Thermal sensitivity of neurons in a rostral part of the rat solitary tract nucleus

Brain Research, 454 (1988) 321-331

321

Elsevier BRE 13736

Thermal sensitivity of neurons in a rostral part of the rat solitary tract nucleus Hisashi Ogawa, Tomio Hayama and Yoshiro Yamashita Department of Physiology, Kumamoto University Medical School, Kumamoto (Japan) (Accepted 28 January 1988)

Key words: Taste; Thermal sensitivity; Mechanoreceptive neuron; Cold neuron; Solitary tract nucleus; Rat; Receptive field

While stimulating the entire oral cavity of anesthetized rats, we recorded 3 types of neurons in the solitary tract nucleus; taste, mechanoreceptive and cold neurons. Most of the taste neurons were sensitive to thermal as well as to mechanical stimulations. Taste neurons predominantly sensitive to sucrose responded to warming and those most excited by NaC1 or HCI were sensitive to cooling, and significant correlations were found between sucrose and warming and between NaCI and cooling. Most of the cold-sensitive taste neurons had receptive fields (RFs) at the anterior tongue and warm-sensitive taste neurons had whole or part of the RFs at the nasoincisor duct. About half the number of mechanoreceptive neurons were sensitive to cooling, producing phasic responses. RFs of some thermosensitive mechanoreceptive neurons and cold neurons were located. Warm-sensitive mechanoreceptive neurons or warm neurons were not evident. Therefore, interaction between thermal and taste sensations in the oral cavity probably takes place in the solitary tract nucleus, as well as in the chorda tympani.

INTRODUCTION It has been reported that subjective sensation of taste stimuli presented on the h u m a n tongue is influenced by the presence of other taste or thermal stimuli on other areas of the tongue 2. As a possible mechanism underlying this interaction between the two modalities, i.e. taste and thermal modalities, the chorda tympani fibers were found to respond to a specific combination of a certain taste quality and cold/warm stimuli in animals 15,16. O n the other hand, other reports 9'12'14 showed a convergence of multimodal inputs in the solitary tract nucleus (NTS), the first taste relay nucleus: Makous et al. 9 reported that cold and taste afferents converge onto single NTS neurons, while Ogawa et al. 12'14found that mechanoreceptive and taste afferents converge on single NTS neurons. In addition, palatal and lingual afferents are known to converge on single NTS n e u r o n s is. Therefore, it is uncertain whether like the chorda tympani fibers, the taste n e u r o n s in the NTS respond to the

same combinations of taste and thermal stimuli. The aim of the present study was to examine to what combination of taste and thermal stimuli neurons in the NTS of rats would respond and to assess the encoding mechanism of taste and thermal information in this nucleus. The responsiveness of NTS neurons to mechanical stimuli and location of their receptive fields (RFs) were also examined. A preliminary report has been published in abstract form 13. MATERIALS AND METHODS

Surgery Thirty-four female albino rats of the S p r a g u e Dawley strain, weighing 240-320 g, were used. The experimental procedures were the same as described 4'14, except for the thermal stimulation of the oral cavity. The animals were anesthetized with amobarbital sodium (80 mg/kg b. wt., i.p.), and anesthesia was m a i n t a i n e d by subsequent i.v. administrations of the drug. The trachea and femoral vein of the

Correspondence: H. Ogawa, Department of Physiology, Kumamoto University Medical School, Honjo 2-2-1, Kumamoto 860, Japan. 0006-8993/88/$03.50© 1988 Elsevier Science Publishers B.V. (Biomedical Division)

322 animal were cannulated, and the animal's head was mounted on a stereotaxic head holder. The posterior portion of the skull was removed, and part of the cerebellum was aspirated to expose the dorsal surface of the medulla and pons. During the experiment, the animal's head was tilted by about 45 ° with the recording side up. The left cheek from the mouth corner to the anterior edge of the mandibular ramus was resected to get a free access to the posterior oral cavity. The tongue was slightly stretched rostroventrally. Rectal temperature was kept at about 37 °C with a water heater. During the recording of neural discharges, the rats were immobilized with d-tubocurarine, and artificially ventilated. The end-tidal CO2 concentration was measured by an 'Expired Gas Monitor' (San-Ei 1H31, Tokyo) and kept between 3.5 and 4.5% by adjustments of the respiratory volume and/or rate. The ECG was monitored throughout the experiment. Glass microelectrodes, filled with 2% Pontamine sky blue in 0.5 M sodium acetate (resistance 10 MQ), were used for recording. An indifferent Ag-AgC1 electrode was placed on the cut muscle surrounding the exposure. Impulse discharges were recorded from the soma of single neurons through conventional physiological equipment, consisting of a pre-amplitier, cathode ray oscilloscope and kymographic camera.

