Skip to main content
NCBI home page
Chemical Senses logoLink to Chemical Senses
. 2020 Mar 10;45(4):249–259. doi: 10.1093/chemse/bjaa015

ENaC-Dependent Sodium Chloride Taste Responses in the Regenerated Rat Chorda Tympani Nerve After Lingual Gustatory Deafferentation Depend on the Taste Bud Field Reinnervated

Enshe Jiang 1,2,3,4, Ginger D Blonde 3,4, Mircea Garcea 3, Alan C Spector 3,4,✉
PMCID: PMC7320219  PMID: 32154568

Abstract

The chorda tympani (CT) nerve is exceptionally responsive to NaCl. Amiloride, an epithelial Na+ channel (ENaC) blocker, consistently and significantly decreases the NaCl responsiveness of the CT but not the glossopharyngeal (GL) nerve in the rat. Here, we examined whether amiloride would suppress the NaCl responsiveness of the CT when it cross-reinnervated the posterior tongue (PT). Whole-nerve electrophysiological recording was performed to investigate the response properties of the intact (CTsham), regenerated (CTr), and cross-regenerated (CT-PT) CT in male rats to NaCl mixed with and without amiloride and common taste stimuli. The intact (GLsham) and regenerated (GLr) GL were also examined. The CT responses of the CT-PT group did not differ from those of the GLr and GLsham groups, but did differ from those of the CTr and CTsham groups for some stimuli. Importantly, the responsiveness of the cross-regenerated CT to a series of NaCl concentrations was not suppressed by amiloride treatment, which significantly decreased the response to NaCl in the CTr and CTsham groups and had no effect in the GLr and GLsham groups. This suggests that the cross-regenerated CT adopts the taste response properties of the GL as opposed to those of the regenerated CT or intact CT. This work replicates the 5 decade-old findings of Oakley and importantly extends them by providing compelling evidence that the presence of functional ENaCs, essential for sodium taste recognition in regenerated taste receptor cells, depends on the reinnervated lingual region and not on the reinnervating gustatory nerve, at least in the rat.

Keywords: amiloride, cross-regeneration, gustatory nerve, taste receptor

Introduction

The chorda tympani (CT) nerve, a branch of the 7th cranial nerve, innervates taste buds of the anterior tongue (AT), and the glossopharyngeal (GL) nerve innervates taste buds of posterior tongue (PT) in rodents. These 2 gustatory nerves can be distinguished by their response properties to prototypical taste stimuli placed on their taste bud fields. For example, the CT responds better than the GL to NaCl (Ninomiya et al. 1982; Boudreau et al. 1983; Frank et al. 1983; Ninomiya and Funakoshi 1988; Frank 1991), whereas the reverse is true when quinine is the stimulus (Shingai and Beidler 1985; Hanamori et al. 1988). In rodents, at least, both nerves have a great proclivity to regenerate after damage or transection and to reinnervate their native receptor fields. The taste buds degenerate after the gustatory nerve innervating them is transected in the rat, but upon regeneration, most of the taste buds will reappear, albeit reduced in number, and nerve function will return to normal as assessed behaviorally or electrophysiologically (St John et al. 1995; Cain et al. 1996; Barry 1999; King et al. 2000; Kopka et al. 2000; Spector 2003; Yasumatsu et al. 2003; Geran et al. 2004). Therefore, the rodent gustatory system can be used as an excellent experimental platform to investigate neural plasticity and its concomitant functions.

Remarkably, the lingual taste nerves can also be surgically cross-wired, at least in rodents (Oakley 1967; Nejad and Beidler 1987; Ninomiya 1998; Smith et al. 1999; King et al. 2008; Spector et al. 2010). That is to say, the central end of the sectioned CT can be sutured to the distal end of a cut GL, and vice versa. The regenerating CT will then innervate the taste buds of the PT instead of the AT, and the regenerating GL will then innervate the taste buds of the AT instead of the PT. Several decades ago, Oakley (1967) first investigated the response properties of the cross-regenerated CT and cross-regenerated GL nerve. He found that the relative taste response properties of the cross-regenerated nerve to an array of tastants placed on a lingual taste bud field change and appear like the responses obtained from the nerve which normally innervated that tongue region. This result suggested that the sensory responses evoked in the CT and GL by taste stimulation were largely dependent on the taste bud field they were innervating rather than the other way around.

Since the time of Oakley’s pioneering experiments, a very distinct difference has been discovered between the nature of the responses of the CT and GL to lingual application of NaCl in the rat. Not only does the CT normally display a more robust response to NaCl than the GL, but that response is significantly suppressed by oral treatment with the epithelial Na+ channel (ENaC) blocker amiloride hydrochloride (Heck et al. 1984; Brand et al. 1985; DeSimone and Ferrell 1985). In contrast, the NaCl responses of the GL appears to be unaffected by the lingual application of the drug, at least in the rat (Formaker and Hill 1991; Kitada et al. 1998). Indeed, the CT fibers that are amiloride sensitive (AS) are narrowly tuned to respond selectively to sodium salts whereas those that are amiloride insensitive (AI) are more generally responsive to electrolytes including NaCl, nonsodium salts, and acids (Heck et al. 1984; Ninomiya and Funakoshi 1988; Halpern 1998; Chandrashekar et al. 2010). The same is true of the mouse CT. Taking advantage of the dichotomous amiloride sensitivity of the CT versus GL with respect to responsiveness to NaCl, Ninomiya (1998) tested the amiloride sensitivity of the cross-regenerated CT and GL in the C57BL/KsJ mice. Surprisingly, he found that the distribution of AS fibers in the CT routed to the PT did not differ from that found in the intact nerve innervating the AT. The GL did not possess many AS fibers, regardless of whether it innervated the PT or AT. These results stand in stark contrast to those of the Oakley experiments in the rat. It appears that, in the mouse, the fibers of the respective nerves are able to find appropriate matching receptor cells such that the response properties of the nerve are maintained irrespective of the lingual taste bud field innervated, at least in regard to amiloride sensitivity.

Here, we replicated the findings of Oakley and importantly extend them by showing that, unlike the mouse, the response properties of the CT in the rat, including amiloride sensitivity, are dictated by the taste receptor field it innervates. Notably, this helps explain why rats that have the CT successfully cross-reinnervating the regenerated taste buds in the PT have difficulty competently performing a psychophysical salt discrimination task (Spector et al. 2010) and suggests that there is a fundamental difference in the reorganizational potential of regenerated nerves in the peripheral gustatory systems of the rat versus the mouse.

