Glutamate Receptor Antagonists Block Gustatory Afferent Input to the Nucleus of the Solitary Tract
Cheng-Shu Li and
David V. Smith
Department of Anatomy and Neurobiology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1509
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ABSTRACT |
Li, Cheng-Shu and David V. Smith. Glutamate receptor antagonists block gustatory afferent input to the nucleus of the solitary tract. J. Neurophysiol. 77: 1514-1525, 1997. The effects of excitatory amino acid (EAA) receptor antagonists in blocking the synaptic transmission between gustatory fibers of the chorda tympani (CT) nerve and taste-responsive neurons within the nucleus of the solitary tract (NST) were examined electrophysiologically in urethan-anesthetized hamsters. Single neurons in the NST were recorded extracellularly and drugs were microinjected into the vicinity of the cell with the use of a multibarrel pipette assembly. The activity of each cell was recorded in response to lingual stimulation with 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid, 0.032 M quinine hydrochloride, and/or 25-µA anodal current pulses. Once a cell was identified as a taste-responsive neuron, one or more EAA receptor antagonists were administered by microinjection. Approximately 27 nl of 50 mM kynurenic acid (KYN), a broad-spectrum EAA receptor antagonist; 0.5 or 2.0 mM DL-2-amino-5-phosphonovalerate (APV), an N-methyl-D-aspartate (NMDA) receptor antagonist; 0.05 or 0.5 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), an
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist; or phosphate-buffered physiological saline was applied to the neuron. Responses to chemical stimulation of the anterior tongue were obtained before and after drug administration and again after recovery; responses to anodal current stimulation (0.1 Hz) were obtained continually throughout the drug administration protocol. Microinjection of KYN completely and reversibly abolished responses elicited by both anodal current and chemical stimulation of the anterior tongue. The excitatory responses of cells in the NST to chemical and electrical stimulation of the anterior tongue were also completely and reversibly blocked by CNQX, implicating the involvement of an AMPA/kainate receptor. Microinjection of APV was generally less effective and partially reduced the responses of some taste-responsive NST cells to chemical stimulation of the anterior tongue. There were no effects following microinjection of a 27-nl bolus of phosphate-buffered saline. None of these EAA receptor antagonists had a differential effect on responses to different taste stimuli. The responses to all tastants were completely blocked by both KYN and CNQX; there was no apparent relationship between the response to any particular tastant and the limited effects of APV. These data implicate glutamate as an excitatory neurotransmitter between CT gustatory fibers and taste-responsive NST cells and suggest that it acts primarily on AMPA/kainate receptors, with some contribution from NMDA receptors. This conclusion is strengthened by other data obtained from in vitro slice preparations, which show that responses of cells in the rostral NST to solitary tract stimulation are blocked by both NMDA and AMPA/kainate receptor antagonists.
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INTRODUCTION |
The rostral portion of the nucleus of the solitary tract (NST) receives topographically organized input from gustatory afferent fibers of the facial, glossopharyngeal, and vagal nerves that terminate in a rostral-caudal sequence (Allen 1923
; Åström 1953
; Beckstead and Norgren 1979
; Bradley et al. 1985
; Hamilton and Norgren 1984
; Hanamori and Smith 1989
). The taste receptors in the fungiform papillae on the anterior portion of the tongue are innervated by the chorda tympani (CT) branch of the facial nerve (Contreras et al. 1982
; Hamilton and Norgren 1984
). Hamster CT gustatory fibers are most responsive to sucrose and NaCl, although they respond to acids and bitter-tasting stimuli as well, particularly at stronger concentrations (Frank 1973
; Frank et al. 1988
; Smith and Frank 1993
). The fibers of the CT terminate onto second-order neurons in the rostral portion of the NST. Taste-responsive neurons of the NST receive converging input from separate subpopulations of taste buds (Sweazey and Smith 1987
; Travers et al. 1986
) and these neurons are more broadly tuned to gustatory stimuli of different qualities than fibers of the CT nerve (Smith and Travers 1979
; Travers and Smith 1979
).
There is some evidence to suggest that glutamate may be an excitatory neurotransmitter between afferent fibers and taste-responsive neurons of the NST. Recent in vitro experiments in rat brain slices have shown that the responses of cells in the gustatory region of the NST to electrical stimulation of the solitary tract are reduced or blocked by antagonists of both N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors (Wang and Bradley 1995
). However, the rostral NST also contains neurons responsive to tactile and thermal stimuli (Travers 1993
; Travers and Norgren 1995
) and so these in vitro data do not directly link excitatory amino acids (EAAs) to gustatory synaptic processing. Although brain slice preparations are ideal for biophysical and pharmacological studies, it is difficult to relate these findings to the gustatory responsiveness of NST neurons because the slices are disconnected from the taste receptors. The present in vivo recording experiments were designed to test the hypothesis that glutamate mediates synaptic transmissionbetween CT gustatory fibers and taste-sensitive neurons inthe NST.
