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

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.

Restoration of quinine-stimulated Fos-immunoreactive neurons in the central nucleus of the amygdala and gustatory cortex following reinnervation or cross-reinnervation of the lingual taste nerves in rats.

Remarkably, when lingual gustatory nerves are surgically rerouted to inappropriate taste fields in the tongue, some taste functions recover. We previously demonstrated that quinine-stimulated oromotor rejection reflexes and neural activity (assessed by Fos immunoreactivity) in subregions of hindbrain gustatory nuclei were restored if the posterior tongue, which contains receptor cells that respond strongly to bitter compounds, was cross-reinnervated by the chorda tympani nerve. Such functional recovery was not seen if instead, the anterior tongue, where receptor cells are less responsive to bitter compounds, was cross-reinnervated by the glossopharyngeal nerve, even though this nerve typically responds robustly to bitter substances. Thus, recovery depended more on the taste field being reinnervated than on the nerve itself. Here, the distribution of quinine-stimulated Fos-immunoreactive neurons in two taste-associated forebrain areas was examined in these same rats. In the central nucleus of the amygdala (CeA), a rostrocaudal gradient characterized the normal quinine-stimulated Fos response, with the greatest number of labeled cells situated rostrally. Quinine-stimulated neurons were found throughout the gustatory cortex, but a "hot spot" was observed in its anterior-posterior center in subregions approximating the dysgranular/agranular layers. Fos neurons here and in the rostral CeA were highly correlated with quinine-elicited gapes. Denervation of the posterior tongue eliminated, and its reinnervation by either nerve restored, numbers of quinine-stimulated labeled cells in the rostralmost CeA and in the subregion approximating the dysgranular gustatory cortex. These results underscore the remarkable plasticity of the gustatory system and also help clarify the functional anatomy of neural circuits activated by bitter taste stimulation.

The consequences of gustatory deafferentation on body mass and feeding patterns in the rat.

The contribution of orosensory signals, especially taste, on body mass, and feeding and drinking patterns in the rat was examined. Gustatory deafferentation was produced by bilateral transection of the chorda tympani, glossopharyngeal, and greater superficial petrosal nerves. Total calories consumed from sweetened-milk diet and oil-chow mash by the nerve-transected rats significantly decreased relative to sham-operated controls, mostly attributable to decreases in bout number, but not size. Nevertheless, caloric intake steadily increased over the postsurgical observation period, but body mass remained below both presurgical baseline and control levels and did not significantly increase over this time. After the sweetened-milk diet/oil-chow mash phase, rats received a series of sucrose preference tests. Interestingly, the nerve-transected rats preferred sucrose, and intake did not differ from controls, likely due to the stimulus sharing some nontaste chemosensory properties with the sweetened-milk diet. The neurotomized rats initiated a greater number of sucrose-licking bouts that were smaller in size and slower in licking rate, compared with control rats, and, unlike in control rats, the latter two bout parameters did not vary across concentration. Thus, in the absence of gustatory neural input, body mass is more stable compared with the progressive trajectory of weight gain seen in intact rats, and caloric intake initially decreases but recovers. The consequences of gustatory neurotomy on processes that determine meal initiation (bout number) and meal termination (bout size) are not fixed and appear to be influenced by presurgical experience with food stimuli coupled with its nongustatory chemosensory properties.

Learning-based recovery from perceptual impairment in salt discrimination after permanently altered peripheral gustatory input.

Rats lacking input to the chorda tympani (CT) nerve, a facial nerve branch innervating anterior tongue taste buds, show robust impairments in salt discrimination demonstrating its necessity. We tested the sufficiency of the CT for salt taste discrimination and whether the remaining input provided by the greater superficial petrosal (GSP) nerve, a facial nerve branch innervating palatal taste buds, or by the glossopharyngeal (GL) nerve, innervating posterior tongue taste buds, could support performance after extended postsurgical testing. Rats presurgically trained and tested in a two-response operant task to discriminate NaCl from KCl were subjected to sham surgery or transection of the CT (CTx), GL (GLx), or GSP (GSPx), alone or in combination. While initially reduced postsurgically, performance by rats with an intact GSP after CTx + GLx increased to normal over 6 wk of testing. Rats with CTx + GSPx consistently performed near chance levels. In contrast, rats with GSPx + GLx were behaviorally normal. A subset of rats subjected to sham surgery and exposed to lower concentrations during postsurgical testing emulating decreased stimulus intensity after neurotomy showed no significant impairment. These results demonstrate that CTx changes the perceptual nature of NaCl and/or KCl, leading to severe initial postsurgical impairments in discriminability, but a "new" discrimination can be relearned based on the input of the GSP. Despite losing ∼75% of their taste buds, rats are unaffected after GSPx + GLx, demonstrating that the CT is not only necessary, but also sufficient, for maintaining salt taste discrimination, notwithstanding the unlikely contribution of the small percentage of taste receptors innervated by the superior laryngeal nerve.