Stimulation Taste stimuli used were 0.1 M NaC1, 0.5 M sucrose, 0.01 N HC1 and 0.02 M quinine-HC1 and were the same as used in our experiments on the chorda tympani fiber responses 15. Taste solutions were delivered to the entire oral cavity through a nozzle containing 5 channels, one for each of the 4 basic taste solutions and rinse water, from a system of overhead funnels via gravity flow at a rate of 3 ml/s. A stream of water or solutions was run to the hard palate, then allowed to flow over an orifice of the nasoincisor duct (ND), posterior oral cavity and anterior tongue. The anterior tongue was the last place where the solutions flowed. After rinsing the oral cavity with distilled water for 15 s, each taste solution was administered into the oral cavity for 10 s followed by another rinse with distilled water for at least 15 s. Both the taste stimuli and rinsing water were administered at room temperature (25-28 °C). Responsiveness of the neurons to

meChanical stimulation was examined by stroking the tongue, palate and perioral structure with a glass rod for light stimulation or by pinching these tissues with a non-serrated forceps for intense, but not noxious, stimulation. Responsiveness of the neurons to thermal stimulation was tested by applying water of 40, 20 and 40 °C successively to the oral cavity. A large thermal stimulation by 20 °C shift in the physiological temperature range (20-40 °C) was used to detect even the slightest thermal sensitivity which NTS neurons might have, since a rapid thermal change is useful to evoke thermal sensation 5 and such a large thermal change has been used in physiological studies 8'15. Water at each temperature was stored in a tank 1.5 m above the animal and allowed to flow into the oral cavity by gravity at a rate of 3 ml/s for 1 1/2 min. Such a long preadaptation time was required until the temperature of the tongue or other tissues of interest reached nearly a steady level 19. A thermister (glass coated, time constant 2 s) was usually placed on the anterior tongue to measure the temperature change. If the receptive field (RF) of the neurons under study had already been determined for thermal stimuli, the thermister was placed on the RF.

Location of receptive fields After determination of the best taste stimulus, a stimulus which produced the maximum response among the 4 basic taste stimuli, the RF for taste was located with a small wisp of cotton soaked in the best stimulus. Sometimes non-best stimuli were also used to locate the RF. The RF for mechanical Stimulation was located with a glass rod and a pair of non-serrated forceps. The RF for thermal stimulation was located with a small thermal stimulator made of brass 6.

Identification of responses A pair of monopolar electrodes were ipsilaterally placed in the parabrachial nucleus and cathodal pulses (max. 300/zA; 0.1 ms) were applied to activate the NTS neurons, antidromically. Antidromic activation of certain neurons was determined according to the following criteria; an invariant latency of spike discharges after the stimulus, capability of the neuron to follow repetitive stimulation of more than 200 Hz, and collision of the antidromic spikes with their orthodromic ones evoked peripherally. Criterion for

323 collision was met when the shortest interval between peripherally evoked spikes and centrally evoked ones equalled twice the latency of the centrally driven spike plus its refractory period 3. Details of the pontine stimulation have been reported 14. For each neuron, a given taste stimulus was tested several times. Taste responses were identified when the following criterion was met; during the first 10 s after stimulus application, changes in discharge rates at least 1.0 s long and 2 S.D. above or below the prestimulus average of discharge rates per 1.0 s during a 10-s water rinsing period. The magnitude of responses was defined as the number of impulses in the first 5 s after the onset of stimulation by subtracting the number of impulses of the spontaneous discharges. Thermal responses were identified when, to cooling and/or warming, neurons produced responses greater by 5 spikes in 5 s than those to water of room temperature applied before taste stimulation. Mechanical responses were recognized when the same magnitude of responses was evoked at the application of preadaptation water, e.g. rinsing water before taste stimulation or 40 °C before cold stimulation. All of the mechanosensitive neurons were then tested for responsiveness for stimulation with a glass rod and non-serrated forceps.

Histology All recording sites were marked by the electrophoretic deposition of dye from the recording electrodes. The stimulation sites in the parabrachial region were marked by passing a cathodal current through the stimulating electrodes. At termination of the experiment, the loci of dye spots and electrolytic lesions were established histologically. RESULTS Seventy neurons were isolated from the rostral part of the NTS. Thirty-seven of these were classified as taste neurons because of their sensitivity to taste stimuli. Twenty-three were classified as mechanoreceptive neurons because they lacked taste sensitivity but showed mechanical sensitivity. The remaining 10 were assumed to be thermoreceptive neurons, specifically cold neurons, because they were exclusively sensitive to cooling and showed tonic as well as phasic

responses to a sudden fall in temperature of the oral cavity.

