group II units are clearly interpolated between the other two low spontaneous activity groups (Fig. 6). Spontaneous activity rates measured under the tongue-out unstimulated condition were similar to tongue-out stimulated rates. It is apparent from Fig. 6 that much of the variability found in the measurements on spontaneous activity was due to the existence of the 3 separate unit groups.
(3) Chemical stimulation In previous work 1,8 on cat geniculate ganglion tongue units involving chemical stimulation with various inorganic salts, it had been found that either the substances did not stimulate units in any reasonable concentration or did not stimulate in a manner that clearly differentiated unit groups. Sodium chloride thresholds for instance were 0.1 M or greater and NaC1 could activate at least some members of all 3 of the unit groups described in this report. Early work had also shown that most cat tongue units could be discharged by weak solutions of various animal components such as chicken breast, cod fillet, pork liver, etc. In an effort to extend our limited list of stimulating substances, to investigate the chemical substrate for the response to foods, and to
CAT 'TONGUE UNITS
167
determine if there existed certain chemical substances which more clearly differentiated between units, a great many chemical substances were tested for their stimulatory effectiveness. An initial group of chemical substances was chosen largely from a list by Solms 12 of the nonvolatile compounds important in human perception of food flavors. This group of substances included amino acids, nucleotides, organic acids and various other compounds. The work on the biochemical substrate of the food response is still going on. In this report we wish to present only chemical data relevant to the classification of chemosensitive tongue units. From 17 to 125 different substances were tested on 31 neurons. From these initial substances, 16 different test chemicals were chosen and were tested on 14 additional neurons. These test chemicals were found to clearly distinguish members of the 3 unit groups which could be identified by spontaneous activity or latency measures. The 'response profiles' of 4 units to the test chemicals are illustrated in Fig, 7. The data from one group I and one group II neuron are shown in this figure. Members of either group I or group II were similar to one another in chemical responsiveness although, as can be seen, the two groups varied markedly from each other. Group III units proved less homogeneous on the chemical measures than the other two groups. Two examples of group III unit chemical responsiveness 'are also presented in Fig. 7. The response profiles shown in this figure proved invaluable in the classification of units. The responses of 42 units to 10 of the test stimuli are presented in Fig. 8. In this figure each neuron is arranged with respect to the shortest latency measured for that cell following papilla removal. The chemical selectivity of each of the 3 unit groups in terms of spikes discharged is clearly indicated in this figure. Short latency units (group I) are most responsive to malic acid, citric acid, and O-phosphorylethanolamine, medium latency units (group II) are among those maximally discharged by the amino acids L-proline and L-cysteine, and only long latency units (group III) discharged appreciably to the nucleotide inosine 5'-monophosphate. Two of the chemicals tested, inosine-5'-triphosphate, and inosine-5'-diphosphate (not shown) stimulated members from all 3 groups whereas some other chemicals stimulated members from two of the 3 unit groups. Thus tetrasodium pyrophosphate stimulated group tI and group III units, and butyryl choline chloride stimulated units in groups I and III. It was possible to utilize the results from the chemical stimulation studies to characterize the 3 groups in terms of their chemical responsiveness. Unit groups I and II proved to be more homogeneous in their responsiveness to the test stimuli than group III units. All of the group I units were activated by malic acid, O-phosphoryletha;nolamine, citric acid and butyryl choline. Group I units were also the only units activated by creatinine. Group II units were air discharged by L-proline and L-cysteine and were usually inhibited by L'trypt0Phan and L-isoleucine. Group III units on the other hand seemed to be divided into two subgroups. One subgroup was activated by the nucleotides ITP, IDP and IMP. The other subgroup was in general insensitive to most of the stimuli used and discharged only to phytic acid and butyryl choline chloride. In practice the responses to the test chemicals presented proved adequate for
t 68
J. C. BOUDREAU AND N. ALEV
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classification of almost all tongue units into 3 categories. Classification was done both in terms of the chemicals to which the neurons were sensitive and the chemicals to which the neurons were insensitive. The responsiveness to the chemicals that maximally discharged neurons from the 3 groups also proved to be the responsiveness most resistant to change.