Rewiring the gustatory system: specificity between nerve and taste bud field is critical for normal salt discrimination.

Forty years have passed since it was demonstrated that a cross-regenerated gustatory nerve in the rat tongue adopts the stimulus-response properties of the taste receptor field it cross-reinnervates. Nevertheless, the functional consequences of channeling peripheral taste signals through inappropriate central circuits remain relatively unexplored. Here we tested whether histologically confirmed cross-regeneration of the chorda tympani nerve (CT) into the posterior tongue in the absence of the glossopharyngeal nerve (GL) (CT-PostTongue) or cross-regeneration of the GL into the anterior tongue in the absence of the CT (GL-AntTongue) would maintain presurgically trained performance in an operant NaCl vs. KCl taste discrimination task in rats. Before surgery all groups were averaging over 90% accuracy. Oral amiloride treatment dropped performance to virtually chance levels. During the first week after surgery, sham-operated rats, GL-transected rats, and rats with regenerated CTs displayed highly competent discrimination performance. In contrast, CT-transected rats were severely impaired (59% accuracy). Both the CT-PostTongue and the GL-AntTongue groups were impaired to a similar degree as CT-transected rats. These initially impaired groups improved their performance over the weeks of postsurgical testing, suggesting that the rats were capable of relearning the task with discriminable signals in the remaining taste nerves. This relearned performance was dependent on input from amiloride-sensitive receptors likely in the palate. Overall, these results suggest that normal competence in a salt discrimination task is dependent on the taste receptor field origin of the input as well as the specific nerve transmitting the signals to its associated circuits in the brain.

Greater superficial petrosal nerve transection in rats does not change unconditioned licking responses to putatively sweet taste stimuli.

The greater superficial petrosal nerve (GSP), innervating taste buds in the palate, is known to be exceptionally responsive to sucrose, especially compared with the responsiveness of the chorda tympani nerve (CT). However, whereas transection of the CT (CTX) alone has little or no effect on unconditioned licking responses to many "sweet" stimuli, the impact of GSP transection (GSPX) alone is equivocal. To further examine the role of the GSP on licking responses to putatively sweet-tasting substances, brief-access taste tests were conducted in nondeprived rats before and after sham surgery (SHAM) or CTX or GSPX. A range of concentrations of sucrose, L-alanine, glycine, and L-serine, with and without 1.0 mM inosine monophosphate (IMP) added, were used. All groups showed significant concentration-dependent increases in licking to all stimuli presurgically and postsurgically. CTX decreased licking responses relative to SHAM rats in the first sucrose test. There was also a group x concentration interaction for L-alanine, but post hoc tests did not reveal its basis. Other than this, there were no significant differences among the surgical groups. Interestingly, rats with GSPX tended to initiate fewer trials than SHAM rats. Overall, after GSPX, the remaining gustatory nerves are apparently sufficient to maintain concentration-dependent licking responses to all stimuli tested here. The disparity between our results and others in the literature where GSPX reduced licking responses to sucrose is possibly related to differences in surgical technique or test trial duration.

Experimentally cross-wired lingual taste nerves can restore normal unconditioned gaping behavior in response to quinine stimulation.

Studies examining the effects of transection and regeneration of the glossopharyngeal (GL) and chorda tympani (CT) nerves on various taste-elicited behaviors in rats have demonstrated that the GL (but not the CT) nerve is essential for the maintenance of both an unconditioned protective reflex (gaping) and the neural activity observed in central gustatory structures in response to lingual application of a bitter substance. An unresolved issue, however, is whether recovery depends more on the taste nerve and the central circuits that it supplies and/or on the tongue receptor cell field being innervated. To address this question, we experimentally cross-wired these taste nerves, which, remarkably, can regenerate into parts of the tongue they normally do not innervate. We report that quinine-stimulated gaping behavior was fully restored, and neuronal activity, as assessed by Fos immunohistochemistry in the nucleus of the solitary tract and the parabrachial nucleus, was partially restored only if the posterior tongue (PT) taste receptor cell field was reinnervated; the particular taste nerve supplying the input was inconsequential to the recovery of function. Thus, PT taste receptor cells appear to play a privileged role in triggering unconditioned gaping to bitter tasting stimuli, regardless of which lingual gustatory nerve innervates them. Our findings demonstrate that even when a lingual gustatory nerve (the CT) forms connections with taste cells in a non-native receptor field (the PT), unconditioned taste rejection reflexes to quinine can be maintained. These findings underscore the extraordinary ability of the gustatory system to adapt to peripherally reorganized input for particular behaviors.