Thermal sensitivity of taste neurons As pointed out previously 14, most of the taste neurons in the NTS (16 of the 28 neurons examined) were sensitive to mechanical stimulation. A much larger proportion of the taste neurons (25 of the 33 neurons examined) showed thermal sensitivity. About half the number of taste neurons examined (13 out of 28) showed both thermal and mechanical sensitivity. Three types of thermal responses were found in the taste neurons. Of the 25 neurons to which both cooling and warming were applied, 12 taste neurons responded phasically to cooling the oral tissue by 20 °C (Fig. 1A), while 5 neurons phasically responded to warming the tissue by 20 °C (Fig. 1B). The remaining 8 neurons responded phasically to both cooling and warming, with increased discharges. Response profiles of the 37 taste neurons to taste and thermal stimuli are shown in Fig. 2, where the neurons are arranged in order of their response magnitudes to 0.1 M NaC1. Average response magnitude to each of the 4 basic taste stimuli and average spontaneous discharge rate are given in the figures. These values are not significantly different from those of our previous study 14. Average responses of taste neurons to quantitative thermal stimulations are also given in the figure. Thermosensitive taste neurons gave much greater thermal responses than those of a whole population of taste neurons, including nonthermosensitive neurons. Average responses of cooling-sensitive taste neurons (n = 16) to cooling were -0.17 + 0.15 impulses/s/°C (mean + S.D.), while those of warming-sensitive taste neurons (n = 10) to warming were 0.23 + 0.14 impulses/s/°C. Six of the 33 taste neurons, whose thermal sensitivity was examined, responded to one or two of the 4 basic stimuli (specific type) and the remaining 27 responded to more than two of the 4 basic stimuli (nonspecific type). Two of the 6 specific type neurons responded to thermal stimuli, while 23 of the 27 nonspecific type neurons responded to the same stimuli. The proportion of taste neurons with thermal sensitivity in these two groups of neurons was significantly different (P < 0.05; Fisher's exact probability test). Breadth of taste responsiveness was measured in

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Fig. 1. Responses of cold-sensitive (A) and warm-sensitive (B) taste neurons in the NTS to the 4 basic taste stimuli, cooling and warming. Four basic taste stimuli used were 0.1 M NaC1, 0.5 M sucrose, 0.01 N HC1 and 0.02 M quinine-HCl; cooling, from 40 to 20 °C; and warming, from 20 to 40 °C. The entire oral cavity was stimulated. In B, the neuron started to respond to warm water before the change of thermister output, probably because the thermister was placed on the anterior tongue where the R F was not located for warming. Downward arrows for taste responses indicate the onset of taste stimulations and upward arrows, the offset of taste stimulations. Downward arrows for thermal responses indicate the time of switching warm (40 °C) to cold water (20 °C) or vice versa. Outputs of thermister, monitoring the temperature of the anterior tongue, is superimposed on the trace of spike discharges. A, unit 605221; B, unit 512172.

terms of entropy (H) 17 and the degree of the breadth of taste responsiveness was quantitatively related to thermal sensitivity (Fig. 3). Some taste neurons with the narrowest breadth had no thermal sensitivity, and thermal-sensitive neurons showed a wide range of taste responsiveness. Based on entropy value, neurons whose thermal sensitivity was examined were divided into two groups; one group with broader taste responsiveness ( H ~> 0.5; n = 24) and the other with narrower taste responsiveness ( H < 0.5; N

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Fig. 2. Response profile of 37 NTS neurons to the 4 basic taste stimuli (0.1 M NaC1, 0.5 M sucrose, 0.01 N HC1 and 0.02 M quinine-HCl) and thermal stimuli (cooling from 40 to 20 °C and warming from 20 to 40 °C) together with their spontaneous discharge rates (spon. dis.). Responses in the first 5 s after the onset of stimulation were used. All the responses of a single unit are arranged vertically one above the other. Symbols of + and - in thermal responses indicate the presence or absence of responses when examined by an application of thermal stimuli with pipettes, while • represents no test of a thermal response. Solid bars indicate the maximum responses among the 4 taste responses in each of the neurons; stars represent neurons projecting to the parabrachial nucleus. Numerals indicate average responses (+ S.D.) and an average rate of spontaneous discharges.

