Differential taste coding of salt and acid by correlative activities between taste-sensitive neuron types in rat gustatory cortex

Neuroscience 144 (2007) 314 –324

DIFFERENTIAL TASTE CODING OF SALT AND ACID BY CORRELATIVE ACTIVITIES BETWEEN TASTE-SENSITIVE NEURON TYPES IN RAT GUSTATORY CORTEX T. YOKOTA,* K. EGUCHI, AND T. SATOH1

been explained by the across-fiber (neuron) pattern theory, which emphasizes relative response magnitudes across individual fibers (neurons) (Erickson, 1963). In our previous studies, we reported that correlative activities of tastesensitive neuron pairs were dependent on relative response magnitudes in the nucleus of solitary tract (NTS; Adachi et al., 1989), parabrachial nucleus (PBN; Yamada et al., 1990) and gustatory cortex (Yokota et al., 1996, 1997). The incidence of correlative activity and the mean frequency of correlated discharges (FC) have been reported to depend on factors such as similarity of taste profiles, temporal patterns, and short inter-neuronal distances (INDs) between paired neurons (Yokota and Satoh, 2001). These findings suggested that, besides the response magnitudes of individual neurons, their correlative firings might be involved in taste quality coding. The importance of correlative activity for taste encoding has been supported by investigations in the gustatory cortex (Nakamura et al., 1997; Katz et al., 2002). Frank (1973) classified chorda tympani (CT) fibers in hamsters by their most responsive taste stimuli, i.e. NaCl (N)-, HCl (H)-, quinine (Q)- and sucrose (S)-best fibers. In contrast, taste-sensitive neurons in rats were frequently responsive not only to salt, but also to acid and other taste stimuli (CT: Pfaffmann, 1955; Ogawa et al., 1968; Ninomiya et al., 1988; NTS: Doetsch and Erickson, 1970; PBN: Norgren and Pfaffmann, 1975; thalamus: Scott and Erickson, 1971; gustatory cortex: Yamamoto, 1984). Those neurons that respond to NaCl, HCl and other electrolytes were termed E-type (Ninomiya et al., 1987) or NaCl-/HCl-generalist (Lundy and Contreras, 1999). E-type neurons are also known to be unaffected by amiloride (a selective Na⫹ channel blocker) applied on the tongue. These properties of E-type neurons distinguish them from N-type neurons, which are highly selective for NaCl and sensitive to amiloride (Ninomiya et al., 1988). It is unknown whether information transmitted by E-type neurons is merged with that by N-type neurons in the higher centers of the brain. If neuronal signals transmitted by E- and N-type fibers in the CT nerve eventually converge on common target neurons in the gustatory cortex, some of these neurons may show a unique response pattern: comparable responses to NaCl and HCl (similar to NH-best neurons in the present study, see below), amiloride sensitivity, and correlated activity with N-best neurons. Since a considerable number of neurons equally responsive to NaCl and HCl (no significant difference by t-test, P⬎0.05) were observed in the present study, a fifth category (NH-best) in addition to Frank’s four categories was used here.

Department of Physiology, School of Dental Medicine, Aichi-Gakuin University, 1-100 Kusumoto, Chikusa, Nagoya, 4648650 Japan

Abstract—Using a multi-electrode recording technique, the present study aimed to elucidate the role of broadly-tuned taste-sensitive neurons in the rat gustatory cortex in discriminating between salt and acid. A majority of taste-sensitive neurons (94/119 neurons; 78%) were classified as NaCl (N)-, HCl (H)- or NaCl and HCl (NH)-best neurons. Of 63 neuron pairs (94 neurons), 31 showed significant peaks and/or troughs in their cross-correlograms (CCs) during taste stimulation periods. During NaCl stimulation, the incidence of significant correlation and the mean frequency of correlated discharges (FC) in the N/N and NH/NH pairs were higher than those in the other best-taste pairs. In contrast, during HCl stimulation both indices in the N/N or H/H pairs were very low, while those in the NH/NH pairs were high. These results suggest that (1) correlated activities between N-best neurons and those between NH-best neurons play a significant role in taste quality coding of salt, and that (2) correlated activities between NH-best neurons may be important for sour taste coding as well. Peak formation in CCs tended to be more frequent in the homo-types (N/N, H/H and NH/NH pairs) than in the hetero-types (N/NH, N/H and H/NH pairs). In contrast, troughs were observed mostly in the hetero-types. Inhibitory interaction in hetero-type pairs together with coactivation in homo-type pairs may enhance taste discrimination by tastesensitive neuron populations. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: best-taste type, cross-correlogram, cell assembly, entropy, synchronization, trough.

Taste-sensitive neurons in rats show vigorous responses to salts and acids. Such broad tuning of taste responsiveness has been reported from peripheral nerves to the CNS in rodents (Pfaffmann, 1955; Ogawa et al., 1968; Doetsch and Erickson, 1970; Norgren and Leonard, 1971; Ganchrow and Erickson, 1972; Frank, 1973; Norgren and Pfaffmann, 1975; Smith et al., 1983; Yamamoto, 1984). Taste quality coding by broadly-tuned neurons has 1

The senior author, Dr. Satoh passed away during the preparation of this paper. *Corresponding author. Tel: ⫹81-52-751-2561; fax: ⫹81-52-752-5988. E-mail address: [email protected] (T. Yokota). Abbreviations: CC, cross-correlogram; CT, chorda tympani; FC, mean frequency of correlated discharges; H-best, HCl-best; Hca, breadth of responsiveness of correlative activity; Htr, breadth of responsiveness of taste responses; IND, inter-neuronal distance; N-best, NaCl-best; NH-best, NaCl and HCl-best; NTS, nucleus of solitary tract; PBN, parabrachial nucleus; Q-best, quinine-best; S-best, sucrose-best; SI, synchronization index.