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METHODS |
Animals and surgery
Young adult male hamsters, Mesocricetus auratus (120-180 g) were deeply anesthetized with urethan (1.7 g/kg ip) and additional anesthetic was given as needed during the course of each experiment. The animal was tracheotomized and mounted in a stereotaxic instrument with the use of a nontraumatic head holder (Erickson 1966
). The snout was angled downward 27° from horizontal to straighten the brain stem and minimize brain movement associated with breathing (Smith et al. 1979
). Body temperature was monitored and maintained at 37°C with a heating pad. A sagittal skin incision was made though the midline overlying the posterior skull and a portion of the occipital bone just dorsal to the foramen magnum was removed to reveal the cerebellum. After the dura was removed, the posterior portion of the cerebellum was aspirated to expose the floor of the fourth ventricle for 3-4 mm anterior to the obex.
Single-unit recording and stimulation
Multibarrel glass micropipette assemblies were used for recording and microinjection of pharmacological agents. The pipettes for extracellular recording (tip diameter 2 µm, resistance 7-10 M
) were filled with 2% (wt/vol) solution of Chicago Blue dye (Sigma Chemical) in 0.5 M sodium acetate. One-barrel or three-barrel glass pipettes (tip diameter ~35 µm) were glued to the recording electrode for microinjection of drugs; the tip of the recording electrode extended 100-120 µm beyond the microinjection pipette. The mean coordinates for taste cell recordings were 2.1 ± 0.37 (SD) mm anterior and 1.2 ± 0.23 mm lateral to obex and between 0.5 and 1.0 mm ventral to the surface of the brain stem. Histological reconstruction of three electrode penetrations showed the electrode tip to be within the NST, medial to the solitary tract; recording sites were not systematically reconstructed. Extracellular potentials were differentially amplified (Bak MDA-4 I), discriminated with a dual time-amplitude window discriminator (Bak DDIS-1), displayed on oscilloscopes, and monitored with an audio monitor. The amplified action potentials were recorded along with voice cues on VCR tape. An IBM computer, configured with Lab Master DMA board (Scientific Solutions) and custom software, controlled chemical stimulus delivery and on-line data acquisition and analysis.
Taste responses were initially identified by a change in neural activity associated with the application of anodal current pulses (50 µA, 0.5 s, 0.33 Hz) to the anterior tongue and confirmed by responses to chemical stimulation of the tongue. In the experiment that examined the effect of EAA antagonists on the responses of NST neurons to anodal tongue stimulation, an anodal current pulse (25 µA, 0.5 s) was delivered to the anterior tongue every 10 s throughout the course of the experiment (Smith and Bealer 1975
). For experiments with taste solutions, the anterior tongue was stimulated with 0.032 M sucrose, 0.032 M sodium chloride, 0.032 M quinine hydrochloride (QHCl), and 0.0032 M citric acid. These concentrations evoke approximately equal multiunit taste responses in the hamster NST (Duncan and Smith 1992
). The tastants were delivered by a gravity flow system composed of a two-way solenoid-operated valve connected via tubing to a distilled water rinse reservoir and a stimulus reservoir. The stimulation sequence, during which the computer acquired data, was a continuous flow initiated by the delivery of 5 s of distilled water, followed by 10 s of stimulus, followed by 5 s of distilled water. The flow rate was 2 ml/s. After each chemical stimulation, the tongue was rinsed with distilled water (>50 ml) and individual stimulations were separated by
2 min to avoid adaptation effects (Smith et al. 1978
).
Pharmacology and microinjections
Each EAA antagonist to be tested was dissolved in buffered physiological saline and microinjected into the NST through the multibarrel pipette assembly. Pressure pulses (30 psi, 10 ms) from a Picospritzer were used to trigger the drug injection; drug volume was estimated from the areas of a series of known volumes (10-40 nl) injected onto filter paper. When drugs were delivered from a single-barrel pipette with a tip diameter of 35 µm, drug volume was 27.0 ± 2.5 (SD) nl, as determined by measuring the volumes produced by a series of 10 pipettes. In some experiments, a triple-barrel microinjection pipette was used, which was also broken to a tip diameter of 35 µm; the 30-psi, 10-ms pressure pulse produced a volume of 20.7 ± 2.3 nl per barrel from these pipettes (n = 11). Kynurenic acid (KYN), a broad-spectrum EAA antagonist (Perkins and Stone 1982
), was delivered at a pipette concentration of 50 mM (pH 7.4 ± 0.1). The NMDA receptor antagonist DL-2-amino-5-phosphonovalerate (APV) (Watkins 1981
) was delivered at pipette concentrations of 0.5 or 2.0 mM (pH 7.4 ± 0.1). The AMPA/kainate receptor antagonist 6-cyano-7-nitroquinoxaline2-,3-dione (CNQX) (Honore et al. 1988
) was microinjected at a pipette concentration of 0.05 or 0.5 mM (pH 8.0 ± 0.1). KYN and APV were obtained from Sigma Chemical and CNQX was obtained from Tocris Cookson.