(4) Location When studying the latency of discharge to electrical stimulation, the receptive field of each unit was also recorded by plotting the location of each unit papilla
170
Group I
J. C. BOUDREAU AND N. ALEV
Group II
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Fig. 9. The approximate locations of the papilla systems of the different unit groups identified in the geniculate ganglion. Each unit is represented by one point - - the locus of the papilla site from which the shortest latency to electrical stimulation was measured. No. of units: 37, group I; 39, group II; 26, group III.
system on a standardized schematic diagram of the tongue. It was found that each of the unit groups projected preferentially to different regions of the tongue although there was much overlap. Group I units innervated the posterior center and side of the tongue; group H units innervated the side and tip of the tongue; and group III units innervated fungiform papillae on the sides of the tongue. This differential innervation of the tongue by the different unit groups is illustrated in Fig. 9. These graphs include the units classified according to the criteria discussed in this report and units classifiable on spontaneous activity measures, latency measurements without papilla removal, and the response to NaC1, citric acid, quinine and often other chemical stimuli. The basis for classifying units on these chemical measures is presented in the discussion of this report. The latency measurements for the 3 unit groups were found to be independent of the cat studied and the locations of the papilla systems innervated. Latency measurements did not vary as a function of distance from the tip of the tongue, rather latency was correlated with group membership (Fig. 10).
(5) Unclass~ed neurons Of the 42 neurons for which spontaneous activity, latency and adequate chemical measures were available, only 3 units could not be readily classified into the 3 unit groups described. These 3 units did not straddle the 3 established categories in such a way as-to render ~ classification invalid; rather they seemed to represent somewhat anomalous units. All 3 of the unclassified units were similar to one another. Two of the latency measurements from those 3 cells were in the group II range, both being 12.5 msec. The latency measurement for the third cell was 14.0 msec. Their unstimulated, tongue-in, spontaneous activity rates were low (6.8, 8.6 and 18.3) and their ISI histograms were of the multimodal group I variety. They were in general not highly responsive to the test chemicals, and what response existed did not clearly indicate a classification.
CAT TONGUE UNITS
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DISCUSSION
The measures presented in this report definitely establish the fact that the chemoresponsive geniculate ganglion units constitute at least 3 separate sensory sub-systems. These 3 sub-systems are composed of neurons that innervate fungiform papillae on different parts of the tongue. The latency measures indicate that the peripheral fibers of the 3 systems are also probably of different fiber diameters. Spontaneous activity measures suggest the possibility that different neural pulse codes are utilized by the 3 separate systems. It is extremely unlikely that there are fewer than 3 unit groups, although the possibility exists that there may be more than 3 groups. Our present measures are inadequate for further subdivisions. Each of the 3 neural groups delimited is not homogeneous however; rather, there are often differences in spontaneous activity and chemical responsiveness among the neurons grouped together. To what extent these differences may represent functional subdivisions within the group is yet to be determined. The measures used to demarcate these 3 separate peripheral neur~tl chemoreceptor groups are at least as valid as measures that have been utilized by other workers in analyzing other sensory systems. In other sensory systems when neurons are functionally grouped with measures similar to those reported here, the peripheral
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sensory systems have often proven to have certain structural correlates. In most cases the different unit groups have been shown to have anatomically distinct peripheral connections, usually different receptor types. Thus muscle afferents may be broken down into fiber groups innervating Golgi tendon organs, primary spindle afferents, secondary spindle afferents and others 13. The fibers present in a nerve innervating the skin innervate anatomically distinct structures such as hair follicles, Vater-Pacini corpuscles, tactile pads, etc. 6. In most cases neural recordings from fibers innervating distinct structures can be distinguished functionally.