0306-4522/07$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.09.013

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T. Yokota et al. / Neuroscience 144 (2007) 314 –324

The present report focuses on the role of N-best, Hbest and NH-best neurons in temporal coding for salt and sour. To investigate how these taste-sensitive neurons, particularly NH-best neurons, encode information about salt and sour tastes, we examined correlative activities between N-, H- and NH-best neurons. A part of this study has appeared in an abstract form (Yokota et al., 1999).

EXPERIMENTAL PROCEDURES Surgical procedures Male Wistar rats (260 – 410 g body weight) were anesthetized initially with a mixture of urethane (0.6 g/kg, i.p.) and pentobarbital sodium (40 mg/kg, i.p.). After a tracheotomy for airway maintenance, the animals were fixed in a stereotaxic apparatus such that the bregma and lambda were level. A craniotomy for electrode insertion was performed over the parietal cortex (anteroposterior ⫹0.8 to ⫹2.2 mm to the bregma, lateral 4 – 6 mm from the midline). In order to minimize damage to the gustatory cortex, we avoided a craniotomy in the skull overlying the gustatory cortex. Recording electrodes were tilted 5 or 8° on the coronal plane such that they approached the gustatory cortex from the dorsolateral to ventromedial direction. The animals were artificially ventilated to maintain 4% end-tidal PCO2. The electrocardiogram was monitored continuously and the rectal temperature was kept at 37 °C with the use of a heating pad. During recording, anesthesia was maintained with urethane (70 mg/kg/h, i.p.), and the animals were immobilized with gallamine triethiodide (90 mg/kg/h, i.p.). All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the guidelines of the Japan Neuroscience Society. Every effort was made to minimize the number of animals used and their suffering.

Gustatory stimulation Four solutions (25–26 °C) were used as taste stimuli: (in M) 0.2 NaCl (N), 0.25 sucrose (S), 0.03 HCl (H) and 0.005 quinine-HCl (Q). Each of them was applied into the oral cavity at a flow rate of 1.1–1.3 ml/s. The solutions, which were ejected from the nozzle placed near the lingual tip, first touched the soft palate, filled the oral cavity, and then drained through the lingual tip. The different test solutions were delivered successively in random order. A stimulation trial consisted of a 6-s presentation, followed by a 14-s rinsing with distilled and deionized water that began 2 s after the end of the tastant presentation. Ten to 12 trials were performed for each tastant with 2 s inter-trial intervals. Data on the first two trials were not included in the analysis because responses in later trials tended to be more stable. In addition to these test trials, water was applied into the oral cavity at two flow rates (the abovementioned flow rate and twice that rate) for 10 trials. If a neuron’s responses depended on the flow rates, the neuron was classified as “tactile” and was excluded from the present study. Moreover, the effects of tactile and thermal components of taste stimulation were minimized by subtracting responses to water stimulation from those to taste stimulation (see below).

Unit recording Two to six glass micropipettes, each with a 1.5-␮m-tip-diameter and 4 – 6 M⍀-impedance were glued together with 50 –200 ␮m distance between contiguous tips. The multi-barreled electrodes were driven with micro-step drivers into the area rostral to the unilateral middle cerebral artery and dorsal to the rhinal fissure. Spike activities were amplified, filtered at 0.3 kHz to 5 kHz and stored on magnetic tape. Single spikes were isolated using a

315

template matching method similar to that in Forster and Handwerker (1990). After simultaneous recording of multiple tastesensitive neurons, Pontamine Sky Blue (2% in 0.5 M Na-acetateadded) was ejected electrophoretically from each recording electrode by applying a 5 ␮A DC current for 10 min. At the end of the experiment, the brain was perfused with Na-citrate-added Ringer solution followed by 10% formalin and stored in a mixture of 30% sucrose and 10% formalin. Serial frozen frontal sections of 20-␮m thickness were prepared and stained with Cresyl Violet.

Cross-correlation analysis The raw cross-correlograms (CCs) (Fig. 1A) were constructed following the conventional technique (Perkel et al., 1967; Yokota et al., 1997). The raw CC (R) was calculated as follows: ⫹T

D

Rxy(k)⫽

兺 兺 x(i)·y(i⫹j) i⫽0 j⫽⫺T

K

CC(R)⫽

兺Rxy(k)

k⫽1

where Rxy(k) is the CC for the k th trial of taste stimulation between a reference neuron (x) and a paired (measured) neuron (y). For each spike (at time 0 in the CC) of a neuron (x) within a stimulation period (D), the relative timings of spikes from a neuron (y) within an analysis time window (⫾T) were allotted to the bins of the histogram and accumulated over all trials (K) per tastant. To examine the stimulus-locked pseudo-synchronous firings in CCs, the expected CCs (Fig. 1B) were constructed on the joint-PSTH (Aertsen et al., 1989). The expected CC (E) was calculated as follows:

ni(x)⫽

1 K

K

兺ni(x)

k⫽1

nij(x, y)⫽ni(x)·nj(y) K

CC(E)⫽

兺nij(x, y)

k⫽1

CC(R)⫺CC(E)⫽

K

K

k⫽1

k⫽1

兺Rxy(k)⫺兺nij(x, y)

where ni(x) is the spike count in the i th bin after the stimulus onset in the averaged PSTH of neuron (x). The bin width is the same as in the raw CC. Thus, nij(x, y) is the CC between the stimuluslocked components in a pair (x and y) (Fig. 1B). The expected CC (E) was subtracted from the raw CC(R) to obtain the CC unaffected by pseudo-synchronous firings (CC(R)⫺CC(E): Fig. 1C). The statistical significance of the peaks and troughs, i.e. the presence of correlative activity, was examined by two methods. (1) Shuffled CCs were obtained by changing randomly the temporal order of the spike trains (trials) in one component neuron of a pair with the original inter-spike-intervals within trials preserved. If the peaks or troughs disappeared or they were strongly attenuated in the shuffled CCs, their presence in the original unshuffled CCs was considered due to neuronal connectivity rather than to stimulus-induced coactivation. (2) The peaks and troughs against the background level in the CCs where the expected CC was subtracted from the raw CC were examined statistically by the following synchronization index (SI) (Wiegner and Wierzbicka, 1987; P⬍0.05).