Materials and methods

Subjects

Sixty-six male Sprague-Dawley rats (Charles River Breeders) weighing between 339 and 547 g on the day of surgery served as subjects. Each rat was individually numbered and housed in a polycarbonate cage, except where noted below, in a room that was maintained on a 12:12 h day:night cycle and where the temperature was automatically controlled. The rats had access to pelleted chow (LabDiet 5001; PMI Nutrition International Inc.) and purified water (filtered RO water; Millipore Elix 10) in the home cage except where noted otherwise. All the experimental manipulations were performed in the light phase. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Florida.

Experimental design

The animals in this experiment were a subset from a postsurgical behavioral experiment, not reported here or elsewhere, that had technical difficulties precluding interpretation. These animals consisted of 6 surgical groups: 1) CTx, in which bilateral CT were transected through a ventral neck approach and a 5-15 mm portion of the nerve was removed to prevent regeneration; 2) GLx, in which the GL was bilaterally transected and an ~8 mm portion of the nerve was removed to prevent regeneration; 3) CTr, in which the CT was transected and allowed to regenerate into AT; 4) GLr, in which the GL was transected and allowed to regenerate into PT; 5) CT-PT, in which the central end of the CT was sutured (see Surgery) into peripheral end of the GL to route regenerating CT fibers into the PT; and 6) Sham, in which the bilateral GL and CT were just exposed and left intact. We also tried to test animals in which the central end of GL was sutured into peripheral end of CT to route regenerating GL fibers into the AT (GL–AT), but because the GL was agglutinated to the surrounding tissue it was very difficult to isolate and we were only able to record successfully from 2 of 11 GL–AT rats and these 2 rats did not meet the preparation stability criterion (see Statistical analysis) for inclusion in the analysis. Some of the animals in the Sham group were allocated to the CTsham group and some were allocated to the GLsham group. To increase the sample size of the CTsham group, we included animals in the GLx group. Likewise, the CTx animals were included in the GLsham group. There were no obvious differences statistically or otherwise between the GL responses in the CTx group and those of the sham rats (all P values >0.140) or between CT responses in the GLx group and those of the sham rats (all P values >0.195). The final sample sizes listed in Table 1 represent the animals that were included in the analysis of the electrophysiological data.

Table 1.

Groups for electrophysiology

Surgical group Status of CT Status of GL Final group size
CTsham Intact Intact or GLx N = 8 (10)a
CTr Normal regeneration to AT Intact N = 7 (8)
GLsham Intact or CTx Intact N = 8 (12)
GLr Intact Normal regeneration to PT N = 5 (8)
CT-PT Cross-regeneration to PT Transected N = 4 (10)
Open in a new tab

aThe numbers in parentheses represent the number of animals for which nerve recording was attempted. AT: anterior tongue; PT: posterior tongue.

Surgery

Rats (70–112 days of age) were anesthetized with an intramuscular injection of a mixture of ketamine hydrochloride (125 mg/kg body mass) and xylazine hydrochloride (5 mg/kg body mass). Supplemental doses were administered as necessary. Additionally, each rat was administered penicillin G Procaine (30 000 units, s.c.) and ketorolac tromethamine (2 mg/kg body mass s.c.) on the day of surgery. We used our already described and validated techniques to perform the nerve surgeries (King et al. 2008; Spector et al. 2010). Briefly, for the CTr rats, the CT was exposed by dissecting the connective tissue between the masseter and the digastric muscles. After the pterygoid muscle and the anterior belly of the digastric muscle were retracted, a small transverse cut was made in the anterior portion of the pterygoid muscle to expose the CT. The nerve was isolated from surrounding connective tissue and transected near its junction with the lingual nerve. The 2 ends were left closely apposed to allow the fibers to normally regenerate into AT. For the rats in CTx group, the CT was exposed as mentioned above. The CT was carefully stretched after its exit from the bulla and before its distal junction with the lingual nerve and a 5–15 mm portion was removed to prevent regeneration. Months later (232–264 days), 40–88 days before electrophysiological testing, the CTx group (and the GL–AT group) had its CT transected in the middle ear as it passed behind the malleus, and the tympanic rim was cauterized to prevent regeneration; this was done to accommodate the behavioral experiment. For the GLr rats, the GL was exposed by making a skin incision in the ventral neck. The sublingual and submaxillary salivary glands and the sternohyoid, omohyoid, and posterior belly of the digastric muscles were retracted to expose the GL. The GL was transected and the 2 ends were placed together such that the fibers would normally regenerate into the PT. For the rats in GLx group, the GL was exposed and transected as mentioned above, and ~8 mm portion of the nerve was removed to prevent regeneration. For the CT-PT group, the CT and GL were exposed as described above. The GL was transected near its exit from the posterior lacerated foramen and the CT was transected near its junction with the lingual nerve. The peripheral portion of the GL was then routed under the mylohyoid muscle into the field of the central portion of the CT and the 2 nerves were connected via a single stitch of 11-0 monofilament suture (Ethicon). For the rats that received sham surgery, the CT and GL were exposed as described above but left intact. In all cases involving neck incision, the wound was closed with nylon suture. Each animal received a subcutaneous injection of penicillin and ketorolac on each of the first 3 days after surgery. Animals were placed in stainless steel hanging cages where they were given special diets to promote recovery. This included daily access to both wet mash (dry powder chow with purified water) and an oil mash diet. Starting on the third postoperative day, a calorically dense nutritional supplement (Nutrical, Evsco Pharmaceuticals) was mixed into the wet mash. These supplemental diets were given to each rat until its body mass increased and stabilized at or above 90% of its presurgical value. The animals were then returned to their plastic cages. Seven animals died or were euthanized during surgery or before electrophysiological recording began.

Stimuli

All solutions were prepared daily with purified water (filtered RO water; Millipore Elix 10) and reagent grade chemicals. The solutions were made and delivered to the tongue at room temperature. Stimuli used during electrophysiological recording were presented in a set with the following order: 0.5 M NH4Cl, NaCl (0.03, 0.1, and 0.3 M) (Fisher Scientific), 0.5 M NH4Cl, NaCl (0.03, 0.1, and 0.3 M) mixed in 0.1 mM amiloride hydrochloride (Sigma-Aldrich), 0.5 M NH4Cl, quinine hydrochloride (0.005, 0.017, and 0.05 M; Sigma-Aldrich), 0.5 M NH4Cl, 0.5 M KCl, 0.1 M citric acid, 1.0 M sucrose (Fisher Scientific), and 0.5 M NH4Cl.