= 9). Neurons with broader taste responsiveness showed a significant tendency toward thermal sensitivity (P < 0.05; Fisher's exact probability test).

Best stimulus categories and thermal sensitivity Taste neurons were classified according to the best stimulus, i.e. the stimulus which produced the largest responses of the 4 basic taste stimuli in the neuron (solid bars in Fig. 2). Thirty-two of the 37 neurons were most responsive to one of the 4 stimuli ('single stimulus type'). The remaining 5 responded equally well to two of the 4 stimuli ('multistimulus type'); i.e. one of the non-best stimuli produced more than 90%

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where Pi represents a proportional response to stimulus i17. Average entropy is given in the figure. of the m a x i m u m responses to the best stimulus. Seven of the 32 'single stimulus type' n e u r o n s were classified as sucrose-best, 16 as NaCl-best, 7 as HCl-best, and 2 as quinine-best. Of the 32 single stimulus type of NTS neurons, 28 (7 sucrose-best, 15 NaCl-best and 6 HCl-best) were subjected to the test of thermal sensitivity. These best stimulus categories of taste neurons showed different thermal sensitivities, as shown in Fig. 4. All of the suc:ose-best n e u r o n s tested, except one without thermal sensitivity, showed sensitivity to warming only (n = 3) or to both warming and cooling (n = 3), while about a half of both the NaCl-best neurons tested (8 of 15) and tile HCl-best neurons tested (3 of 6) responded to cooling only. Some NaCl-best (n = 2) and HCl-best (n = 1) n e u r o n s responded to both cooling and warming. None of the quinine-best neurons were tested for thermal sensitivity. Three of the 5 neurons of the multistimulus type were best excited by HC1 and quinine ( H Q type), and one of the remaining two was of sucrose-NaC1 (SN) type and the other of NaC1-HC1 (NH) type. All were tested for thermal sensitivity and 3 showed thermal sensitivity; an N H type n e u r o n was sensitive to cooling only, and each of the SN and H Q type n e u r o n s was sensitive to both cooling and warming. Correlations among thermal responses, taste re-

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Fig. 4. Best stimulus categories of taste neurons and thermal responses. Taste neurons were classified into single stimulus types, such as sucrose-best, NaCl-best, HCl-best and quininebest, and multistimulus type, according to the stimulus which produced the largest responses among the 4 basic taste stimuli (best-stimulus). Tuning curves to the 4 basic taste stimuli, plotted with solid lines, represent neurons whose theimal responses were examined: solid lines with filled circles mean cold-sensitive neurons, those with open circles warm-sensitive ones, and those with half-filled circles neurons sensitive to both cooling and warming. Solid lines with x's indicate neurons without thermal responses. Tuning curves of small filled circles connected with broken lines indicate neurons whose thermal sensitivity was not tested. S, N, H and Q mean sucrose, NaCI, HCI and quinine, respectively. Multi indicates multistimulus type.

sponses and spontaneous discharges, were calculated by means of the p r o d u c t - m o m e n t correlation coefficient between every pair of responses and all pairs of responses and spontaneous discharges. The results are shown in Table I. Significant correlations were obtained among NaC1, HC1, quinine and spontaneous discharges, between sucrose and warming, be-

326 TABLE I Correlation coefficients between pairs of responses to 4 basic taste solutions and thermal stimulation, and spontaneous discharge rates (n = 37)

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tween NaC1 and cooling and b e t w e e n cooling and spontaneous discharges. No significant correlation was found between HC1 and cooling, although most HCl-best neurons r e s p o n d e d to cooling. Receptive fields o f thermosensitive taste neurons RFs f o r taste stimuli. R e c e p t i v e fields of the 20 taste neurons (17 thermosensitive and 3 n o n - t h e r m o -

sensitive neurons) were e x a m i n e d using the best taste stimuli. A m o n g the 17 thermosensitive neurons, 8 neurons (7 NaCl-best, 1 HCl-best) had R F s at the anterior tongue only; 5 neurons (2 sucrose-best, 2 HCl-best, 1 multiple stimulus type) at the N D only; two neurons (1 NaCl-best and I sucrose-best) at both anterior tongue and N D ; and the remaining two (both sucrose-best) had R F s at wide regions of the tongue and palate. Relations of thermal sensitivities and locations of the R F s for taste are shown in Table II. Six of the 9 cold-sensitive taste neurons had R F s confined to the tongue, particularly at the anterior tongue, and all of the 3 warm-sensitive neurons had their R F s exclusively or partly on the palate, particularly at the N D . Of the 3 non-thermosensitive neurons, two (both NaCl-best) had R F s on an area over the Geschmacksstreifen (a n a r r o w strip of taste buds at the b o u n d a r y between the hard and soft palate) 1° and soft palate. The remaining one (sucrose-best) had an R F on the N D only.