Data analysis
The responses of each cell to chemical stimulation of the tongue were accumulated over three consecutive time periods: 1) 5 s of prestimulus water rinse, 2) 10 s of stimulus flow, and 3) 5 s of poststimulus water rinse. The net response was calculated as the number of action potentials during the first 5 s of chemical stimulation minus the number of action potentials during the 5-s prestimulus water rinse (Vogt and Smith 1993
). An effective chemical response was defined as an unadjusted impulse frequency that exceeded twice the spontaneous discharge rate measured during the 5-s prerinse. To compare the effects of the pharmacological manipulations, the means of all effective responses to chemical stimulation in the control condition were compared with the mean to the same stimuli in the same cells under the drug condition. For the single-barrel experiments, the control condition was tastant stimulation without microinjection; none of the cells that were tested with 27-nl saline injections showed any change in their firing rates. For the triple-barrel experiments, the responses during microinjection of the vehicle were used for statistical comparison (paired t-tests). The responses to all effective stimuli (i.e., responses to sucrose, NaCl, citric acid, and/or QHCl) were combined to provide an adequate number for statistical comparison. As seen in the responses of individual cells (Figs. 2-4, 6, and 7), the different tastants were not differentially affected by these agents. For statistical comparison of the effects of these agents on the responses of cells to anodal current stimulation, the mean firing rate in each cell over a 2-min period before drug administration was compared with the mean firing rate during a comparable period following the drug. These differences were statistically analyzed with the use of a Wilcoxon signed-ranks test because of the relatively small number of cells tested under each condition (n = 5 or 7). The Wilcoxon test was also used to compare the effects of the drugs on spontaneous activity of the cells because these distributions were highly skewed.

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| FIG. 2.
Peristimulus-time histograms of the responses of a single NST neuron to 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid, and 0.032 M quinine hydrochloride (QHCl) before and immediately after microinjection of a single 27-nl pulse of 50 mM kynurenic acid into the vicinity of the cell and again 40 min after the injection. A distilled water rinse was applied to the anterior tongue during the 5 to 0 s interval and again at the 10-s point; the stimulus flowed from 0 to 10 s.
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| FIG. 6.
Peristimulus-time histograms of the responses of a single NST neuron to 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid, and 0.032 M QHCl before (Control) and immediately after microinjection of a single 21-nl pulse of the saline vehicle, 2.0 mM APV, or 0.5 mM CNQX into the vicinity of the cell; another control stimulation was given ~20 min after the last drug injection. A distilled water rinse was applied to the anterior tongue during the 5 to 0 s interval and again at the 10-s point; the stimulus flowed from 0 to 10 s. This cell responded exclusively to sucrose; its response was eliminated by CNQX and reduced somewhat by APV.
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| FIG. 7.
Peristimulus-time histograms of the responses of a single NST neuron to 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid, and 0.032 M QHCl before (Control) and immediately after microinjection of a single 21-nl pulse of the saline vehicle, 2.0 mM APV, or 0.5 mM CNQX into the vicinity of the cell; another control stimulation was given ~20 min after the last drug injection. A distilled water rinse was applied to the anterior tongue during the 5 to 0 s interval and again at the 10-s point; the stimulus flowed from 0 to 10 s. This cell responded to NaCl, citric acid, and QHCl, but not to sucrose; its response was eliminated by CNQX but APV had no measurable effect.
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RESULTS |
Effects of KYN, CNQX, and APV on the responses of NST cells to anodal current stimulation of the anterior tongue
Electrical stimulation of the tongue through an anode at 1-50 µA produces responses in gustatory fibers of the CT nerve by the activation of electrolyte-sensitive taste receptor mechanisms (Herness 1987
; Smith and Bealer 1975
). Therefore we used anodal stimulation of the tongue to repetitively drive gustatory input to the NST to investigate the time course of the effects of several EAA antagonists on this peripherally evoked response.