Electrophysiology

The postoperative survival time (from the initial surgery in the CTx group) for the rats included in the analysis was 243–355 days and the age range was 343–449 days. The rat was initially anesthetized by intraperitoneal injection of sodium pentobarbital (75 mg/kg). The level of anesthesia was monitored by frequently checking the pedal withdrawal reflex. Supplemental doses were administered as necessary. Body temperature was monitored and regulated at 36–37 °C by an electric heating pad under the body. Tracheal cannulation was made to facilitate the breathing before the rat was secured in a non-traumatic headholder. The hypoglossal nerves were transected bilaterally in order to prevent movements of the tongue during the recording. For the rats in the CTr, CTsham, and CT-PT groups, the CT nerve was exposed through the mandibular approach. The nerve was cut near its entrance to the tympanic bulla after it was dissected free from the surrounding connective tissue. For the rats in the GLr and GLsham groups, the GL nerve was exposed through the ventral approach. The nerve was dissected free and cut near its exit from the posterior lacerated foramen. The free end of the nerve was desheathed carefully with two microforceps and put on the hook of a platinum/iridium electrode. The indifferent electrode was placed in the underlying muscle tissue close to the recording electrode. Whole-nerve neural activity was band-pass filtered (0.3–5 kHz), differentially amplified (Differential AC Amplifier, Model 1700, A-M Systems INC), displayed, and recorded on a computer using Cambridge Electronic Design (CED) hardware and software (Spike-2). Taste stimuli were presented to the AT or PT via a custom-designed fluid delivery system (DiLog Instruments) at a rate of 0.3 mL/s for 20-s after 30-s water prerinse and followed by a 40-s water postrinse using the purified water (Filtered RO water; Millipore Elix-10) that was used to mix the solutions. The flow rate and the time were controlled by a customized program written using Spike-2 software. There was a 95-s rest between 2 stimuli. During NaCl with amiloride presentations, 0.1 mM amiloride hydrochloride was used as the prerinse and postrinse solution. A 0.5 M NH4Cl solution was applied periodically to assess the stability of the preparation. This salt solution is commonly used as a standard in the electrophysiological studies of the peripheral gustatory nerves, because it elicits robust and reliable responses from the rat CT and GL. We ran as many stimulus sets as possible. We would suspend data collection during a stimulus set when noise became an issue, and, once it was resolved, we would restart the nerve recording until a complete stimulus set tested. At times, for some animals we could only test 1 complete stimulus set. Other times, we could get 2 or more complete stimulus sets tested without significant noise. In such cases, we would calculate the mean value of the nerve responses to the 2 or more complete stimulus sets as the final nerve response to the stimulus for the rats and only complete stimulus sets tested were used in the data analysis. Two rats died while preparing the animal for nerve recording.

Histology

Immediately after the recording session, the animal was deeply anesthetized with supplemental sodium pentobarbital (≥50 mg/kg body mass, intraperitoneally) and was transcardially perfused with saline followed by 10% buffered formalin. The whole tongue of the rat was removed and stored in 10% buffered formalin. The AT was cut into 2 halves along the midline after it was dipped into a 0.5% methylene blue solution for 1 min and rinsed with water. The epithelium of each half was tightly pressed between 2 glass slides using adhesive tape after the underlying muscle was largely removed. A light microscope was used to count the total number of the fungiform papillae and taste pores. The latter appear as dark blue dots in the center of the paler staining fungiform papillae, in rats, each fungiform papilla houses only a single taste bud. The circumvallate papilla was embedded in paraffin and cut into 10 µm sections. Then, the sections were mounted on glass slides, stained with hematoxylin and eosin, and the number of taste pores were counted. All tissues were coded so that the counters were blind to the surgical condition.

Statistical analysis

One rat in the CT-PT group which was included in the electrophysiological analysis had no histological data due to tissue loss during processing and was removed from the CT-PT group in the histological analysis. Tissue from the CTsham and GLsham rats that did not have a nerve transection (i.e., either CTx or GLx) were combined into a single Sham group (n = 5) for the histological comparison with CT-PT, CTr, and GLr groups.

The repeated presentation of 0.5 M NH4Cl allowed us to measure and control for the stability of the preparation. First, if one of the 0.5 M NH4Cl presentations differed from the mean of the five 0.5 M NH4Cl in a complete stimulus set by ≥35%, the animal was discarded (n = 5) from the data analysis. Second, the most recent NH4Cl presentations before and after a test stimulus presentation were used to standardize the response across rats and across the test session. We discarded some animals from the analysis for which the nerve had no response to the stimuli (n = 10), likely because of damage that occurred during the isolation of the nerve from surrounding tissue (especially for the rats in group of GL–AT), and discarded other animals for which we failed to test a complete stimulus set for the nerve recording (n = 6). There were also some animals that were discarded due to excessive noise contaminating gustatory responses (n = 4). The final sample sizes for the electrophysiological analysis can be found in Table 1. For the statistical analysis, we first measured the area under the curve (AUC) of the rectified and averaged signal (smooth function, Spike 2, time constant = 2 s) for the first 10 s of stimulus application and then divided this value by the average AUC of the rectified and averaged signal (smooth function, Spike 2, time constant = 2 s) for the most recent applications of 0.5 M NH4Cl before and after the stimulus. In all cases, the AUC of the last 10 s of the water prerinse was first subtracted from the stimulus AUC in the above calculations. The ratio scores of the AUC were first analyzed with a 2-way mixed analysis of variance (ANOVA, group [3 levels] × stimulus [12 levels]) across the groups of CT-PT, GLr, and GLsham and across the groups of CT-PT, CTr, and CTsham. Then, the ratio scores of the AUC were analyzed with a series of 1-way ANOVAs for KCl, citric acid and sucrose, a 2-way mixed ANOVA (group × concentration) for quinine, and a 3-way mixed ANOVA for NaCl (group × amiloride treatment × concentration) such that we were able to compare the properties of the cross-regenerated CT (CT-PT) with the normally regenerated (CTr) and the intact (CTsham) CT as well as with the normally regenerated (GLr) and intact (GLsham) GL. We also conducted a 2-way ANOVA (concentration × amiloride) for analysis of NaCl responses in all groups. Of course, because the sample sizes are small, especially in the CT-PT group, the outcomes of the statistical tests should be viewed with some caution. Accordingly, we also treated each animal in the regeneration groups as a replication and calculated their Z-scores for each AUC ratio score for each stimulus relative to the mean and SD of the GLsham and CTsham group. The rejection criterion (e.g., alpha) for all statistical tests was set at the conventional value of 0.05.

Results

Histology

A reliable morphological proxy of nerve regeneration is the presence or absence of taste buds from the denervated gustatory receptor field. In rats, taste buds completely disappear when the GL is transected and reappear upon restitution of the nerve supply (Vintschgau and Honingschmid 1876; Whiteside 1926; Guth 1957; St John et al. 2003). When the CT is transected, the number of taste buds in the AT drops by >80% and many, but not all, of these taste buds reappear upon reinnervation of the regenerated CT (Cheal and Oakley 1977; Ganchrow and Ganchrow 1989; Segerstad et al. 1989; Barry and Frank 1992; Oakley et al. 1993; St John et al. 1995). The failure for regeneration to completely restore the normal complement of AT taste buds in this case is likely due to a decrease in the number of fungiform papilla targets, some of which degenerate when the CT is transected (Oakley et al. 1993; St John et al. 1995).