The measures utilized (1) Spontaneous activity. It is often possible to classify a geniculate ganglion unit simply on the basis of its spontaneous activity. Thus most group II units and some group III units could be clearly identified on the basis of rate and ISI measures. In other cases spontaneous activity measures for neurons from different groups did not clearly indicate group membership. Spontaneous activity patterns of geniculate ganglion tongue units are extremely complex and it is possible that more precise measures of these spike trains would more clearly differentiate units. (2) Latency measures. The latency measures proved to be among the most reliable measures for grouping cells. The fact that the threshold latencies measured for stimulating the intact papillae system often differ markedly from those measured by stimulating the site of the excised papilla indicates that there exists more than one electrically excitable component in the peripheral system. Since latencies almost invariably shorten when the fungiform papillae are removed, it is likely that the end organs for the neural units are in most cases located in the fungiform papillae. Latency measures for members of each neural group do not vary as a function of position along the tongue, suggesting that greater distances are compensated for by larger diameter fibers. This adjustment for distance insures that within a group information from different parts of the tongue arrives centrally at the same time. (3) Chemical measures. The results from the studies on chemical responsiveness of geniculate ganglion tongue units suggest that each neural group has a different chemical stimulus dimension. That members of all 3 groups discharge to substances normally present in animal tissues the cat is likely to ingest (at least in part) suggests that the 3 systems are concerned with different aspects of feeding and nutrition. Studies in the neurophysiology of oral chemoreception have been dominated by the so-called '4 basic tastes' derived from human psychophysical studies. Certain chemical substances are blandly assumed to elicit these 4 basic sensations. The substances used are normally NaCI, HCI, sucrose and quinine. Neurophysiological work has been dominated by these stimuli, even though any relationship between human psychophysics and first order neural discharge from another species must be highly speculative. We have investigated in some detail the response of geniculate ganglion units to NaCI, quinine, acids and sugars 1,s. We have found that there is little or no response to sugars from any neural group. The other 3 types of stimuli have been limited in their effectiveness in delimiting the different groups. The thresholds for NaCI are in general
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TABLE I SUMMARY OF THE RESPONSIVENESS OF GENICOLATE GANGLION TONGUE UNITS TO N a C I ,
ACIDS AND
QUININE
NaCI
Acids
Quinine
Group I
Variable, some units relatively insensitive
All units highly responsive
Variable, units either insensitive or excited
Group II
Most units highly responsive
Variable, many units excited
Most units inhibited
Group III
Variable,some units excited
Variable,some units excited
Variable,many units excited
above 0.1 M and although group II units are more responsive to NaC1 than other units, NaC1 will activate members of the other two groups also. Acids, both organic and inorganic (HCI), will excite members of all 3 groups although in general group I units are preferentially activated. Quinine normally inhibits group II neurons but will excite some members of group I and group III. The response of units from the 3 groups to NaC1, acid and quinine is summarized in Table I. The data summarized in this table will be used to compare our cat geniculate ganglion with published work on cat chorda tympani units. In this comparison, it must be realized that the experimental conditions differed markedly among investigators. Often, for instance, only the responses to a single concentration of a few chemicals were used in the different studies and the concentration of any chemical substance often differed from experimenter to experimenter.
Comparison with chorda tympani studies The peripheral processes of geniculate ganglion tongue neurons leave the ganglion with the facial nerve, depart from the facial nerve in the chorda tympani, and join the lingual nerve to innervate the tongue. There have been several neurophysiological studies on cat chorda tympani fibers. Unfortunately these studies were in most cases not quantitative and did not include many of the measures presented in this report. In almost all cases the measures taken were spike discharge to solutions of NaC1, quinine and HC1. As we have seen, these measures by themselves are often inadequate for distinguishing in the geniculate ganglion the unit groups presented here. The one attempt to determine whether chorda tympani fibers can be quantitatively grouped on the basis of their responses to NaCI, quinine and HC1 found a great deal of overlap among the response measures 10. These results were essentially in agreement with our conclusions 1: the responses of units to NaCl, quinine and acids are often inadequate criteria for distinguishing neural groups. Investigators who have extensively studied chorda tympani fibers in the cat have tended to parcel them out into groups, even though these groups were often only qualitatively described. The fiber groups distinguished by 3 different teams of investi-
174
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TABLE 1I COMPARISONOF CAT CHORDATYMPANIFIBERGROUPSWITHCAT GENICULATEGANGLIONGROUPS Investigators
Characteristics o f groups
Probably included in geniculate ganglion group
1. Pfaffmann 11
1. Acid sensitive Water response labile 2. Acid and NaCI sensitive 3. Acid and quinine sensitive
1
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2. Cohen et al. 4 A. 4 groups
3. Nagaki et al. x°
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I, I1, 11I I, Ill
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1. NaCI sensitive, no water response II, lIl, I 2. Water sensitive, not NaC1 1 sensitive 3. Quinine sensitive 111
gators are presented in T a b l e II. The fiber groups o f Pfaffmann 11 a n d C o h e n et al. 4 are the g r o u p s distinguished by these investigators. The groups of N a g a k i et al. ~° are our i n t e r p r e t a t i o n o f their data. I n every case we have indicated the p r o b a b l e c o r r e s p o n d ing unit g r o u p or groups in the geniculate ganglion based on o u r experience with the response o f the different groups to stimuli as indicated in Table 1. The first g r o u p i n g o f cat c h o r d a t y m p a n i fibers was a t t e m p t e d by Pfaffmann ~ , who g r o u p e d t h e m a l m o s t exclusively on the basis o f their responses to a few chemical stimuli. As can be seen, his g r o u p i n g w o u l d in m o s t cases include m o r e t h a n one g r o u p as d e t e r m i n e d by geniculate ganglion classification. O u r g r o u p I units could be classified in all 3 o f Pfaffmann's groups. The tentative groupings derived f r o m the d a t a o f N a g a k i et al. 1° include all 3 o f o u r geniculate ganglion groups. The closest c o r r e s p o n dence between c h o r d a t y m p a n i fiber groups a n d geniculate ganglion g r o u p s can be seen in the c o m p a r i s o n with the g r o u p s o f C o h e n et al. 4. Three o f their 4 groups corres p o n d to o u r 3 groups. Even the s p o n t a n e o u s activity rates o f the first two groups closely c o r r e s p o n d . Their f o u r t h g r o u p m a y consist o f g r o u p I units that are relatively insensitive to NaC1 a n d quinine or have lost sensitivity to these chemicals. The response o f units to distilled water is frequently used as a criterion for grouping. In o u r experience only g r o u p I units will discharge to distilled water. As o t h e r investigators 4,~1 have observed, however, this response is extremely labile. W e find the response o f g r o u p I units to deionized, glass-distilled water to be extremely weak a n d occasionally absent.