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counts/bin

NaCl

water

(A) raw CC

(D) raw CC

(B) expected CC

(E) expected CC

(C) raw - expected CC

(F) raw - expected CC

20 0 -10 -75

0

+75

-75

0

+75 ms

Fig. 1. Statistically significant peaks in CCs. (A) Raw CC of a neuron pair during NaCl stimulation. (B) The expected CC for (A). (C) The expected CC (B) subtracted from the raw CC (A). The ordinate indicates the spike counts per a bin in the pair. The abscissa is the time of the measured neuron’s firings that occurred before (left) or after (right) the reference neuron. Arrowheads indicate the consecutive bins over the gray line (the 5% significance level) forming a significant peak. The zero base line indicates the background level. (D) Raw CC of the same pair as (A) during water application. (E) The expected CC during water application. (F) The expected CC (E) subtracted from the raw CC (D). When a significant peak (an arrowhead) was detected during distilled water application, the correlation counts over the level zero in this peak during water application were subtracted from those during NaCl stimulation for the calculation of FC (counts/s). Bin width is 3 ms. j

SI⫽

兺

¯) ⁄ T (bi⫺b

i⫽1

where bi is the number of spike counts within the peak (J bins), b៮ is the mean counts in non-peak regions, and T is the total number of counts in the CC. The significance level (5%) is indicated by a solid gray line (Fig. 1C, F). The consecutive bins beyond this level around time zero were considered significant. SI standardized the firing rates of paired neurons with respect to the total spike counts, thereby allowing the firing-rate independent evaluation of a peak or trough in CCs. The magnitude of peaks in CCs was quantified by counting the number of spikes in excess of the background level (count zero on the ordinate in Fig. 1C). The magnitude of troughs was quantified by counting the number of spikes to be added for reaching the background level. The number of spikes was divided by the product of the total taste stimulation period and the number of trials, giving the FC (correlation spikes/s) as an index expressing the intensity of correlation (see Yokota et al., 1997). This index has been reported to represent the common input strength, and to be independent of the firing rates of paired neurons (Nordstrom et al., 1992; Ushiba et al., 2002). In cases where significant peaks or troughs also appeared during application of distilled water, the subtraction procedure as used in taste stimulations was performed (Fig. 1D–F). FC for taste stimulation (Fig. 1C) was calculated by subtracting water-induced FC (Fig. 1F) on the corresponding bins of peaks or troughs. In most cases, the bin width in the range of 1–5 ms was used. In other cases longer bin widths (6 –14 ms) were needed to accumulate sufficient numbers of spikes from slowly firing neurons, especially for detecting troughs in CCs (Melssen and Epping, 1987). Rhythmically firing neurons showing periodic peaks in their autocorrelograms were excluded

from the present analysis because the peaks in CCs from these neurons did not necessarily represent neuronal connectivity (Moore et al., 1970).

Estimation of taste response and best-stimulus type The number of spikes discharged during each stimulation period (6 s) was counted and averaged over eight or 10 trials for each tastant. In order to obtain the net taste response unaffected by somatosensory or thermal aspects of the taste solutions, the average response to water application was subtracted from the above average. The mean water response was calculated by averaging the number of spikes during the last 3 s of each 14 s water application for all water trials. The reason for using only the last portion of water application was to minimize the potential effects of persistent or off responses to taste stimuli carried over from the preceding trial in the initial portion of the water application. The significance of a response was examined by comparing the spikes during taste stimulation and those during distilled water application (t-test). P values ⬍0.05 were considered statistically significant. The best-tastant was defined as the most effective stimulus of four basic tastants for each neuron (Frank, 1973). In the present study, the best-tastant was determined by comparing statistically each neuron’s responses to the four tastants (t-test, P⬍0.05). For example, when a neuron’s response to NaCl stimulation was significantly stronger than that to any of the other three taste stimuli, the neuron was classified into the N-best type. On the other hand, when a neuron’s responses to NaCl and HCl stimulation were not statistically different from each other and were stronger than those to any of the other two tastants, the neuron was classified into the NH-best type (Table 1). Moreover the overall difference in the taste responses between the N-best,

T. Yokota et al. / Neuroscience 144 (2007) 314 –324

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Table 1. Mean response magnitude of taste-sensitive neurons Best-stimulus type

Number of neurons (%)

N-best neurons H-best neurons NH-best neurons Others Total

50 (42) 19 (16) 25 (21) 25 (21) 119

Number of spikes per s (mean⫾SE) NaCl

HCl

Quinine

Sucrose

5.1⫾0.7 0.8⫾0.3 2.0⫾0.7 0.3⫾0.4

0.6⫾0.2 3.9⫾0.6 2.0⫾0.7 0.4⫾0.2

⫺0.1⫾0.1 0.5⫾0.2 0.3⫾0.1 1.3⫾0.4

0.0⫾0.1 0.3⫾0.2 0.1⫾0.1 1.0⫾0.5

Mean response magnitude is the mean number of spikes per s during distilled water application subtracted from that during each taste stimulation. “Others” consists of neurons with the following best-tastants: Q, 2; S, 2; QN, 6; HS, 1; HQ, 1; QS, 1; NHS, 5; NQH, 3; SQN, 1; QHSN, 3. In parentheses, % of the total.