In the present study, there was no difference across all the groups in the number of taste buds in the circumvallate papilla (F(3,16) = 1.725, P = 0.202; Table 2). This confirms that the PT was successfully reinnervated in the GLr and CT-PT groups. There was a significant difference between the groups in the number of fungiform taste pores (F(3,16) = 158.474, P < 0.001), the proportion of fungiform papillae containing a pore (F(3,15) > 1000, P < 0.001), and the number of fungiform papillae (F(3,16) = 66.364, P < 0.001; Table 2). Bonferroni-corrected paired comparisons revealed that, on all 3 measures, the CT-PT group had significantly lower values than the Sham rats (all P values ≤0.001), which, in turn, did not differ from either the CTr or GLr groups (P values ≥0.235). This confirms that the CT-PT had a denervated AT, as expected, because the CT cross-reinnervated the PT.

Table 2.

The number of taste buds in AT and circumvallate papillae across groups

Group Number of intact fungiform papillae Pores in AT % Intact fungiform papillae containing pore Number of taste buds in circumvallate papillae
CT-PT (n = 3)a 56.67 ± 7.06 7.00 ± 1.53 12.26 ± 2.34 451.33 ± 3.76
CTr (n = 7) 133.57 ± 2.43 126.86 ± 2.73 94.97 ± 1.01 441.57 ± 8.96
GLr (n = 5) 157.40 ± 4.49 150.00 ± 4.72 95.27 ± 0.55 427.40 ± 7.42
Sham (n = 5)b 144.00 ± 6.06 138.60 ± 6.05 96.22 ± 0.31 420.40 ± 12.25
Open in a new tab

aOne rat that was included in the electrophysiological analysis was removed due to the tissue damage during the histological processing.

bFor the Sham group, only animals that had CT and GL intact and were part of the electrophysiology analysis were included. There were 5 rats in CTsham-GLx subgroup and 6 rats in GLsham-CTx subgroup included in the statistical analysis electrophysiological data and were not included in the histological analysis.

Electrophysiology

Because we were testing whether the cross-reinnervated CT (i.e., CT-PT) would take on the response properties of the intact or regenerated CT as opposed to the intact or regenerated GL, we divided the analysis such that CT-PT group was included in ANOVAs with the GLsham and GLr groups as well as in separate ANOVAs with the CTsham and CTr groups.

Figure 1 provides representative traces from 3 different preparations: CT-PT, GLr, and CTr. As will be supported statistically below, notwithstanding some idiosyncratic differences, it is clear that the response properties of the CT reinnervating the PT emulate those of the regenerated (and intact) GL (Figure 2) much more than they do with those of the regenerated (and intact) CT (Figure 3). This is especially true for NaCl responsiveness as well as its suppression by amiloride treatment.

Figure 1.

Figure 1.

Open in a new tab

Representative continuous traces of rectified and integrated CT responses (see Statistical analysis) from 3 different rats with CT-PT, GLr, and CTr. Ami, amiloride hydrochloride (100 µM); Q, quinine.

Figure 2.

Figure 2.

Open in a new tab

Mean (±SE) responses of the CT in CT-PT rats and the GL in rats with GLr and GLsham. The data points represent the AUC of the rectified and integrated response (time constant = 2 s) for the first 10 s of stimulus application divided by the rectified and integrated response to the most recent application of 0.5 M ammonium chloride before and after the stimulus (see Statistical analysis). In all cases, the response of the last 10 s of the water prerinse was subtracted out of stimulus AUC in the calculation. Ami, amiloride hydrochloride (0.1 mM); Q, quinine.

Figure 3.

Figure 3.

Open in a new tab

Mean (±SE) responses of the CT in CT-PT rats and rats with CTr and CTsham. The data points represent the AUC of the rectified and integrated response (time constant = 2 s) for the first 10 s of stimulus application divided by the rectified and integrated response to the most recent application of 0.5 M ammonium chloride before and after the stimulus (see Statistical analysis). In all cases, the response of the last 10 s of the water prerinse was subtracted out of stimulus AUC in the calculation. Ami, amiloride hydrochloride (0.1 mM). *CT-PT versus CTr and CTsham, P < 0.05; #CT-PT versus CTsham, P < 0.05.

CT-PT versus GLr and GLsham

We first tested whether there were any differences in nerve responses to the entire stimulus set between the CT-PT group and the GLr and GLsham groups, by subjecting the AUC ratio scores to a 2-way mixed ANOVA (group × stimulus). Although there were differences between the stimuli (F(11,154) = 64.313, P < 0.001), there was no main effect of group (F(2,14) = 0.216, P = 0.808) nor a group × stimulus interaction (F(22,154) = 1.423, P = 0.111). We also conducted a series of ANOVAs testing the AUC ratio scores of the CT-PT group with those of the GLr group and those of the GLsham group (Table 3). To test whether amiloride sensitivity differed between the CT-PT group and the GLr or GLsham group, the AUC ratio scores for each group was subjected to a 3-way mixed ANOVA (group × NaCl concentration × amiloride treatment). In these ANOVAs the CT-PT group never differed from either the GLr or GLsham group, nor were there any group-related interactions. As expected, amiloride treatment had no effect on NaCl responsiveness in any of these 3 groups, which all displayed a significant monotonic increase as concentration was raised (Table 3 and Figure 2). A 2-way mixed ANOVA (group × concentration) was conducted on the AUC ratio scores associated with quinine stimulation (Table 3 and Figure 2). The CT-PT group did not differ from the GLr or GLsham groups and only the concentration factor was significant. All 3 groups increased responding to quinine in a concentration-dependent manner (Table 4). The AUC ratio scores of the CT-PT group for KCl, citric acid, sucrose did not differ from those for either the GLr or GLsham groups (Table 3 and Figure 2).

Table 3.