To examine the hypothesis that glutamate mediates synaptic transmission between first-order gustatory fibers and taste-responsive NST cells, we first tested the influence of KYN, a broad-spectrum EAA antagonist (Perkins and Stone 1982
). Microinjection of 27 nl of KYN from a single-barrel pipette at a concentration of 50 mM completely blocked the responses of NST neurons to anodal current stimulation of the anterior tongue in all seven cells tested. Across seven cells, the mean firing rate over a 2-min period before drug injection was 0.92 imp/s, which decreased significantly to 0.01 imp/s over a 2-min period at the peak of the effect of KYN (Wilcoxon signed-ranks test, Z = 2.37, P = 0.018). The responses to anodal current began to reappear 15-20 min after KYN microinfusion and completely recovered by 25-30 min after drug administration. The time course of the response of an NST unit to 0.1-Hz anodal current stimulation before and after administration of 50 mM KYN is shown in Fig. 1A; the response of another cell (5 tested) during vehicle injection is shown in Fig. 1C. Administration of vehicle alone had no effect on any of the five cells tested; the mean firing rate of these cells was 1.41 imp/s before vehicle and 1.28 imp/s after (Z = 0.73, P = 0.47).

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| FIG. 1.
Impulse histograms of the responses of 4 rostral nucleus of the solitary tract (NST) neurons to repetitive anodal current stimulation of the anterior tongue (25 µA, 0.5 s, 0.1 Hz) before and after microinjection of 50 mM kynurenic acid (A), 0.5 mM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (B), buffered saline (C), or 2.0 mM DL-2-amino-5-phosphonovalerate (APV) (D). Drug application began at the arrow and consisted of a single pulse with a volume of ~27 nl microinjected 120 µm dorsal to the tip of the recording electrode.
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To begin to determine the subtype of postsynaptic EAA receptors, we infused CNQX, a selective non-NMDA (AMPA/kainate) receptor antagonist (Honore et al. 1988
), adjacent to the recorded cells of the NST. The application of 0.5 mM CNQX totally abolished the responses to anodal current stimulation of the anterior tongue in all five cells tested. Across five cells, the mean firing rate over a 2-min period before drug injection was 3.16 imp/s, which decreased significantly to 0.14 imp/s over a 2-min period at the peak of the effect of CNQX (Wilcoxon signed-ranks test, Z = 2.02, P = 0.043). The anodal current-evoked responses began to recover in ~5 min and totally recovered 10-15 min after the CNQX infusion, as shown by the response of one such cell in Fig. 1B. In contrast, the application of 2.0 mM APV, a selective NMDA receptor antagonist (Watkins 1981
), produced no detectable change in the responses of five NST cells to anodal current stimulation at 0.1 Hz; the mean firing rate of these cells was 4.14 imp/s before vehicle and 4.03 imp/s after (Z = 0.0, P = 1.0). The response of one cell during microinjection of 2.0 mM APV is shown in Fig. 1D.
Effects of KYN, CNQX, and APV on the responses of NST cells to chemical stimulation of the anterior tongue
In the second experiment we tested the influence of EAA receptor antagonists delivered from single-barrel microinjection pipettes on the responses of NST cells to chemical stimulation of the anterior tongue. The responses of NST neurons to NaCl, sucrose, citric acid, and QHCl were all completely and reversibly blocked by administration of 50 mM KYN in all 18 cells tested. The responses evoked by all four tastants in a broadly tuned NST cell are depicted in Fig. 2. The responses to all tastants were blocked by KYN in this cell. In fact, all taste-evoked responses were blocked in all cells. The means of 34 responses to stimulation with four basic tastants in 18 cells before and after the KYN application are shown in Fig. 3A. These means included 13 responses to NaCl, 3 to sucrose, 6 to citric acid, and 12 to QHCl. The mean difference in firing rate before and after KYN administration was statistically significant (2-tailed paired t-test, t = 7.169, df = 33, P < 0.0001). Although not producing a striking effect in the cell shown in Fig. 2, KYN also significantly reduced the spontaneous activity occurring in the initial 5 s before stimulus application. Across the same 18 neurons, the mean spontaneous activity was 3.82 imp/s before KYN and was reduced to 2.15 imp/s after KYN (Wilcoxon signed-ranks test, Z = 3.615, 2-tailed P < 0.001).

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| FIG. 3.
Mean ± SE of the responses to chemical stimulation of the anterior tongue before, during, and after recovery from several excitatory amino acid (EAA) antagonists. Responses in each graph are the average of all responses evoked in several neurons by the 4 taste stimuli (NaCl, sucrose, citric acid, and QHCl); only those responses that were greater than twice the spontaneous discharge rate during the control condition were included in these means (see text). The number of responses included in each mean is indicated in the figure; the number of neurons and the number of responses to each stimulus are indicated in the text. Asterisks: significant differences between control responses and those following microinjection of the antagonist (see text for details).