The relative effects of transection of the gustatory branches of the seventh and ninth cranial nerves on NaCl taste detection in rats.

Chorda tympani nerve (CT) transection in rats severely impairs NaCl taste detection. These rats can detect higher concentrations of NaCl, however, suggesting that remaining oral nerves maintain some salt sensibility. Rats were tested in a gustometer with a 2-response operant taste-detection task before and after sham surgery (n = 5), combined transection of the CT and the greater superficial petrosal nerves (GSP; 7x, n = 6), or transection of the glossopharyngeal nerve (GL; 9x, n = 4). Thresholds did not significantly change after sham surgery. Although the GL responds to NaCl and innervates nearly 60% of total taste buds, 9x surgery had no effect. However, 7x surgery increased NaCl detection threshold by approximately 2.5 log(10) units, greater than that reported for CT transection alone. These results suggest that the GSP contributes to NaCl sensitivity in rats and also demonstrate that the GL and perhaps the superior laryngeal and lingual nerve proper can maintain some NaCl detectability at high concentrations. These findings confirm the primacy of the 7th nerve relative to the 9th nerve in sensibility of NaCl in the rat model.

Effects of selective lingual gustatory deafferentation on suprathreshold taste intensity discrimination of NaCl in rats.

In rats, chorda tympani nerve transection (CTX) greatly increases the detection threshold of sodium chloride (NaCl) and severely disrupts salt discriminability. Here it is shown that CTX has surprisingly little effect, if any, on suprathreshold intensity discrimination. Glossopharyngeal nerve transection (GLX), which has no reported effect on salt sensibility, also did not affect performance. Rats were tested in a 2-response, operant taste intensity discrimination task. Difference thresholds for CTX rats were only slightly higher (-0.15 log/10 unit) than those for GLX and sham-transected rats, when 0.05 M served as the standard, and did not significantly differ when 0.1 M NaCl was the standard. Although the perceived intensity of NaCl might be reduced by CTX, input from remaining taste nerves sufficiently maintains the relative discriminability of suprathreshold NaCl concentrations.

Nerve regeneration-induced recovery of quinine avoidance after complete gustatory deafferentation of the tongue.

The concentration-dependent decrease in quinine licking by rats is substantially attenuated by combined bilateral transection of the chorda tympani (CT) and glossopharyngeal (GL) nerves, but transection of either nerve alone produces marginal impairments at most. Here we tested whether regeneration of one or both of these nerves after combined transection would result in recovery of taste avoidance. Water-restricted rats were presented with a series of brief-access (5 s) taste trials (water and 0.003-3.0 mM quinine-HCl) in a 5-day test block of 40-min sessions both before nerve transection and starting 75-77 days after transection. Licking avoidance returned to presurgical levels when both nerves were allowed to regenerate. When only the GL was allowed to regenerate, performance did not differ from that of sham-transected animals. This suggests that even after considerable gustatory deafferentation, regeneration has the capacity to restore normal taste-guided behavior. Surprisingly, when only the CT was allowed to regenerate, avoidance behavior was severely impaired and was not different from that of rats in which regeneration of both nerves was prevented. Taking into account prior findings, it appears that the absence of the GL in the presence of an intact CT is fundamentally different from the absence of the GL in the presence of a regenerated CT with respect to some taste functions. This represents the first reported instance to our knowledge in which the capacity of a regenerated nerve to maintain taste-guided behavior was distinctly different from that of an intact nerve in a rodent model.

Amiloride is an ineffective conditioned stimulus in taste aversion learning in C57BL/6J and DBA/2J mice.

The epithelial sodium channel (ENaC) blocker amiloride has been shown to increase the behaviorally measured NaCl detection threshold in mice. In this study, a conditioned taste aversion (CTA) paradigm was used to examine whether 100 microM amiloride has a perceptible taste that could contribute to this observed decrease in behavioral responsiveness. Eighty-four C57BL/6J (B6) and 64 DBA/2J (D2) mice were divided into eight groups (n=8-12 per group), in which half received an injection of 0.15 M LiCl (2 mEq/kg) and the other half an equivalent saline injection, in three conditioning trials. The four conditioned stimuli were 100 microM amiloride hydrochloride, water, 0.1 and 0.3 M NaCl. Neither strain demonstrated acquisition of a CTA to amiloride in a brief-access (BA) taste test (5 s trials in the gustometer). Although 0.3 M NaCl is inherently aversive, its pairing with LiCl led to significantly further decreases in licking during the BA test on salt trials in both strains. The D2 strain clearly avoided 0.1 M NaCl, whereas avoidance of this stimulus was more equivocal in B6 mice. The inefficacy of amiloride to serve as a conditioned stimulus in taste aversion learning involving three LiCl pairings suggests that the effects of this ENaC blocker on taste-related behavioral responses to NaCl are likely due to its pharmacological interference with sodium taste transduction.