Summary of ANOVA results for NaCl, quinine, KCl, citric acid, and sucrose comparing CT-PT with GLr, GLsham, CTr, and CTsham groups

Effects CT-PT versus GLr CT-PT versus GLsham CT-PT versus CTr CT-PT versus CTsham
NaCl
 Group F (1,7) = 0.321 F (1,10) = 1.393 F (1,9) = 152.212 F (1,10) = 37.720
P = 0.589 P = 0.265 P < 0.001 P < 0.001
 Ami F (1,7) = 0.387 F (1,10) = 0.238 F (1,9) = 58.376 F (1,10) = 38.787
P = 0.554 P = 0.636 P < 0.001 P < 0.001
 Ami × Group F (1,7) = 1.876 F (1,10) < 0.001 F (1,9) = 52.936 F (1,10) = 35.185
P = 0.213 P = 0.991 P < 0.001 P < 0.001
 Conc F (2,14) = 50.270 F (2,20) = 71.510 F (2,18) = 176.870 F (2,20) = 99.736
P < 0.001 P < 0.001 P < 0.001 P < 0.001
 Conc × Group F (2,14) = 3.202 F (2,20) = 1.952 F (2,18) = 47.370 F (2,20) = 23.259
P = 0.072 P = 0.168 P < 0.001 P < 0.001
 Conc × Ami F (2,14) = 0.189 F (2,20) = 0.625 F (2,18) = 66.219 F (2,20) = 31.764
P = 0.830 P = 0.545 P < 0.001 P < 0.001
 Conc × Ami × Group F (2,14) = 0.093 F (2,20) = 0.721 F (2,18) = 71.700 F (2,20) = 35.019
P = 0.911 P = 0.605 P < 0.001 P < 0.001
Quinine HCl
 Group F (1,7) = 0.607 F (1,10) = 0.145 F (1,9) = 0.105 F (1,10) < 0.001
P = 0.462 P = 0.711 P = 0.754 P = 0.993
 Conc F (2,14) = 28.775 F (2,20) = 27.988 F (2,18) = 38.743 F (2,20) = 28.282
P < 0.001 P < 0.001 P < 0.001 P < 0.001
 Group × Conc F (2,14) = 1.000 F (2,20) = 0.786 F (2,18) = 0.539 F (2,20) = 0.767
P = 0.393 P = 0.469 P = 0.593 P = 0.487
KCl
 Group F (1,7) = 1.219 F (1,10) = 0.022 F (1,9) = 0.398 F (1,10) = 13.883
P = 0.306 P = 0.884 P = 0.544 P = 0.004
Citric acid
 Group F (1,7) = 3.771 F (1,10) = 1.262 F (1,9) = 3.579 F(1,10) = 4.326
P = 0.093 P = 0.288 P = 0.091 P = 0.064
Sucrose
 Group F (1,7) = 10.30 F (1,10) = 0.434 F (1,9) = 0.362 F (1,10) = 0.131
P = 0.344 P = 0.525 P = 0.562 P = 0.725
Open in a new tab

Ami, amiloride; Conc, concentration. P-values in bold indicate statistically significant differences.

Table 4.

Summary of 2-way ANOVA with repeated measures (NaCl concentration × amiloride) results for NaCl response and 1-way ANOVA (concentration) results for quinine response by groups

Effect CT-PT GLr GLsham CTr CTsham
NaCl
 Ami F (1,3) = 0.335 F (1,4) = 1.878 F (1,7) = 0.130 F (1,6) = 107.560 F (1,7) = 79.977
P = 0.603 P = 0.242 P = 0.729 P < 0.001 P < 0.001
 Conc F (2,6) = 9.575 F (2,8) = 58.844 F (2,14) = 124.742 F (2,12) = 508.658 F (2,14) = 175.777
P = 0.014 P < 0.001 P < 0.001 P < 0.001 P < 0.001
 Ami × Conc F (2,6) = 0.424 F (2,8) = 0.149 F (2,14) = 0.962 F (2,12) = 142.324 F (2,14) = 75.611
P = 0.673 P = 0.864 P = 0.406 P < 0.001 P < 0.001
Quinine HCl
 Conc F (2,6) = 20.683 F (2,8) = 11.764 F (2,14) = 14.234 F (2,12) = 22.822 F (2,14) = 15.168
P = 0.002 P = 0.004 P < 0.001 P < 0.001 P < 0.001
Open in a new tab

Ami, amiloride; Conc, concentration. P-values in bold indicate statistically significant differences.

As is evident from Figure 4 which displays the Z-scores of the AUC ratio scores relative to the mean and SD of the GLsham group of every animal, the rats in the GLr and CT-PT group for the most part fall in the distribution of scores for the GLsham group. In contrast, the GLsham, GLr, and CT-PT groups have AUC ratio scores for the midrange and high concentration of NaCl that are on the fringe of the distribution hovering around 3 SDs from the mean of the CTsham group (Figure 5).

Figure 4.

Figure 4.

Open in a new tab

Z-scores of the AUC ratio scores for all animals in each of the 5 groups relative to the mean and SD of the GLsham group. The red dashed lines represent ±3 SD. The green curve line represents the animals for which all of the NH4Cl responses were less than 20% of the mean of all 5 NH4Cl responses. The blue curve represents the animals for which the variation of some of the NH4Cl responses were between 20% and 35% of the mean of all 5 NH4Cl responses.

Figure 5.

Figure 5.

Open in a new tab

Z-scores of the AUC ratio scores for all animals in each of the 5 groups relative to the mean and SD of the CTsham group. The red dashed lines represent ±3 SD. The green curve line represents the animals for which all of the NH4Cl responses were less than 20% of the mean of all 5 NH4Cl responses. The blue curve represents the animals for which the variation of some of the NH4Cl responses were between 20% and 35% of the mean of all 5 NH4Cl responses.

CT-PT versus CTr and CTsham

After finding that the cross-reinnervated CT (CT-PT) had similar response properties to the intact and regenerated GL, we also compared this group to the CTsham and CTr groups. A 2-way mixed ANOVA (group × stimulus) revealed a significant main effect of group (F(2,16) = 15.863, P < 0.001) and stimulus (F(11,176) = 104.950, P < 0.001), and a significant group × stimulus interaction (F(22,176) = 12.453, P < 0.001; Figure 3). To test whether amiloride sensitivity differed between the CT-PT group and the CTr or CTsham group, the AUC ratio scores for each group was subjected to a 2-way mixed ANOVA (NaCl concentration × amiloride treatment) and a 3-way mixed ANOVA (group × NaCl concentration × amiloride treatment). Tables 3 and 4 present the outcomes of those ANOVAs demonstrating that while each group displayed a monotonic increase in responsiveness, the nerve responses to NaCl concentrations were significantly decreased by amiloride treatment only in the CTr and CTsham groups (Figure 3); as we had shown above, the CT-PT group did not display amiloride sensitivity.

A 2-way mixed ANOVA (group × concentration) was conducted on the AUC ratio scores associated with quinine stimulation (Table 3 and Figure 3). The CT-PT group did not differ from the CTr or CTsham groups and only the concentration factor was significant. Each of these groups displayed a concentration-dependent increase in nerve responsiveness to quinine (Table 4). Although the lower AUC ratio scores of the CT-PT group relative to the CTr group for KCl did not reach statistical significance, they were nonetheless significantly different from the CTsham group (Table 3 and Figure 3). The citric acid and sucrose ratio scores for the CT-PT group did not differ from either the CTr or CTsham group (Table 3 and Figure 3).