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Microinjection of 0.5 mM CNQX completely and reversibly blocked the 20 tastant-induced responses in all 10 NST cells tested with the use of single-barrel microinjection pipettes (Fig. 3E); this included 5 responses to NaCl, 2 to sucrose, 5 to citric acid, and 8 to QHCl. The mean responses before and after 0.5 mM CNQX administration were significantly different (2-tailed paired t-test, t = 4.144, df = 19, P < 0.001). The responses of one such cell to NaCl, sucrose, citric acid, and QHCl are shown in Fig. 4. This cell responded to NaCl and QHCl but not to sucrose or citric acid. CNQX blocked not only the responses to chemical stimulation of the tongue but also effectively blocked the spontaneous firing in the absence of chemical stimulation (compare times
5 through 0, Fig. 4). Across all 10 cells, mean spontaneous activity was reduced from 3.45 to 0.12 imp/s after 0.5 mM CNQX (Wilcoxon signed-ranks test, Z = 2.712, 2-tailed P < 0.01). At a reduced pipette concentration (0.05 mM), CNQX produced no effect on 18 responses evoked in six NST cells (Fig. 3D): 5 responses to NaCl, 6 to sucrose, 4 to citric acid, and 3 to QHCl (2-tailed paired t-test, t = 0.597, df = 17, P = 0.558).

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| FIG. 4.
Peristimulus-time histograms of the responses of a single NST neuron to 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid, and 0.032 M QHCl before and immediately after microinjection of a single 27-nl pulse of 0.5 mM CNQX into the vicinity of the cell and again 15 min after the injection. A distilled water rinse was applied to the anterior tongue during the 5 to 0 s interval and again at the 10-s point; the stimulus flowed from 0 to 10 s.
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Administration of APV at the same concentration at which CNQX produced a robust reduction in the responses of NST cells (0.5 mM) produced no effect on the 20 taste-evoked responses of six NST neurons (Fig. 3B): 6 responses to NaCl, 4 to sucrose, 5 to citric acid, and 5 to QHCl. However, there was some indication of an effect of APV when the pipette concentration was increased to 2.0 mM. As shown in Fig. 3C, the mean of 16 responses (6 to NaCl, 2 to sucrose, 3 to citric acid, and 5 to QHCl) in seven cells was reduced somewhat after 2.0 mM APV administration, although this difference was not statistically significant (2-tailed pairedt-test, t = 1.720, df = 15, P = 0.105). However, closer inspection of these data showed that of the 16 taste-evoked responses in these seven cells, 10 showed a decrease after 2.0 mM APV and 8 of these decreases were >40%; of these, there were 4 responses to NaCl, 1 to sucrose, 2 to citric acid, and 1 to QHCl. The mean of these eight responses was significantly less after APV administration (1-tailed paired t-test, t = 2.210, df = 7, P < 0.05). The responses of one such cell are shown in Fig. 5. This cell responded somewhat to all four tastants and the response to each was partially (>40%) and reversibly reduced by 2.0 mM APV.

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| FIG. 5.
Peristimulus-time histograms of the responses of a single NST neuron to 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid, and 0.032 M QHCl before and immediately after microinjection of a single 27-nl pulse of 2.0 mM APV into the vicinity of the cell and again 20 min after the injection. A distilled water rinse was applied to the anterior tongue during the 5 to 0 s interval and again at the 10-s point; the stimulus flowed from 0 to 10 s. The response to each of these stimuli was reduced by >40%.
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Triple-barrel pipettes: effects of CNQX and APV on the same cells
The results presented above were all obtained with the use of single-barrel pipettes to deliver EAA antagonists to the NST, limiting our studies to a single antagonist per cell. We also used a triple-barrel pipette, which allowed us to present more than one agent to a recorded cell. We employed this triple-barrel assembly to address the question of whether the synaptic responses of individual taste-responsive NST cells consist of both AMPA/kainate and NMDA receptor components. For this experiment, a three-barrel micropipette, broken to obtain a tip diameter of ~35 µm, was glued 120 µm away from the tip of the recording electrode. The three barrels were filled with 0.5 mM CNQX, 2.0 mM APV, or buffered physiological saline, respectively.
As in the single-barrel experiments, microinjection of 0.5 mM CNQX into the NST completely and reversibly abolished the responses of NST cells to chemical stimulation of the anterior tongue. The responses of two different NST cells to sucrose, NaCl, citric acid, and QHCl under several drug conditions are shown in Figs. 6 and 7. The responses in the absence of drug infusion (control) are shown before and after the series of drug microinjections. The responses during vehicle administration are also shown for each cell. For both cells (Figs. 6 and 7) the responses to all tastants were completely eliminated after 0.5 mM CNQX administration. The effects of 2.0 mM APV were, as in the single-barrel experiments, more variable. For the sucrose-sensitive cell in Fig. 6, CNQX eliminated the response to sucrose and APV produced a partial suppression of this response, although not as dramatically as in the cell shown in Fig. 5. The cell depicted in Fig. 7 responded well to citric acid, QHCl, and NaCl. All of these responses were eliminated by CNQX; APV had no discernible effect. The responses of both cells were the same during vehicle administration as in control trials in which no drug was administered.