H-best and NH-best neuron groups was examined by the KruskallWallis test (significance level, P⬍0.001), and the paired comparisons between the groups were made by the Mann-Whitney U test (P⬍0.01). A neuron pair’s best-stimulus types were described as, for example N/H (N-best and H-best neuron pair).

Tuning breadth of taste response and correlative activity In order to examine the selectivity of taste responses and correlative activity, we used an equation for entropy (Smith and Travers, 1979). The breadth of responsiveness of taste responses (Htr) was defined as follows. 4

兺Pi log Pi

Htr⫽⫺1.661

i⫽1

where Pi is the number of spikes in response to the i th taste stimulus divided by the total number of spikes. The value of Htr ranges from 0 to 1.0. When a neuron was responsive to only one of the four stimuli, Htr⫽0. When a neuron was equally responsive to the 4 stimuli, Htr⫽1.0. When there was no response to one or more tastants, Pi log Pi was calculated as zero. The breadth of responsiveness of correlative activity (Hca) was defined as follows.

neurons were recorded simultaneously. Based on the injected-dye marks and in consultation with the brain atlas of Paxinos and Watson (1986), the locations of 115 of these 119 neurons were identified histologically as follows: the dysgranular (n⫽83), granular (n⫽21) and agranular (n⫽11) areas, distributed in the laminae V (n⫽53), IV (n⫽29), III (n⫽29) and II (n⫽4). The recording sites of the remaining four neurons were unavailable due to dye ejection failures. Best-stimulus types of individual neurons Of the 119 taste neurons, 94 (78%) were classified as N-, H- or NH-best types according to their most responsive stimuli (Table 1). The remaining neurons (n⫽25, 21%), i.e. Q-, S-best, etc., classified here as “others” were not analyzed further. Fig. 2A shows peri-stimulus time histograms of a representative N-best neuron, whose responses to NaCl were more intensive than those to the other three taste stimuli. An NH-best neuron responded equally to NaCl and HCl with minimal or no responses to quinine or sucrose (Fig. 2B). An H-best neuron responded exclusively to HCl (Fig. 2C).

4

兺Ci log Ci

Hca⫽⫺1.661

i⫽1

where Ci is the FC value for the i th taste stimulus divided by the total FC value. When a neuron pair synchronized during only one of the four stimuli, Hca⫽0. When a neuron pair synchronized equally during each of the four stimuli, Hca⫽1.0. When no peaks or troughs in CCs during one or more taste stimuli were detected, Ci log Ci was calculated as zero.

Statistical analysis Relative incidences of correlative activity during NaCl and HCl stimulations were examined using the ␹2-test. Comparisons of FC, tuning breadth of taste responses and correlative activities between pairs with different combinations of best-taste categories were performed using the Mann-Whitney U test. The numbers of peaks and/or troughs in homo- (N/N, H/H and NH/NH) and heterotype (N/NH, N/H and H/NH) pairs were examined by Fisher’s exact probability test. In these analyses, P values ⬍0.05 were considered statistically significant.

RESULTS A total of 119 taste neurons were recorded from the gustatory cortex in 42 animals. In each animal two to four

Incidence of correlative activities and combination of best-taste categories in paired neurons From the 94 neurons categorized as N-, H- or NH-best types, the following 63 pairs were obtained and analyzed: N/NH (21), N/N (17), NH/NH (8), N/H (8), H/NH (5) and H/H (4). Of these 63 pairs, 31 showed significant peaks and/or troughs in their CCs (CA-pairs: pairs with correlative activity). The incidence of correlative activity was defined as the number of CA-pairs divided by the total number of CA- and non-CA-pairs during NaCl or HCl stimulation (Fig. 3). Incidences depended on both the pair types and tastants as shown in Fig. 3. During NaCl stimulation, the N/N (10/ 17⫽0.59) or NH/NH (5/8⫽0.63) pairs tended to show a higher incidence of correlative activity than any of the N/NH (9/21⫽0.43), N/H (2/8⫽0.25), H/NH (1/5⫽0.20) and H/H (0/4⫽0) pairs (Fig. 3A). In contrast, during HCl stimulation the incidence of correlative activity in the N/N pairs was very low (2/17⫽0.12), and that in the NH/NH (4/8⫽0.50) or H/NH (3/5⫽0.60) pairs was higher (Fig. 3B). In the N/N pairs, the incidence of correlative activity (0.59) during NaCl stimulation was significantly higher than that (0.12) during HCl stimulation (P⬍0.05, Fisher’s exact probability

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T. Yokota et al. / Neuroscience 144 (2007) 314 –324

(A) N-best

(B) NH-best

spikes/bin 64

(C) H-best

-2 0

NaCl

6s

HCl

quinine

sucrose

Fig. 2. Peristimulus time histograms (PSTH) of representative neurons (N-, H- and NH-best neurons) during application of four tastants (NaCl, HCl, quinine, and sucrose). (A) N-best neuron. The response to NaCl is significantly larger than that to any of the other three tastants. (B) NH-best neuron. The responses to NaCl and HCl are equally effective and significantly larger than those to quinine or sucrose. (C) H-best neuron. The response to HCl is significantly larger than that to any of the other three tastants. Gray horizontal bars indicate taste stimulation periods: (in M) NaCl (0.2), sucrose (0.25), HCl (0.03), and quinine-HCl (0.005). Each taste solution was applied and followed by rinsing water. Each PSTH is based on 10 trials. Bin width is 200 ms.