As is evident from Figure 5 which displays the Z-scores of the AUC ratio scores relative to the mean and SD of the CTsham group of every animal, the rats in the CTr group for the most part fall within in the distribution of scores for the CTsham group. In contrast, the CTsham and CTr groups have AUC ratio scores for all concentrations of NaCl, regardless of amiloride treatment, that either all fall outside of the distribution or hover around 3 SDs of the mean of the GLsham group (Figure 4). Taken as a whole, the profile of responsiveness across the stimulus set when the CT reinnervated the PT was notably different from that seen with the regenerated and intact CT innervating the AT, especially with respect to NaCl and amiloride sensitivity.

Discussion

The cross-reinnervated CT responses to the stimuli tested here in CT-PT rats generally emulated those seen in the intact and regenerated GL (i.e., GLsham and GLr). This is consistent with prior work showing that the response properties of the unilaterally cross-anastomosed CT were similar to those obtained from the nerve that normally innervated the targeted tongue region (Oakley 1967; Nejad and Beidler 1987); that is to say, the cross-regenerated CT responded like a GL. The findings here not only replicate the prior work of Oakley, but extends it in a very important way by clearly demonstrating that amiloride treatment had no effect on the NaCl responses of the cross-regenerated CT nerve, but normally suppressed responsiveness to this salt in the regenerated CT when it reinnervated the AT. This strongly suggests that the presence of functional ENaCs in regenerated taste receptor cells is dependent on the reinnervated lingual epithelium and not on the reinnervating gustatory nerve.

Our findings contrast with those from Ninomiya (1998) in the mouse model. Using C57BL/KsJ mice, Ninomiya (1998) formed a cross-anastomosis between the central end of the CT and the peripheral end of the GL similar to our CT-PT group. In other mice, he formed a cross-anastomosis of the central end of the GL to the peripheral end of the CT so that the regenerating fibers reinnervated the AT. The NaCl response of the nerve with and without the amiloride treatment was tested and it was found that the distribution of the AS and AI types of fibers in the CT and GL nerve was not altered by cross-regeneration of the two gustatory nerves into the reverse tongue regions. Accordingly, it appeared that the regenerated taste axons could selectively recouple with the appropriate type of receptor cell regardless of whether they innervated the front or the back of the tongue. As such, the amiloride sensitivity of the nerve was maintained regardless of whether it innervated the anterior or PT. Although we did not characterize the responsiveness of single fibers in the CT-PT, it is not unreasonable to assume that the ineffectiveness of amiloride to have any effect on responsiveness of the nerve to NaCl reflects the absence of an appreciable expression of functional ENaCs on the PT taste receptor cells they are innervating. Ninomiya (1998) did not test for responsiveness to stimuli other than NaCl, but, with the data available at hand, it would appear that the rules governing taste receptor cell regeneration and/or the mechanism underlying the functional coupling of such cells with reinnervating nerve fibers fundamentally differ between the two rodent models. General differences in morphometric characteristics and the relative expression of some signaling molecules in taste receptor cells between the mouse and the rat have been reported (Ma et al. 2007). One possibility entertained by Ninomiya (1998) to explain the species difference in the electrophysiological outcomes of his cross-regeneration experiments and prior studies in the rat is that although there is a small proportion of AS taste cells in the mouse PT, there is a far greater number of taste buds than the AT, and thus there were a sufficient number of AS taste receptor cells to serve as targets for the cross-regenerating AS CT fibers. Perhaps in the rat, the number of matching receptor cell types as well as the branching patterns of the cross-regenerated fibers differs from the mouse to favor a more tissue-specific response. Indeed, it could be that there are simply no or an exceptionally low number of functional ENaC-expressing taste receptor cell targets in the rat PT. Nevertheless, a better understanding of this interesting species difference awaits further investigation.

Our results here help provide functional insight into the inability of rats that have the CT bilaterally routed into the PT, leaving the central GL transected and the AT deafferented of gustatory innervation, to competently distinguish between NaCl and KCl in a behavioral salt discrimination task (Spector et al. 2010). Under normal circumstances, performance in this psychophysical assay is severely impaired by amiloride treatment likely due to the silencing of the AS narrowly tuned sodium fibers (Spector et al. 1996; Geran and Spector 2000a, 2000b; Eylam and Spector 2002, 2003, 2005; Spector et al. 2010). The fact that in the rat, at least, the NaCl responsiveness of the cross-regenerated CT does not display amiloride sensitivity, suggests that there is no longer a distinguishable signal arising from the peripheral gustatory system to support the discrimination. It would be worthwhile for future work to confirm the prediction that there is a paucity of fibers narrowly tuned to sodium in the rats in which the CT cross-reinnervates the PT. Further, it would be instructive to test the prediction based on Ninomiya’s (1998) findings, that CT-PT mice should be able to discriminate NaCl from KCl and that amiloride treatment should be effective at disrupting the discrimination.

In conclusion, our findings are consistent with the results from the Oakley (1967) and Nejad and Beidler (1987) experiments showing that the response properties of a cross-regenerated nerve to taste stimuli in the rat depends on the taste bud field reinnervated. Furthermore, the presence of functional ENaCs in regenerated taste receptor cells in the rat appears to be an inherent property of the lingual region reinnervated rather than the nerve reinnervating them. In the rat, the fact that cross-regenerated nerves appear to take on the response properties normally associated with the oral fields of taste buds they innervate, suggests that animals surgically prepared in this way have inappropriate signals being channeled through gustatory circuits in the brain, notwithstanding the possibility of major central reorganizational events. In the case of long-term preference tests with basic taste stimuli, animals with cross-reinnervated nerves do not appear to differ from their intact counterparts (Oakley 1969). However, long-term intake tests are not considered optimal measures of taste function. Indeed, as discussed above, rats with the CT cross-anastomosed into the PT with the AT left deafferented of gustatory innervation are unable to competently discriminate NaCl from KCl (Spector et al. 2010). Interestingly, rats that have the GL cross-anastomosed to the AT with the PT left deafferented of gustatory innervation are also unable to perform this discrimination. In the first case, we can conclude that the failure for the rats to discriminate was due to the absence of a distinguishable signal. In the latter case, assuming that discriminable signals are present, something we were unfortunately statistically unable to test here, such signals are apparently unable to support the behavioral discrimination, perhaps because they are channeled through circuits that do not play a significant role in sensory-discriminative function (Spector and Grill 1992; St John and Spector 1998; Spector 2003; Blonde et al. 2010; Spector et al. 2010). Rats with the CT routed to the PT display normal gaping to intraorally delivered quinine, whereas rats that have the GL routed to the AT do not. This may be because the PT is thought to have a stronger response to quinine than the AT (King et al. 2000; Geran et al. 2004), but such did not seem to be the case in our study.