CAT TONGUE UNITS
175
I n no case has a c h o r d a t y m p a n i unit g r o u p been identified t h a t c a n n o t be e q u a t e d with at least one o f the 3 geniculate g a n g l i o n groups. The criteria established in o u r study for g r o u p i n g the m a j o r i t y o f g e n i c u l a t e ganglion c h e m o r e s p o n s i v e tongue units should also be a p p l i c a b l e for classifying c h o r d a t y m p a n i fibers in the cat. ACKNOWLEDGEMENTS This study was s u p p o r t e d in p a r t by U.S. Public H e a l t h Service G r a n t NB06547. W e t h a n k S a n d r a M c C a r t n e y , Jackie Truxell a n d J o s e p h Oravec for technical assistance, a n d M a r y P e t r u s k a a n d Lillian G w y n n for secretarial assistance.
REFERENCES 1 BOUDREAU,J. C., BRADLEY,B. E., BIERER,P. R., KRUGER,S., AND TSUCHITANI,C., Single unit recordings from the geniculate ganglion of the facial nerve of the cat, Exp. Brain Res., 13 (1971) 461-488. 2 BURGESS,P. R., AND CLARK, F. J., Characteristics of knee joint receptors in the cat, J. Physiol. (Lond.), 203 (1969) 317-335. 3 BURGESS,P. R., PETIT, D., AND WARREN,R. M., Receptor types in cat hairy skin supplied by myelinated fibers, J. NeurophysioL, 31 (1968) 833-848. 4 COHEN, M. J., HAGIWARA,S., AND ZOTTERMAN,Y., The response spectrum of taste fibers in the cat: A single fiber analysis, Actaphysiol. scand., 33 (1955) 316-332. 5 HUNT, C. C., Relation of function to diameter in afferent fibers of muscle nerves, J. gen. Physiol., 38 (1954) 117-131. 6 IGGO, A., Somesthetic sensory mechanisms. In M. J. SWENSON (Ed.), Dukes' Physiology of Domestic Animals, Cornell University Press, Ithaca, N.Y., 1970, pp. 947-990. 7 KIANG, N. Y.-S., PFEIFFER,R. R., WARR,W. B., AND BACKUS,A. S. N., Stimulus coding in the cochlear nucleus, Ann. Otol. (St. Louis), 74 (1965) 463-485. 8 KRUGER,S., ANDBOUDREAU,J. C., Responses of cat geniculate ganglion tongue units to some salts and physiological buffer solutions, Brain Research, 47 (1972) 127-145. 9 MATTHEWS,P. B. C., AND STEIN,R. B., The regularity of primary and secondary muscle spindle afferent discharges, J. Physiol. (Lond.), 202 (1969) 59-82. 10 NAGAKI,J., YAMASHITA,S., AND SATO, M., Neural response of cat to taste stimuli of varying temperatures, Jap. J. Physiol., 14 (1964) 67-89. 11 PFAFFMANN,C., Gustatory afferent impulses, J. cell. comp. Physiol., 17 (1941) 243-258. 12 SOLMS,J., Nonvolatile compounds and the flavor of foods. In G. OHLOFFAND A. F. THOMAS (Eds.), Gustation and Olfaction, an International Symposium, Academic Press, New York, 1971, pp. 92-110. 13 STACEY,M. J., Free nerve endings in skeletal muscle of the cat, J. Anat. (Lond.), 105 (1969) 231-254.