test). In pairs with the other combinations of best-taste categories, no significant difference was observed in the incidence of correlative activity between NaCl and HCl stimulations (P⬎0.05, Fisher’s exact probability test). The average of INDs for the CA-pairs was 105 ␮m (range: 0 –270 ␮m), and that for the non-CA-pairs was 179 ␮m (range: 0 – 410 ␮m). As is apparent from Fig. 4A–D showing CCs of a representative N/N pair, the significant peak (arrowheads) was formed only during NaCl stimulation (Fig. 4A). In an NH/NH pair, significant peaks in CCs were observed during both NaCl and HCl stimulations (Fig. 4E, F). Correlative activities during NaCl or HCl stimulation periods In Fig. 5 the FC during NaCl or HCl stimulation are shown for the 31 neuron pairs with significant peaks and/or troughs in CCs. In this figure the neuron pairs are arranged in order of magnitude of FC from left to right in each combination of best-taste categories. During NaCl stimulation, FC values in the N/N (0.76⫾0.24 correlation counts/s: mean⫾S.E., n⫽10 pairs) or NH/NH (0.75⫾0.34, n⫽5) pairs tended to be higher than those in the N/NH (0.21⫾0.12, n⫽9), N/H (⫺0.12⫾0.06, n⫽3) or H/NH (⫺0.20⫾0.20, n⫽3) pairs (Fig. 5A). The FC in the N/N pairs during NaCl stimulation was significantly higher than that during HCl stimulation (Fig. 5A, B: P⬍0.05, MannWhitney U test). In the pairs with the other combinations of best-taste categories, no difference between NaCl and HCl stimulations was detected (P⬎0.05, Mann-Whitney U test). During HCl stimulation, the FC in the NH/NH (0.89⫾0.47, n⫽5) pairs was significantly higher than that in the N/N (0.24⫾0.23, n⫽10) pairs (Fig. 5B: P⬍0.05, Mann-Whitney U test). These results indicate that the correlative activities depended on the best-stimulus types of pairs, and the temporal activities changed according to taste qualities.

Selectivity of taste qualities in correlative activities The tuning breadth of taste responses in the N/N pairs was the narrowest (0.41⫾0.03: mean⫾S.E., n⫽20) and that in the NH/NH pairs the broadest (0.66⫾0.03, n⫽10) among all the combinations of best-taste categories (Table 2). Similarly, the tuning breadth of correlative activity was narrow in the N/N (0.36⫾0.08, n⫽10) pairs and broad in the NH/NH (0.65⫾0.06, n⫽5) pairs. In both taste responses and the correlative activity, the breadth of tuning in N/N pairs was significantly narrower than that in the NH/NH pairs (P⬍0.05, Mann-Whitney U test). In the N/N pair shown in Fig. 3A–D, the correlative activity occurred only during NaCl stimulation (Hca⫽0.19). The Htr was 0.30 and 0.34 for the two N-best neurons of the pair (Fig. 3A–D). These values indicate that the neurons responded to one or two tastants. However, in the NH/NH pair shown in Fig. 3E–H, the peaks were formed in CCs during NaCl and HCl stimulations (Hca⫽0.63), and the two NH-best neurons responded to two or four tastants (Htr⫽0.64, 0.58). The tuning breadth of the correlative activity was parallel with that of taste responses. Peak or trough formation, and best-stimulus types of neuron pairs Neuron pairs with correlative activity during NaCl or HCl stimulation were classified as homo- (N/N, H/H and NH/ NH) or hetero- (N/NH, N/H and H/NH) type (Table 3). Of 39 CCs with significant peaks, peaks were located around the center (time zero) in 34 cases (average peak width, 6 ms) and shifted away from the center in the remaining five cases (average peak width, 6 ms; average peak onset latency from time zero, 3 ms). This indicates that the coactivation of a neuron pair rather than one neuron driving the other neuron was a predominant feature of these

T. Yokota et al. / Neuroscience 144 (2007) 314 –324

16

0.8

12

0.6

8

0.4

0.2

4

Incidence of CA

Number of pairs

(A) NaCl

0

0 N/N

N/NH

N/H

NH/NH H/NH

H/H

16

0.8

12

0.6

8

0.4

4

0.2

Incidence of CA

Number of pairs

(B) HCl

0

0 N/N

N/NH

N/H

NH/NH H/NH

H/H

Combination of best tastes in neuron pairs Fig. 3. Frequencies and incidences of correlative activities (CAs) during (A) NaCl and (B) HCl stimulation. Combinations of best-taste types of paired neurons are shown on abscissa. (A) During NaCl stimulation, the number of CA-pairs and that of non-CA pairs are shown in black and white columns, respectively (left ordinate). The incidence of CA, i.e. the ratio of CA-pairs to the sum of CA- and non-CA pairs, is shown in solid line (right ordinate). (B) During HCl stimulation, the number of CA-pairs and that of non-CA pairs are shown in gray and white columns, respectively. The incidence of CA is shown in solid line (right ordinate).