R F s f o r thermal stimuli. In 7 taste neurons, R F s for thermal stimuli were located. T h r e e of the 4 cold-sensitive neurons (2 NaCl-best, one HCl-best) had R F s common for these two modalities on the anterior tongue only (Fig. 5Bb,c) and all 3 showed mechanical sensitivity on this area. The o t h e r neuron (HCl-best) had an R F for taste and cooling on the N D and an inhibitory R F for mechanical stimulation on the soft palate. On the other hand, a p r e d o m i n a n t l y warmsensitive neuron (sucrose-best), which was classified as a neuron sensitive to both warming and cooling, had an R F on the N D for taste, warming and mechanical stimulation (Fig. 5Ba). The remaining two neurons (NaCl-best) were sensitive to both cooling and warming, and one was p r e d o m i n a n t l y sensitive to cooling. These two neurons had R F s for taste, cool-

TABLE II Thermal sensitivity of NTS taste neurons and locations of RFs for taste Locations of RFs for taste

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Fig. 6. Thermal response of a mechanoreceptive neuron to a sudden fall and rise in temperature of the oral cavity (A), and its receptive field for mechanical and thermal stimulation (B). This neuron which shows spontaneous activity is affected by temperature. A: a continuous record. Arrows indicate the time of switching from warm (40 °C) to cold water (20 °C) and vice versa. Following switching from cold to warm water, a transient mechanical response is noticed. B: a shaded area represents the RF for mechanical (M) and thermal (T) stimulation.

ate and I M E (Fig. 5Cb) , and the remaining one on both the N D and hard palate. No mechanoreceptive

Thermosensitivity of mechanoreceptive neurons and thermoreceptive neurons Of the 23 mechanoreceptive neurons, 18 were ex-

n e u r o n was sensitive to warming. All the 10 thermoreceptive n e u r o n s were sensitive to cooling only. They responded to cooling with both

cited by stroking the tissue with a glass rod (low threshold, or LT type) and 5 by pinching the tissue with a non-serrated forceps (high threshold, or H T

phasic and tonic discharges (Fig. 7A). The average

type). Thirteen n e u r o n s (9 LT and 4 HT) responded phasically to cooling with increased discharges (Fig. 6A). Although non-thermosensitive mechanorecep-

rate of steady responses at 20 °C was 53.5 +_ 73.8 impulses/5 s after adaptation for 1 min, which was significantly larger than the average steady discharge of

tive n e u r o n s did not produce a noticeable rate of

thermosensitive taste n e u r o n s (0.01 < P < 0.05, M a n n - W h i t n e y U-test) but did not differ from that

spontaneous discharges, thermosensitive n e u r o n s showed a considerable rate of spontaneous discharges at 20 °C (41.5 _+ 53.2 impulses/5 s after adaptation for 1 min, n = 12) (Fig. 6A), this being significantly larger than that of thermosensitive taste neurons (5.6 + 7.5 impulses/5 s at 20 °C, n = 19; 0.01 < P

responses of the 7 thermoreceptive n e u r o n s to cooling were -1.51 + 1.56 impulses/s/°C, and the average

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< 0.05, M a n n - W h i t n e y U-test). In 4 thermosensitive units, spontaneous discharge rates at 30 °C were also obtained; they were 0.8, 17.2, 34.8 and 107.1 impulses/5 s. The average thermal responses were - 1 . 3 8 + 1.26 impulses/s/°C (n = 12), which was about 8 times as large as those of the taste neurons. The RFs for both mechanical and thermal modalities were located for 5 n e u r o n s and were present on the same region. Three n e u r o n s had RFs on the anterior tongue (Figs. 5C a and 6B), one on both the hard pal-

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328 of thermosensitive m e c h a n o r e c e p t i v e neurons (0.05 < P). In all of the 7 neurons fully studied, bursting discharges were noted at the tonic Phase of responses at 20 °C, as in the case of cutaneous cold afferents of cats s, dogs and m o n k e y s 7. The spontaneous discharge rates at 30 °C were counted in 4 neurons; the average rate being 45.2 + 52.3 impulses/5 s. Receptive fields were located in 3 cold units; two units had RFs on the anterior tongue only (Figs. 7B and 5Da) and the remaining one had RFs on both the intermolar eminence ( I M E ) and hard palate (Fig. 5Db).