The responses to all four tastants were examined under these drug conditions in 10 NST neurons. These stimuli produced 16 responses in these cells: 8 to NaCl, 2 to sucrose, 3 to citric acid, and 3 to QHCl. The means of these 16 responses under each drug condition are shown in Fig. 8. Again, 0.5 mM CNQX produced a complete elimination of the response to chemical stimulation of the tongue compared either with the initial control response (2-tailed paired t-test, t = 3.33, df = 15, P < 0.005), with the response during vehicle administration (t = 3.17, P < 0.01), or with the control response after recovery (t = 3.38, P < 0.005). As in the single-barrel experiments, 2.0 mM APV did not produce overall a significant decrement in the evoked responses to chemical stimulation; compared with the vehicle, 8 of 16 responses were reduced by 2.0 mM APV, although onlyone response (to sucrose) was reduced by >40%. Overthese eight APV-sensitive responses, the average reduction was 20.7%.

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| FIG. 8.
Mean ± SE of 16 responses to chemical stimulation of the anterior tongue before, during, and after recovery from several EAA antagonists in 10 NST neurons. Only those responses that were greater than twice the spontaneous discharge rate during the control condition were included in these means (see text). The number of responses to each stimulus (NaCl, sucrose, citric acid, or QHCl) are indicated in the text. Asterisks: significant differences between control (or vehicle or recovery) responses and those following microinjection of the antagonist (see text for details).
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DISCUSSION |
Glutamatergic excitatory transmission onto taste-responsive cells
The results presented here suggest strongly that glutamate is a major excitatory neurotransmitter between gustatory fibers in the CT nerve and taste-responsive neurons in the NST. Microinjection of KYN or CNQX into the NST completely and reversibly blocked excitatory input stimulated either by anodal current or gustatory stimuli applied to the anterior tongue. The blocking effect of CNQX or KYN on taste-evoked activity in the NST completely recovered 15-30 min after application of the antagonist and there was no effect of the vehicle, indicating that the reduced discharge in these cells was not due to other factors such as pressure or toxicity. This finding suggests that glutamate release from CT afferent fibers mediates the synaptic transmission between these fibers and taste-responsive NST cells. Although we have not determined that the input to these cells is monosynaptic and cannot rule out the possibility that these antagonists are acting on glutamatergic synapses between medullary interneurons and the recorded cells, it is unlikely that every cell recorded from the NST would be separated from the CT afferent terminals by an interneuron. Nevertheless, a glutamatergic synapse is clearly located somewhere between the afferent input and every taste-responsive cell recorded from the NST, because the responses of all cells were blocked by glutamate receptor antagonists. Thus the present results suggest that glutamate may be an excitatory neurotransmitter at this first central gustatory synapse. Further experiments are needed to show that glutamate meets the requirements for a neurotransmitter at this synapse, including the presence of glutamate in primary afferent fibers and its release after CT nerve stimulation.
Whole cell recording in an in vitro slice preparation has also implicated glutamate in excitatory transmission between solitary tract fibers and neurons in the rostral gustatory portion of the rat NST (Wang and Bradley 1995
). Excitatory postsynaptic potentials produced by electrical stimulation of the solitary tract were blocked or reduced by both AMPA/kainate and NMDA receptor antagonists. Electrical stimulation of the solitary tract would undoubtedly drive taste-sensitive afferent input to the rostral NST, but axons in the solitary tract carry tactile and temperature information as well (Travers 1993
; Travers and Norgren 1995
). The sensory properties of these afferent fibers are not determinable in an in vitro preparation. However, the present in vivo experiment makes it possible to implicate glutamate in excitatory transmission between afferent CT fibers responding to taste stimulation of the tongue and known taste-responsive NST neurons. All taste-evoked activity in these NST cells was eliminated by KYN or CNQX and was often reduced by APV. In addition to activity evoked by chemical or anodal stimulation of the tongue, the spontaneous activity of cells in the NST was also reduced by KYN and CNQX. The almost complete elimination of activity by CNQX (Figs. 4, 6, and 7) suggests that most of the excitatory input to these cells is mediated via glutamate. Some of that excitation may arise from peripheral fibers, because it has been shown that treatment of the tongue with amiloride, which blocks taste receptor Na+-channel activity, reduces the spontaneous discharge of NST neurons by ~50% (Smith et al. 1996
).