CCs. Of six CCs with significant troughs, troughs were shifted away from the center in all six cases (average trough width, 8 ms; average trough onset latency from time zero, 3 ms). Fig. 6 shows the CCs of a homo-type (NH/NH pair) and a hetero-type (NH/H pair) derived from three simultaneously recorded neurons (a common reference neuron is marked with an asterisk in Fig. 6A–D and E–H). The salient peaks were formed in all the CCs of the NH/NH (homo-type) pair during the four taste stimulations (Fig. 6A–D, pair no. 23 in Fig. 5). In contrast, the CCs of the NH/H (hetero-type) pair contained marked troughs during NaCl and HCl stimulations (Fig. 6E, F, pair no. 30 in Fig. 5), but those during quinine and sucrose stimulations were less marked. The NH/NH pair had a significant peak duration of 2– 6 ms (Fig. 6A–D), while the NH/H pair had a significant trough duration of 6 ms with the onset latency of 1 ms (Fig. 6E, F). In the remaining pair (i.e. NH/H) from the three neurons in Fig. 6, no significant peak or trough was

319

detected (data not shown). The number of peaks in CCs were slightly more frequent in the homo-types (22 [56%] in Table 3) than in the hetero-types (17 [44%]). In contrast, most of the troughs were observed in the hetero-types (5 [83%]).

DISCUSSION Correlative activity and FC during NaCl and HCl stimulation Our data indicated that both the incidence of correlative activity and the FC depended on best-taste combinations. During NaCl stimulation, both of these indices in the N/N and NH/NH pairs were higher than those in the other combinations of best-taste categories (Figs. 4A, 5A). In contrast, the indices in the N/N pairs were very low during HCl stimulation (Figs. 4B, 5B). Salient correlative activities were observed frequently for the NH/NH pairs during HCl stimulation. The N/NH pairs were most frequently recorded among all pairs, and these paired neurons were commonly responsive to NaCl. However, both the incidence of correlative activity and the FC in the N/NH pairs were lower than those in the N/N or NH/NH pairs during NaCl stimulation. These results indicate that the correlative activities depend partly on taste-sensitive neuron types, and thus vary with taste qualities. Although previous studies (Yokota et al., 1997; Nakamura and Ogawa, 1997) reported that taste-sensitive pairs with correlative activity tended to belong to similar best-taste categories, this tendency was not strong. Since these studies had not examined the possible difference in response magnitude between NaCl and HCl stimulations, some of the N/H pairs in these studies might have been classified as NH/NH pairs. It seems likely, therefore, that the number of CA-pairs which belong to the same best-stimulus type was underestimated in these studies. In the N/N pairs, both the incidence of correlative activity and the FC during NaCl stimulation were significantly higher than those during HCl stimulation (Figs. 4A, B and 5A, B). These results suggest that a group of N-best neurons which fire synchronously is recruited during NaCl stimulation, and such synchrony is nearly lost during HCl stimulation. The cell assembly has been defined as a functional group of sparsely distributed neurons, under dynamic construction and reconstruction, that partially overlap with other assemblies (Sakurai, 1999; Wickelgren, 1999). Since correlated discharges in N/N pairs occurred mainly during NaCl stimulation, these synchronously firing neurons may serve as a cell assembly for salt taste coding. The present study also provides evidence supporting sparse overlapping coding for taste discrimination between salt and acid. As found for both the incidence of correlative activity and the FC, spike discharges of the NH/NH pairs synchronized during both NaCl and HCl stimulations (Fig. 3 and Fig. 5). Thus, the NH/NH pair can be considered as a common component of different cell assemblies during NaCl and HCl stimulations. It has been reported that individual neurons belong to different cell assemblies in the

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counts/bin

N/N

NH/NH

(A) NaCl

(E) NaCl

(B) HCl

(F) HCl

20 0

-10

counts/bin

46

0 -15

(C) quinine

(G) quinine

(D) sucrose

(H) sucrose

-50

0

#C142RT

0 #C030RM

+50 ms

Fig. 4. CCs of representative N/N and NH/NH pairs during taste stimulation. (A–D) N/N pair. A significant peak (⫹1⬃⫹5 ms) in CCs was formed only during NaCl stimulation (FC⫽0.55 correlation counts/s). The peak width is 4 ms. Entropy for correlative activities: Hca⫽0.19. (E–H) NH/NH pair. Significant peaks in CCs were formed during NaCl (FC⫽1.42) and HCl (2.44), but not during quinine or sucrose. (E) The peak ranges from ⫺5 to ⫹1 ms. The peak width is 6 ms. (F) The peak ranges from ⫺5 to ⫹5 ms. The peak width is 10 ms. The peak width is 2 ms. Hca⫽0.63. The zero baseline indicates the background level; the expected CCs have already been subtracted from the raw CCs. Arrowheads indicate bins forming significant peaks. Gray lines indicate the significance level (5%). Bin width is 2 ms.

visual (Arieli et al., 1995) and motor (Riehle et al., 1997) cortices, and whisker-related barreloids in the thalamus (Nicolelis et al., 1993). During NaCl stimulation, the mean of FC in the N/N (0.76⫾0.24) or NH/NH (0.75⫾0.34) pairs were higher than those in the N/NH, N/H or H/NH pairs (Fig. 5A). During HCl stimulation, however, the FC in the NH/NH (0.89⫾0.47) pairs was significantly higher than that in the N/N or N/NH pairs (Fig. 5B). The correlative activities in the homo-type pairs (N/N and NH/NH) were relatively more prominent than those in the hetero-type pairs (N/NH, N/H and H/NH), except for the H/H, of which only a small number of H/H pairs were identified. Two reasons for this observation may be noted: First, the percentage of H-best neurons in the present study (16%, 19 of 119 taste-sensitive neurons) was less than that in the previous studies (23%; Yamamoto et al., 1985). This may be due to the fact that H-best neurons were distinguished from NH-best neurons in the present study. Second, a low rate of encounters with H/H pairs may be due to the lack of aggregations of H-best neurons (i.e. their evenly sparse distributions) within the gustatory cortex (Yamamoto et al., 1985), which should increase the average INDs for these neurons.