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Projections of NTS neurons to parabrachial nucleus Projections to the parabrachial nucleus were tested in 29 taste, 23 m e c h a n o r e c e p t i v e and 8 cold neurons. A m o n g them, 11 taste (5 sucrose-best, 5 NaCl-best, 1 HCl-best) and two m e c h a n o r e c e p t i v e (1 LT and 1 H T ) neurons were activated antidromically from the parabrachial region, with a latency of 0.9-13.0 ms (Fig. 8); thus, they were identified as solitario-parabrachial relay neurons. The p r o p o r t i o n of best categories of taste relay neurons is consistent with that r e p o r t e d earlier t4. In 8 of these taste relay neurons (4 sucrose-best, 4 NaCl-best), R F s were located with the best stimulus. Two sucrose-best neurons had RFs on the N D only, and the other two sucrose-best had R F s on the tongue and palate. O n e NaCl-best neuron had an R F on the anterior tongue only, another two had R F s on the palate, and the remaining two had RFs on the tongue and palate. O n e mechanoreceptive (1 H T ) and 9 taste relay neurons were thermosensitive. Five of these 9 taste neurons responded to both cooling and warming and two each

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Fig. 9. Locations of 3 types of neurons in the NTS. Recording sites are plotted on 4 representative coronal sections of the NTS (at the levels of 0.2, 0.4, 0.6 and 0.8 mm from the caudal margin of the acoustic tubercle at 1.5 mm lateral to the midline). A, taste neurons. Large filled circles mean thermosensitive taste neurons, large open circles non-thermosensitive ones; dots, neurons whose thermal sensitivities were not tested. B: mechanoreceptive neurons with or without thermal sensitivity (solid or open triangles) and cold units (solid circles). CR, corpus restiformis; CUL, nucleus cuneatus lateralis; DM, nucleus dorsomedialis, Astrom; MVN, nucleus vestibularis medialis; NTS, nucleus tractus solitarii; NTSV, nucleus tractus spinalis n. trigemini; TSV, tractus spinalis n. trigemini.

of the remaining 4 r e s p o n d e d only to either cooling or warming. T h e r m a l sensitivity of the taste relay neurons did not differ from that of the non-relay neurons. Cold neurons were not activated antidromically from the parabrachial region.

Location of three types of neurons in the N T S As noted previously a4, the m a j o r i t y of taste neurons was c o n c e n t r a t e d at the central part of the NTS at the most rostral two planes, while both mechanoreceptive and cold neurons were diffusely located at the second and third-most rostral planes (Fig. 9). Thermosensitive taste and m e c h a n o r e c e p t i v e neurons occupied no particular region of the NTS and intermingled with the non-thermosensitive neuron groups, DISCUSSION

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Fig. 8. Distribution of antidromic latencies of NTS neurons to stimulation of the parabrachial region. Stars indicate neurons with thermal responsiveness.

Thermosensitive taste neurons In the present experiment, we p r e a d a p t e d the oral cavity to a certain t e m p e r a t u r e for 1 1/2 min before stimulation by changing the t e m p e r a t u r e . This proce-

329 dure is essential to obtain a desired degree of temperature rise or fall (20 °C difference) in the tissue 15. Although a shorter preadaptation time of 10 or 15 s is used, a similar preadaptation has been used in a series of our experiments to produce taste stimulation free of thermal stimulation 4,14. Cold-sensitive taste neurons are present in the chorda tympani 15 as well as in the NTS 9. The present study seems to be the first to reveal the presence of warm-sensitive taste neurons in the NTS of rats. These warm-sensitive neurons tended to have RFs for taste at the ND, an area not innervated by the chorda tympani, whereas a majority of the cold-sensitive taste neurons, including those sensitive to both cooling and warming (10/14), had RFs exclusively or partly at the anterior tongue innervated by the chorda tympani (see Table II). Therefore, it is suggested that in rats, the greater superficial petrosal nerve contains warm-sensitive taste fibers. Some taste neurons responsive to both cooling and warming had RFs on the anterior tongue (Table II), though such taste fibers were not detected in the chorda tympani of rats. W h e n neurons produced a small magnitude of phasic responses to thermal changes in both directions, usually they were considered to be mechanical responses to applied solution; however, some of these responses were too large to be attributed to mechanical responses only (Fig. 2).