Different gustatory fiber types within the CT do not appear to employ different excitatory neurotransmitters or receptors at the level of the NST, i.e., CNQX affected all NST responses similarly. Many individual neurons in the NST respond to taste solutions representing more than one taste quality (McPheeters et al. 1990
; Travers and Smith 1979
), as is often the case in single afferent taste fibers (Frank 1973
; Frank et al. 1988
; Smith and Frank 1993
). Neurons in the hamster NST are more broadly tuned than the peripheral fibers of the CT nerve (Smith and Travers 1979
; Travers and Smith 1979
). These and other data showing convergence onto NST cells from different subpopulations of taste buds (Sweazey and Smith 1987
; Travers and Norgren 1991
; Travers et al. 1986
) suggest that individual neurons in the NST receive afferent input from two or more different peripheral nerve fibers. Microinjection of CNQX into the NST abolished the responses to stimuli representing each of the four basic taste qualities. These data suggest that all of the gustatory neuron types in the NST are excited via AMPA/kainate receptors regardless of their profiles of taste sensitivity or patterns of convergence.
Glutamate receptor subtypes
EAA receptors can be categorized into several subtypes, which include the ligand-gated ion channel (iontotropic) receptors: the NMDA and AMPA/kainate receptors (Watkins et al. 1990
). Fast excitatory synaptic transmission at many sites within the CNS is mediated by iontotropic glutamate receptors. APV is considered to be a specific antagonist for the NMDA receptor (Davies and Watkins 1982
; Evans et al. 1982
), whereas CNQX blocks AMPA/kainate receptors (Honore et al. 1988
).
The reversible elimination of the responses of NST neurons to both anodal current and chemical stimulation of the tongue by the selective AMPA/kainate receptor antagonist CNQX provides direct evidence that AMPA/kainate receptors are involved in the transmission of taste information within the NST. Microinjection of APV, an NMDA receptor antagonist, reduced the responses of some taste-responsive NST neurons to chemical stimulation of the anterior tongue; it was ineffective for many other responses. In at least one instance (Fig. 6), both CNQX and APV reduced the responses of the same cell. In whole cell recordings from rostral NST cells in a rat slice preparation, both CNQX and APV were effective in reducing the responses of these cells (Wang and Bradley 1995
); CNQX blocked or reduced excitatory postsynaptic potentials (EPSPs) and APV reduced them in NST cells maintained in normal Mg2+ concentrations. The results of the present investigation are similar to those obtained in vitro, except for the relative effectiveness of these antagonists. One limitation of this kind of experiment in vivo is the unknown concentration of the drugs at the recorded cell. We determined the pipette concentrations empirically: 0.05 mM CNQX was without effect and 0.5 mM produced a complete block of NST cell activity, whereas 0.5 mM APV was without effect and 2.0 mM APV was partially effective. It is possible that a pipette concentration of 0.5 mM CNQX produces a dose at the cell that is strong enough to block both AMPA/kainate and NMDA receptors (Kessler et al. 1989
) and that 2.0 mM APV results in a minimally effective dose for the NMDA receptor. Thus it is difficult in vivo to reveal the relative contribution of these two receptor subtypes in gustatory afferent transmission. However, these data and those obtained in vitro from the rat (Wang and Bradley 1995
) suggest that both receptor types are involved.
In the rat slice preparation (Wang and Bradley 1995
), in Mg2+-free saline, APV blocked a CNQX-resistant component of the EPSP, suggesting that excitatory transmission in some neurons in the rostral NST involves both NMDA and AMPA/kainate receptor activation. In our in vivo preparation, we saw some evidence for both receptors on the same cell (Fig. 6), but it is probably more difficult to see the NMDA component in these responses because the NMDA channel would likely be blocked by Mg2+ except at high rates of impulse discharge. Combined with the possibility that the dose of APV might have been somewhat low, it is difficult to draw quantitative conclusions about the role of the NMDA receptor in these data. Clearly, however, the AMPA/kainate receptor plays a major role in the transmission between CT afferent fibers and rostral NST gustatory-responsive cells.