Since HCl stimulation induced correlative activities more frequently in NH/NH pairs than in H/H pairs, NH-best neurons may play a predominant role in sour taste coding that requires synchronous discharges (e.g. temporal coding). In our previous study (Yokota and Satoh, 2001), the incidence of correlative activity was restricted to small INDs (⬍0.1 mm) and was higher when the paired neurons had similar taste profiles or temporal firing patterns. Therefore, we hypothesize that taste-sensitive neurons with correlative activity may serve as functional units in taste quality coding. These functional units may not be densely packed in the gustatory cortex, since taste-sensitive neurons were found intermingled with tactile or non-responsive neurons (Yamamoto et al., 1984; Kosar et al., 1986; Ogawa et al., 1992; Yokota et al., 1997; Wang and Ogawa, 2002) and the probability of encountering a taste-sensitive neuron, even in the gustatory cortex, is reported to be approximately 5% (Ogawa et al., 1992). Furthermore, the existence of columnar organization was suggested for mechanosensitive neurons, but not for taste-sensitive ones, in the gustatory cortex (Wang and Ogawa, 2002). In the present study, the high incidence of correlative activity in the N/N or NH/NH pairs was not due to the

T. Yokota et al. / Neuroscience 144 (2007) 314 –324

Table 3. Number of peaks or troughs during NaCl or HCl stimulation in CCs

FC (counts/s) 3 (A) NaCl

Best-stimulus type pairs

2

Homo-types (N/N, H/H or NH/NH) Hetero-types (N/NH, N/H or H/NH) Total

1 0 -1

H/H

(B) HCl

no.1

10 11

N/N

19 20 22 23

N/NH

27 28 30 31

N/H NH/NH H/NH

Combination of best-tastes in neuron pairs Fig. 5. Magnitude of FC during (A) NaCl and (B) HCl stimulation. Paired neurons are grouped by best-taste combinations and arranged in order of magnitude of FC (correlation counts/s) from left to right in each group. Neurons 1–10 are N/N pairs, neurons 11–19 N/NH pairs, neurons 20 –22 N/H pairs, neurons 23–27 NH/NH pairs, neurons 28 –30 H/NH pairs and neuron 31 an H/H pair. FC values in troughs are shown as negative. The black bar for one of the N/N pairs straddles the abscissa (zero line) because its CC had both a peak and a trough. The groups of neuron pairs are separated from each other by one empty space.

smaller INDs between these neuron pairs, because no significant difference in the mean INDs between any beststimulus types of pairs was observed (126 ␮m for N/N; 116 ␮m for NH/NH; P⬎0.05, Mann-Whitney U test [cf. 109 ␮m for all pairs]). Taste selectivity in N/N pairs In both taste responses and correlative activity, the breadth of tuning in the N/N pairs (mean⫾S.E., 0.41⫾0.03 and 0.36⫾0.08, respectively) pairs was significantly narrower than that in the NH/NH pairs (0.66⫾0.03 and 0.65⫾ 0.06, respectively; see Table 2). The N/N pairs showed high selectivity not only in taste responses to NaCl but also in the correlative activity (a sample pair in Fig. 3A–D). In hamsters, taste responses of CT nerves were narrowly tuned as compared with those of NTS (Travers and Table 2. Breadth of tuning for taste response and correlative activity Type pairs

N/N N/NH N/H NH/NH H/NH

321

Htr

Hca

Mean⫾SE

(n)

Mean⫾SE

(n)

0.41⫾0.03* 0.54⫾0.06 0.47⫾0.07 0.66⫾0.03* 0.58⫾0.03

(20) (18) (6) (10) (6)

0.36⫾0.08§ 0.59⫾0.08 0.59⫾0.19 0.65⫾0.06§ 0.41⫾0.08

(10) (9) (3) (5) (3)

*,§ P⬍0.05. Mann-Whitney U-test.

Peaks

Troughs

22 (56) N 15 H 7

1 (17) N 1 H 0

17 (44) N 9 H 8 39 N 24 H 15

5 (83) N 3 H 2 6 N4H2

In parentheses, % of the total.

Smith, 1979). Amiloride reduced the response to NaCl in N- and S-best neurons with no effects on H-best neurons in NTS, suggesting that N-fibers of CT nerves converged on S-best neurons in NTS (Boughter and Smith, 1998). In anesthetized or unanesthetized rats, taste-sensitive neurons (fibers) in CT nerves, NTS, PBN, and the gustatory cortex have been shown to be broadly tuned and the average entropy for these neurons was comparable (Yamamoto et al., 1984; Nishijo and Norgren, 1990; Nakamura and Norgren, 1991). CC studies can provide information addressing how particular response patterns are maintained or modified along the gustatory sensory pathways. Among NTS–PBN pairs with significant CCs, some showed a similar taste response but others showed different responses (Di Lorenzo and Monroe, 1997). These findings suggest that selective connections between neurons of the same taste-response types can explain only part of their response profiles. Moreover, in our preliminary study, amiloride pre-treatment reduced taste responses not only in N-best neurons but also in H-best or NH-best neurons in the gustatory cortex. Although the NH-best neurons resemble the E-type in taste profile, the NH-best neurons might have a different characteristic (partly amiloride sensitive) from the E-type (Ninomiya and Funakoshi, 1988) in the CT nerve. This observation suggests that the taste selectivity of responsive neurons tends to be reduced along the ascending pathway to the cortex (i.e. the NHbest neurons might receive inputs from both N-type and E-type), and that the NH-best neurons may not necessarily be connected only to those with the same taste response. However, it should be noted that in most of the N/N pairs the salient correlative activity occurred only during NaCl stimulation. This result suggests that convergent inputs from common source neurons with different taste responses are not so synchronous as to produce a peaked CC in target neuron pairs. The correlative activity of pairs of the same best-taste type may be functionally important in that precisely correlated firings can drive target cells efficiently (Alonso et al., 1996). N- and NH-best neurons may belong to separate cell assemblies, because both the incidence of correlative activity and the FC in the N/NH pairs tended to be lower than those in the N/N or NH/NH pairs. This suggests that common inputs to N-best and NH-best neurons are rather limited despite the fact that these two neuron types tend to coexist in close proximity (i.e. an average IND of 131 ␮m) within the gustatory cortex as indicated by their higher