Best stimulus category and thermal sensitivity Thermosensitivity of taste neurons was related to their best stimulus categories: warm responses were found in the sucrose-best neurons, and cold responses were noted in the NaCl-best or HCl-best neurons. Significantly high correlation coefficients were obtained between sucrose and warming and between NaC1 and cooling, but not between HC1 and cooling. High correlations between sucrose and warming have also been found in the chorda tympani of hamsters 15 and monkeys 16. The absence of a significant correlation between HC1 and cooling in the NTS, despite its presence in the chorda tympani 15, can be attributed to the convergence of taste fibers, perhaps HCl-best ones, with various thermal sensitivities on single neurons in the NTS. This is supported from the finding that some neurons predominantly sensitive to HC1 responded to warming only or to both cooling and warming (Fig. 4). Most NaCl-best

neurons responded to cooling of the tongue in the NTS, and a significant correlation was found between NaC1 and cooling. A concomitant appearance of NaC1 responses with responses to cooling was not found in the chorda tympani of S p r a g u e - D a w l e y rats 15. The appearance of a concomitant response of neurons to NaC1 and cooling in the NTS may be due to convergence of NaCl-best afferents and thermosensitive afferents in the intermediate and/or trigeminal nerves. According to the dual taste theory of von Bekesy 2, basic taste and thermal senses of the human tongue are categorized into two groups; salty, sour and cooling in one group, and sweet, bitter and warming in the other. Based on significant correlations between a pair of taste and thermal responses (Table I) as well as the finding that 3 of 5 HCl-best units were cold sensitive, we considered that NaC1, HC1, quinine and cooling belong to one group, and sucrose and warming to another group, in the chorda tympani fibers, HC1, quinine and cooling form one group, but neither NaCI nor sucrose make a group 15. In the chorda tympani fibers, the non-specific type of neurons had a tendency to show a large thermal sensitivity compared to the specific type of neurons 15. A similar finding was obtained in the NTS in the present study. This was also true when taste neurons were divided into two groups based on whether their entropy values were larger or less than 0.5. A n entropy value of 0.5 was used as a reference to classify neurons, since, when a neuron produces responses to only two of the 4 basic solutions with equal magnitudes, the entropy value is 0.5, the highest among specific neurons. However, this criterion is not consistent with the one used to classify taste neurons into specific and non-specific types, because entropy values of a non-specific type of neurons vary greatly between 0 and 1 depending on the relative magnitudes of responses evoked by 4 basic taste solutions 17.

Characteristics of taste responses evoked in the palate In the present study we found that many taste neurons had RFs exclusively or partly on the palate and that some of these were sucrose-best. The present findings are consistent with those of Travers et al. 18 in intact rats and by H a y a m a et al. 4 in decerebrate rats, but they are not in good agreement with the findings

330 of Ogawa and H a y a m a in intact rats 12, in which RFs of only a few neurons were detected on the palate. The reasons for this discrepancy may relate to either a possible inadequate artificial ventilation of animals in earlier experiments without monitoring of the endtidal CO 2 concentration, or to a technical failure in locating RFs at the palate. Small responses evoked from the palate, as evident in NTS neurons of decerebrate rats 4, maybe disappear under conditions of poor ventilation. We obtained evidence for numerous cold-sensitive taste neurons together with cold-sensitive mechanoreceptive neurons and cold neurons among NTS neurons with RFs on the palate. We also found warmsensitive taste neurons, which were often sucrosebest. In one of these taste neurons, the RFs for warming were located at the ND. The present findings suggest that the greater superficial petrosal nerve innervating the hard palate of rats contains warm-sensitive fibers associated with sucrose sensitivity, as noted in the chorda tympani fibers of the hamster 15 and m o n k e y 16. Some HCl-best neurons were also warm-sensitive or sensitive to both cooling and warming. Since no warm-sensitive taste fibers were found in the chorda tympani 15, it is likely that warmsensitive HCl-best neurons receive a main input from the palate. Recently, it was reported that the greater superficial petrosal nerve is different from the chorda tympani, in sensitivity to monovalent chloride salts and sweet substances n, although both nerves are branches of the intermediate nerve. The present study suggests that taste afferents in the greater superficial petrosal nerve are thermosensitive. Thus, different combinations of oral sensory information are sent to the NTS, mainly by two different taste nerves; the chorda tympani and greater superfi-