The role of NMDA receptors in synaptic transmission has been well described in the hippocampus (Collingridge and Bliss 1987
; Madison et al. 1991
). NMDA receptor-mediated currents generally do not contribute appreciably to spikes elicited by low levels of synaptic input, but instead are recruited only when the postsynaptic cell depolarizes beyond a critical threshold (Feldman and Knudsen 1994
). The presence of Mg2+ at normal membrane potentials has been shown to suppress NMDA receptors (Mayer et al. 1984
; Nowak et al. 1984
) and suprathreshold depolarization relieves NMDA receptor channels of a chronic, voltage-sensitive Mg2+ blockade (Ascher and Nowak 1988
; Feldman and Knudsen 1994
; Nowak et al. 1984
). Until this blockade is overcome, NMDA receptors contribute little, if at all, to normal synaptic transmission in the hippocampal slice. A relatively minor role for NMDA receptor currents has been observed in both in vitro and in vivo studies of several sensory systems (Artola and Singer 1987
; Herron et al. 1986
; Hickmott and Constantine-Paton 1993
; Roberts et al. 1991
; Sutor and Hablitz 1989
; Udin et al. 1992
), although there is now evidence that NMDA receptors may make a larger contribution to sensory transmission in the neocortex (Armstrong-James et al. 1993
; Miller et al. 1989
).
Glutamatergic transmission in the caudal NST and the olfactory bulb
Synaptic transmission between primary vagal afferent fibers and second-order cells in the NST may be mediated by glutamate (Andresen and Yang 1990
, 1994
; Drewe et al. 1990
; Felder and Mifflin 1994
; Glaum and Miller 1992
; Vardhan et al. 1993
). Several EAA agonists, including kainate, quisqualate, and NMDA, elicit cardiovascular responses when injected into the medial NST, mimicking the effects of stimulation of baroreceptor afferent fibers, and these responses are blocked by the appropriate antagonists (Guyenet et al. 1987
; Kubo and Kihara 1988
; Le Galloudec et al. 1989
; Leone and Gordon 1989
; Talman 1989
). Microinjection of the metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) depolarizes cells in the medial NST, implicating metabotropic receptors in baroreceptor reflexes as well (Glaum and Miller 1992
). Studies on cells isolated from the baroreceptive area of the dorsal medial NST, Drewe et al. (1990)
and Drewe and Kunze (1994)
have demonstrated the presence of NMDA, kainate, and quisqualate receptors on these cells. In a brain stem slice preparation, Andresen and Yang (1990)
reported that non-NMDA receptors mediate visceral sensory afferent input to baroreceptor areas in the dorsal medial portion of the NST. Application of CNQX resulted in a rapid dose-dependent suppression of EPSPs recorded in vitro in response to electrical stimulation of the solitary tract; APV had only a small effect and only at high doses. These authors (Andreson and Yang 1990, 1994) concluded that non-NMDA receptors mediate primary, monosynaptic, fast afferent synaptic transmission in these cells, and that other glutamate receptor subtypes play some yet undefined role.
In addition to a role in the NST, glutamate has been shown to be a neurotransmitter at many other synapses in the CNS (Mayer and Westbrook 1987
; Stone and Burton 1988
; Watkins 1981
). Recent evidence from studies of the rat olfactory bulb in vitro show that afferent synaptic transmission between the olfactory nerve fibers and mitral cells is mediated by both NMDA and non-NMDA receptors (Ennis et al. 1996
). Electrical stimulation of the olfactory nerve layer evokes a short-latency excitation, inhibition, and a second epoch of excitation. The two excitatory components were differentially blocked by glutamate receptor antagonists: the early excitation was blocked by 6,7-dinitroquinoxaline-2,3-dione, an AMPA/kainate antagonist, and the delayed excitatory response was blocked by APV. More recent experiments have shown that these two components are monosynaptically driven from the olfactory nerve (Aroniadou-Anderjaska et al. 1997
). Thus taste and olfactory transmission have in common the use of EAAs at their first central synapse.
Conclusions
In conclusion, the present study offers the first direct functional evidence that glutamate mediates excitatory afferent synaptic transmission between primary CT fibers and taste-responsive NST neurons and that this transmission is mediated primarily by the AMPA/kainate receptor, with some contribution from NMDA receptors. AMPA/kainate and NMDA receptors are colocalized on the some of the same NST cells (see also Wang and Bradley 1995
), but NMDA receptors may play a secondary role under normal conditions.
 |
ACKNOWLEDGEMENTS |
The authors thank Drs. Barry Davis, Matthew Ennis, and Mark B. Vogt for valuable comments on the manuscript.
This work was supported in part by National Institute of Deafness and Other Communications Disorders Grant DC-00066 to D. V. Smith. A portion of these results was presented at the 1995 meeting of the Association for Chemoreception Sciences in Sarasota, FL and at the 1995 meeting of the Society for Neuroscience in San Diego, CA.
 |
FOOTNOTES |
Address for reprint requests: D. V. Smith, Dept. of Anatomy and Neurobiology, University of Maryland School of Medicine, 685 W. Baltimore St., Baltimore, MD 21201-1509.
Received 9 July 1996; accepted in final form 3 December 1996.
 |
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