Clearly, there is more work that must be done. The contemporary molecular toolbox may provide unique strategies for nonsurgically rewiring targeted portions of the peripheral gustatory system. For example, Lee et al. (2017) took advantage of the differential distribution semaphorins, which are involved in axonal guidance, in different classes of taste receptor cells to apply knockout and transgene strategies to redirect the axons of the taste nerves to inappropriate taste bud cells in mice. In one experiment, Lee et al. (2017) misdirected ganglion cells normally innervating the T2R-expressing taste receptor cells (i.e., activated by bitter ligands) to innervate T1R3-expressing taste receptor cells (activated by amino acids or sweeteners). As a result, this changed the profile of tuning among the population of “bitter-responsive” geniculate ganglion units in the direction of broader tuning and shifted concentration-licking curves for bitter ligands, as measured in a brief access test, rightward. The study by Lee et al. (2017) and our study here, as well as those of others (Oakley 1967, 1969; Nejad and Beidler 1987; Ninomiya 1998; Smith et al. 1999; King et al. 2008; Spector et al. 2010), highlight the remarkable utility of the rodent peripheral gustatory system to serve as a platform for revealing the properties of plasticity and reorganization in the nervous system.

Acknowledgments

We would like to thank Angela Newth, Tiffany von Hartmann, and Nathalie Anderson for their help in all phases of this experiment.

Funding

This work was supported by the National Institute on Deafness and Other Communication Disorders of the National Institutes of Health under (grant number R01DC01628 to A.C.S.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Authors’ contributions

E.J. and A.C.S. designed research; E.J., G.B., and M.G. performed research; E.J. and A.C.S. analyzed data; E.J. and A.C.S. wrote the paper.

Conflict of interest

The authors declare no competing financial interests.