REFERENCES 1 Amerine, M.A., Pangborn, R.M. and Roessler, E.B., Principles of Sensory Evaluation of Food, Academic, New York, 1965, pp. 223-241. 2 Bekesy, G. von, Duplexity theory of taste, Science, 145 (1964) 834-835. 3 Fuller, J.H. and Schlag, J.D., Determination of antidromic excitation by the collision test: problems of interpretation, Brain Research, 112 (1976) 283-298. 4 Hayama, T., Ito, S. and Ogawa, H., Responses of solitary tract nucleus neurons to taste and mechanical stimulations of the oral cavity in decerebrate rats, Exp. Brain Res., 60

cial petrosal nerve. The different patterns of taste and somatosensory inputs from two taste nerves converge onto single neurons in the NTS, so that correlation coefficients between a certain pair of stimuli, which are significant in one of these nerves, may become blurred in the NTS. Complexity o f taste coding in the N T S The present study confirmed that numerous taste neurons project from the NTS to the parabrachial nucleus, the second taste relay nucleus of rats, but few mechanoreceptive neurons do so. In addition, almost none of the thermoreceptive neurons projected to the parabrachial nucleus. The present findings are consistent with the generally accepted idea that the rostral part of the NTS is the first taste relay nucleus. Although a single group of NTS neurons, i.e. taste neurons, mainly project to the second relay, most of these taste neurons respond to both thermal and mechanical stimulation. It has been suggested that both thermal and mechanical aspects of food influence taste sensation during eating and drinking 1. Our study shows that the NTS is one of the key relay nuclei where thermal and mechanical information originating from the oral cavity interact with information related to taste.

ACKNOWLEDGEMENTS We express our gratitude to Dr. M. Sato (Brain Science Foundation) for helpful advice during preparation of this report and to M. Ohara (Kyushu University) for comments. This research was supported by a Grant-in-Aid (59570061) from the Japanese Ministry of Education, Science and Culture.

(1985) 235-244. 5 Hensel, H., The time factor in thermoreceptor excitation, Acta Physiol. Scand., 29 (1953) 109-116. 6 Iggo, A., Cutaneous thermoreceptors in primates and subprimates, J. Physiol. (Lond.), 200 (1969) 403-430. 7 Iggo, A. and Young, D.W., Cutaneous thermoreceptors and thermal nociceptors. In H.H. Kornhuber (Ed.), The Somatosensory System, Thieme, Stuttgart, 1975, pp. 5-22. 8 Kenshalo, D.R., Hensel, H., Graziadei, P. and Fruhstorfer, H., On the anatomy, physiology and psychophysics of the cat's temperature-sensing system. In R. Dubner and Y. Kawamura (Eds.), Oral-facial Sensory and Motor Mechanisms, Appleton-Century-Crofts, New York, 1971, pp.

331 23-45. 9 Makous, W., Nord, S., Oakley, B. and Pfaffmann, C., The gustatory relay in the medulla. In Y. Zotterman (Ed.), Ol-

faction and Taste, Werner-Gren Center International Symposium Series Vol. 1, Pergamon, Liverpool, 1963, pp. 381-393. 10 Miller, I.J., Jr. and Spangler, K.M., Taste bud distribution and innervation on the palate of the rat, Chem. Senses, 7 (1982) 99-108. 11 Najad, M.S., The neural activities of the superficial petrosal nerve of the rat in response to chemical stimulation of the palate, Chem. Senses, 11 (1986) 283-293. 12 Ogawa, H. and Hayama, T., Receptive fields of solitarioparabrachial relay neurons responsive to natural stimulations of the oral cavity in rats, Exp. Brain Res., 54 (1984) 359-366. 13 Ogawa, H., Hayama, T. and Yamashita, Y., Thermal sensitivity of taste neurons in solitary tract nucleus of rats, J. Physiol. Soc. Jpn., 48 (1986) 309. 14 Ogawa, H., Imoto, T. and Hayama, T., Responsiveness of

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solitario-parabrachial relay neurons to taste and mechanical stimulations applied to the oral cavity in rats, Exp. Brain Res., 54 (1984) 349-358. Ogawa, H., Sato, M. and Yamashita, S., Multiple sensitivity of chorda tympani fibers of the rat and hamster to gustatory and thermal stimuli, J. Physiol. (Lond.), 199 (1968) 223-240. Sato, M., Ogawa, H. and Yamashita, S., Response properties of macaque monkey chorda tympani fibers, J. Gen. Physiol., 66 (1975) 781-810. Smith, D.V. and Travers, J.B., A metric for the breadth of tuning of gustatory neurons, Chem. Senses, 4 (1979) 215-229. Travers, S.P., Pfaffmann, C. and Norgren, R., Convergence of lingual and palatal gustatory neural activity in the nucleus of the solitary tract, Brain Research, 365 (1986) 305-320. Yamashita, S. and Sato, M., The effect of temperature on gustatory response of rats, J. Cell. Comp. Physiol., 66 (1965) 1-18.