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* NH /NH (A) NaCl

* NH /H (E) NaCl

(B) HCl

20

counts/bin

115

(F) HCl

0 0

-16

(C) quinine

(G) quinine

(D) sucrose

(H) sucrose

-50

0

#C140RM

0 #C140MT

+50 ms

Fig. 6. CCs of homo-type (NH/NH) and hetero-type (NH/H) pairs. These pairs were derived from three neurons recorded simultaneously. The NH-best neuron with an asterisk is the same (common) reference neuron. (A–D) homo-type (NH/NH) pair. The significant peaks (NaCl: ⫺5⬃⫹1, HCl and quinine: ⫺3⬃⫹1, sucrose: ⫺1⬃⫹1 ms: arrowheads) were located around time zero in all CCs during four taste stimulations. Prominent coactivation was detected in the CCs during NaCl and HCl stimulations (FC⫽1.72 and 1.52). Hca⫽0.88. (E–H) hetero-type (NH/H) pair. The significant trough (⫹1⬃⫹7 ms: between two arrowheads) in CCs were formed during HCl and NaCl stimulations (FC⫽⫺0.76 and ⫺0.61). Hca⫽0.55. The troughs in the right side during HCl (G) and NaCl stimulation (E) suggest a possibility that the NH-best neuron monosynaptically inhibited the H-best neuron. The zero baseline indicates the background level. Gray lines indicate the significance level (5%). Bin width is 2 ms.

encounter rate (21/63) than the other pair types. In our experiments, the taste responses of N-best neurons in the gustatory cortex were dependent on NaCl concentrations, while a few NH-best neurons tested were not (data not shown). A similar result was reported in which NaCl-generalists neurons, but not NaCl-specialists neurons, in the rat geniculate ganglion showed NaCl-concentration dependent responses (Lundy and Contreras, 1999). The existence of these different neuronal populations with the distinct response patterns for salt may allow complex processing of this taste quality. Relation between peak/trough in CCs and best-taste homology The number of peaks in CCs tended to be larger in the homo-types (22 [56%] in Table 3) than in the hetero-types (17 [44%]). This finding is consistent with our previous result that the homo-type pairs synchronized more frequently than hetero-type pairs (Yokota and Satoh, 2001). Correlative firings between neurons with best responses to

the same taste stimulus may represent a spatiotemporal pattern of multineuronal activities in the gustatory cortex, which facilitate the transmission of the same taste information to target neurons within and possibly beyond the gustatory cortex. In contrast, most of the troughs were found in the hetero-types (5 [83%] in Table 3). Troughs in CCs (an indicator of inhibition) between two neurons with different response properties have been observed in the visual cortex (Toyama et al., 1981) and auditory dorsal cochlear nucleus (Voigt and Young, 1990). Inhibitory connections have been suggested to enhance response contrast among neurons in the barrel cortex (Kyriazi et al., 1996; Brumberg et al., 1996) and in the gustatory NTS (Smith and Li, 1998). In Fig. 6 the reference (NH-best) neuron fired synchronously with a homo-type partner (NHbest), but inhibited a hetero-type partner (H-best) with a monosynaptic delay (1 ms) (Mason et al., 1991). In the gustatory cortex, the inhibitory influence was detected mainly between the hetero-type pairs, which may strengthen a contrast generated by excitatory correlative activities in the

T. Yokota et al. / Neuroscience 144 (2007) 314 –324

homo-type pairs. In previous studies, inhibitory inputs were reported to be concerned with the modification of taste responses (Ogawa et al., 1998), and neural activities in taste-sensitive neurons (fibers) were shown to be suppressed by the application of mixed taste solutions (Hyman and Frank, 1980a,b; Travers and Smith, 1984; London and Wehby, 1994). Moreover, Hasegawa et al. (2003) reported that neurons with the best taste response to both NaCl and HCl (probably corresponding to NH-best neurons in the present study) which were not suppressed by the mixed solutions may inhibit neurons with other taste properties. These observations suggest the existence of complex interactions between homo-type and hetero-type pairs during mixed taste stimulations. In normal food intake behavior, when afferent inputs of various tastes flow into the gustatory cortex, inhibitory circuits among neurons with different best-taste responses may play an important role in taste quality discrimination. Only a small number of troughs have been reported because of the low sensitivity of troughs in cross-correlation analysis (Melssen and Epping, 1987). Further research is required to elucidate the function of inhibitory interactions in the gustatory cortex. Acknowledgments—This work was partly supported by Grants-inAid from the Ministry of Education, Science, Sports and Culture of Japan to T.Y. We thank Dr. Katsunari Hiraba for valuable discussions and suggestions. Thanks are also due to Ms. Tomoe Kawamura and Ms. Kaori Ishii for their skillful technical contributions.

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(Accepted 12 September 2006) (Available online 19 October 2006)