References

  1. Barry MA. 1999. Recovery of functional response in the nucleus of the solitary tract after peripheral gustatory nerve crush and regeneration. J Neurophysiol. 82(1):237–247. [DOI] [PubMed] [Google Scholar]
  2. Barry MA, Frank ME. 1992. Response of the gustatory system to peripheral nerve injury. Exp Neurol. 115(1):60–64. [DOI] [PubMed] [Google Scholar]
  3. Blonde G, Jiang E, Garcea M, Spector AC. 2010. Learning-based recovery from perceptual impairment in salt discrimination after permanently altered peripheral gustatory input. Am J Physiol Regul Integr Comp Physiol. 299(4):R1027–R1036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Boudreau JC, Hoang NK, Oravec J, Do LT. 1983. Rat neurophysiological taste responses to salt solutions. Chem Senses. 1:131–150. [Google Scholar]
  5. Brand JG, Teeter JH, Silver WL. 1985. Inhibition by amiloride of chorda tympani responses evoked by monovalent salts. Brain Res. 334(2):207–214. [DOI] [PubMed] [Google Scholar]
  6. Cain P, Frank ME, Barry MA. 1996. Recovery of chorda tympani nerve function following injury. Exp Neurol. 141(2):337–346. [DOI] [PubMed] [Google Scholar]
  7. Chandrashekar J, Kuhn C, Oka Y, Yarmolinsky DA, Hummler E, Ryba NJ, Zuker CS. 2010. The cells and peripheral representation of sodium taste in mice. Nature. 464(7286):297–301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cheal M, Oakley B. 1977. Regeneration of fungiform taste buds: temporal and spatial characteristics. J Comp Neurol. 172(4):609–626. [DOI] [PubMed] [Google Scholar]
  9. DeSimone JA, Ferrell F. 1985. Analysis of amiloride inhibition of chorda tympani taste response of rat to NaCl. Am J Physiol. 249(1 Pt 2):R52–R61. [DOI] [PubMed] [Google Scholar]
  10. Eylam S, Spector AC. 2002. The effect of amiloride on operantly conditioned performance in an NaCl taste detection task and NaCl preference in C57BL/6J mice. Behav Neurosci. 116(1):149–159. [PubMed] [Google Scholar]
  11. Eylam S, Spector AC. 2003. Oral amiloride treatment decreases taste sensitivity to sodium salts in C57BL/6J and DBA/2J mice. Chem Senses. 28(5):447–458. [DOI] [PubMed] [Google Scholar]
  12. Eylam S, Spector AC. 2005. Taste discrimination between NaCl and KCl is disrupted by amiloride in inbred mice with amiloride-insensitive chorda tympani nerves. Am J Physiol Regul Integr Comp Physiol. 288(5):R1361–R1368. [DOI] [PubMed] [Google Scholar]
  13. Formaker BK, Hill DL. 1991. Lack of amiloride sensitivity in SHR and WKY glossopharyngeal taste responses to NaCl. Physiol Behav. 50(4):765–769. [DOI] [PubMed] [Google Scholar]
  14. Frank ME. 1991. Taste-responsive neurons of the glossopharyngeal nerve of the rat. J Neurophysiol. 65(6):1452–1463. [DOI] [PubMed] [Google Scholar]
  15. Frank ME, Contreras RJ, Hettinger TP. 1983. Nerve fibers sensitive to ionic taste stimuli in chorda tympani of the rat. J Neurophysiol. 50(4):941–960. [DOI] [PubMed] [Google Scholar]
  16. Ganchrow JR, Ganchrow D. 1989. Long-term effects of gustatory neurectomy on fungiform papillae in the young rat. Anat Rec. 225(3):224–231. [DOI] [PubMed] [Google Scholar]
  17. Geran LC, Garcea M, Spector AC. 2004. Nerve regeneration-induced recovery of quinine avoidance after complete gustatory deafferentation of the tongue. Am J Physiol Regul Integr Comp Physiol. 287(5):R1235–R1243. [DOI] [PubMed] [Google Scholar]
  18. Geran LC, Spector AC. 2000a. Amiloride increases sodium chloride taste detection threshold in rats. Behav Neurosci. 114(3):623–634. [DOI] [PubMed] [Google Scholar]
  19. Geran LC, Spector AC. 2000b. Sodium taste detectability in rats is independent of anion size: the psychophysical characteristics of the transcellular sodium taste transduction pathway. Behav Neurosci. 114(6):1229–1238. [PubMed] [Google Scholar]
  20. Guth L. 1957. The effects of glossopharyngeal nerve transection on the circumvallate papilla of the rat. Anat Rec. 128(4):715–731. [DOI] [PubMed] [Google Scholar]
  21. Halpern BP. 1998. Amiloride and vertebrate gustatory responses to NaCl. Neurosci Biobehav Rev. 23(1):5–47. [DOI] [PubMed] [Google Scholar]
  22. Hanamori T, Miller IJ Jr, Smith DV. 1988. Gustatory responsiveness of fibers in the hamster glossopharyngeal nerve. J Neurophysiol. 60(2):478–498. [DOI] [PubMed] [Google Scholar]
  23. Heck GL, Mierson S, DeSimone JA. 1984. Salt taste transduction occurs through an amiloride-sensitive sodium transport pathway. Science. 223(4634):403–405. [DOI] [PubMed] [Google Scholar]
  24. King CT, Garcea M, Spector AC. 2000. Glossopharyngeal nerve regeneration is essential for the complete recovery of quinine-stimulated oromotor rejection behaviors and central patterns of neuronal activity in the nucleus of the solitary tract in the rat. J Neurosci. 20(22): 8426–8434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. King CT, Garcea M, Stolzenberg DS, Spector AC. 2008. Experimentally cross-wired lingual taste nerves can restore normal unconditioned gaping behavior in response to quinine stimulation. Am J Physiol Regul Integr Comp Physiol. 294(3):R738–R747. [DOI] [PubMed] [Google Scholar]
  26. Kitada Y, Mitoh Y, Hill DL. 1998. Salt taste responses of the IXth nerve in Sprague-Dawley rats: lack of sensitivity to amiloride. Physiol Behav. 63(5):945–949. [DOI] [PubMed] [Google Scholar]
  27. Kopka SL, Geran LC, Spector AC. 2000. Functional status of the regenerated chorda tympani nerve as assessed in a salt taste discrimination task. Am J Physiol Regul Integr Comp Physiol. 278(3):R720–R731. [DOI] [PubMed] [Google Scholar]
  28. Lee H, Macpherson LJ, Parada CA, Zuker CS, Ryba NJP. 2017. Rewiring the taste system. Nature. 548(7667):330–333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ma H, Yang R, Thomas SM, Kinnamon JC. 2007. Qualitative and quantitative differences between taste buds of the rat and mouse. BMC Neurosci. 8:5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Nejad MS, Beidler LM. 1987. Taste responses of the cross-regenerated greater superficial petrosal and chorda tympani nerves of the rat. Ann NY Acad Sci. 510:523–526. [Google Scholar]
  31. Ninomiya Y. 1998. Reinnervation of cross-regenerated gustatory nerve fibers into amiloride-sensitive and amiloride-insensitive taste receptor cells. Proc Natl Acad Sci U S A. 95(9):5347–5350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ninomiya Y, Funakoshi M. 1988. Amiloride inhibition of responses of rat single chorda tympani fibers to chemical and electrical tongue stimulations. Brain Res. 451(1–2):319–325. [DOI] [PubMed] [Google Scholar]
  33. Ninomiya Y, Tonosaki K, Funakoshi M. 1982. Gustatory neural response in the mouse. Brain Res. 244(2):370–373. [DOI] [PubMed] [Google Scholar]
  34. Oakley B. 1967. Altered temperature and taste responses from cross-regenerated sensory nerves in the rat’s tongue. J Physiol. 188(3):353–371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Oakley B. 1969. Taste preference following cross-innervation of rat fungiform taste buds. Physiol Behav. 4:929–933. [Google Scholar]
  36. Oakley B, Lawton A, Riddle DR, Wu LH. 1993. Morphometric and immunocytochemical assessment of fungiform taste buds after interruption of the chorda-lingual nerve. Microsc Res Tech. 26(3):187–195. [DOI] [PubMed] [Google Scholar]
  37. Segerstad CHA, Hellekant G, Farbman AI. 1989. Changes in number and morphology of fungiform taste buds in rat after transection of the chorda tympani or chordalingual nerve. Chem Senses. 14:335–348. [Google Scholar]
  38. Shingai T, Beidler LM. 1985. Response characteristics of three taste nerves in mice. Brain Res. 335(2):245–249. [DOI] [PubMed] [Google Scholar]
  39. Smith DV, Som J, Boughter JD Jr, St John SJ, Yu C, Christy RC. 1999. Cellular expression of alpha-gustducin and the A blood group antigen in rat fungiform taste buds cross-reinnervated by the IXth nerve. J Comp Neurol. 409(1):118–130. [DOI] [PubMed] [Google Scholar]
  40. Spector AC. 2003. The functional organization of the peripheral gustatory system: lessons from behavior. In: Fluharty SJ, Grill HJ, editors. Progress in psychobiology and physiological psychology. San Diego (CA): Academic Press; p. 101–161. [Google Scholar]
  41. Spector AC, Blonde G, Garcea M, Jiang E. 2010. Rewiring the gustatory system: specificity between nerve and taste bud field is critical for normal salt discrimination. Brain Res. 1310:46–57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Spector AC, Grill HJ. 1992. Salt taste discrimination after bilateral section of the chorda tympani or glossopharyngeal nerves. Am J Physiol. 263(1 Pt 2):R169–R176. [DOI] [PubMed] [Google Scholar]
  43. Spector AC, Guagliardo NA, St John SJ. 1996. Amiloride disrupts NaCl versus KCl discrimination performance: implications for salt taste coding in rats. J Neurosci. 16(24):8115–8122. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. St John SJ, Garcea M, Spector AC. 2003. The time course of taste bud regeneration after glossopharyngeal or greater superficial petrosal nerve transection in rats. Chem Senses. 28(1):33–43. [DOI] [PubMed] [Google Scholar]
  45. St John SJ, Markison S, Spector AC. 1995. Salt discriminability is related to number of regenerated taste buds after chorda tympani nerve section in rats. Am J Physiol. 269(1 Pt 2):R141–R153. [DOI] [PubMed] [Google Scholar]
  46. St John SJ, Spector AC. 1998. Behavioral discrimination between quinine and KCl is dependent on input from the seventh cranial nerve: implications for the functional roles of the gustatory nerves in rats. J Neurosci. 18(11):4353–4362. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Vintschgau MV, Honingschmid J. 1876. Nervus glossopharyngeus und schmeckbecker. Arch Physiol. 14:443–448. [Google Scholar]
  48. Whiteside B. 1926. The regeneration of the gustatory apparatus in the rat. J Comp Neurol. 40:33–45. [Google Scholar]
  49. Yasumatsu K, Katsukawa H, Sasamoto K, Ninomiya Y. 2003. Recovery of amiloride-sensitive neural coding during regeneration of the gustatory nerve: behavioral-neural correlation of salt taste discrimination. J Neurosci. 23(10):4362–4368. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Chemical Senses are provided here courtesy of Oxford University Press

RESOURCES