Effects of gustatory nerve transection and regeneration on quinine-stimulated Fos-like immunoreactivity in the parabrachial nucleus of the rat.

The distribution of quinine-stimulated Fos-like immunoreactivity (FLI) in several subdivisions of the parabrachial nucleus (PBN) known to be responsive to gustatory stimulation was examined in rats in which the chorda tympani nerve (CT) and/or glossopharyngeal nerve (GL) was transected (Experiment 1) and in rats in which the GL was transected with regeneration promoted or prevented (Experiment 2). We confirmed previous findings in the literature by demonstrating that rats intraorally infused with 3 mM quinine showed a robust population of FLI in the waist area and the external lateral (EL) and external medial (EM) subdivisions of the PBN (Yamamoto et al. [1994] Physiol Behav 56:1197-1202; Travers et al., [ 1999] Am J Physiol 277:R384-R394). In the waist area, only GL transection significantly decreased the number of FLI-neurons elicited by intraoral infusion of quinine compared with water-stimulated controls. In the external subdivisions neither neurotomy affected the number of FLI-neurons. The effect of GL transection in the waist area was enduring for rats in which the GL did not regenerate (up to 94 days), but regeneration of the GL after 52 days restored quinine-stimulated FLI to control values. In these same GL-transected animals, there were parallel decreases in the number of gapes elicited by intraoral quinine stimulation that recovered, but only subsequent to regeneration of the GL. These data provide support for the role of the waist area in the brainstem processing that underlies oromotor rejection behaviors and also help substantiate the hypothesis that the CT and GL are relatively specialized with regard to function. Moreover, when the quinine-induced pattern of neural activity in the second central gustatory relay, as assessed by FLI, is substantially altered by the loss of peripheral gustatory input from the GL, it can be restored upon regeneration of the nerve.

The time course of taste bud regeneration after glossopharyngeal or greater superficial petrosal nerve transection in rats.

We previously have published data detailing the time course of taste bud regeneration in the anterior tongue following transection of the chorda tympani (CT) nerve in the rat. This study extends the prior work by determining the time course of taste bud regeneration in the vallate papilla, soft palate and nasoincisor ducts (NID) following transection of either the glossopharyngeal (GL) or greater superficial petrosal (GSP) nerve. Following GL transection in rats (n = 6 per time point), taste buds reappeared in the vallate papilla between 15 and 28 days after surgery, and returned to 80.3% of control levels (n = 12) of taste buds by 70 days postsurgery. The first appearance and the final percentage of the normal complement of regenerated vallate taste buds after GL transection resembled that seen previously in the anterior tongue after CT transection. However, in the latter case, regenerated taste buds reached asymptotic levels by 42 days after surgery, whereas within the time frame of the present study, a clear asymptotic return of vallate taste buds was not observed. In contrast to the posterior (and anterior) tongue, only 25% of the normal complement of palatal taste buds regenerated by 112 days and 224 days after GSP transection (n = 9). The difference in regenerative capacity might relate to the surgical approach used to transect the GSP. These experiments provide useful parametric data for investigators studying the functional consequences of gustatory nerve transection and regeneration.

Transecting the gustatory branches of the facial nerve impairs NH(4)Cl vs. KCl discrimination in rats.

Ammonium and potassium chloride share a common taste quality and an amiloride-insensitive route of transduction. An amiloride-sensitive pathway might also be partially activated by these salts, although very few studies have reported effects of amiloride on nonsodium salt perception. This experiment was designed to determine 1) whether rats could discriminate KCl from NH(4)Cl and, if discrimination was evident, whether performance was impaired with 2) amiloride or 3) gustatory nerve transection. Rats were trained to discriminate KCl from NH(4)Cl (n = 8) and NaCl from NH(4)Cl (n = 8). Amiloride (100 microM) impaired NaCl vs. NH(4)Cl but not KCl vs. NH(4)Cl performance, whereas both groups showed significant impairments after transection of the chorda tympani (CT) and greater superficial petrosal (GSP) branches of the facial nerve. This suggests that rats can discriminate between KCl and NH(4)Cl and that this discrimination does not rely on an amiloride-sensitive mechanism but does depend on the CT and/or GSP nerves. This experiment supports the hypothesis that the facial nerve is important for salt taste recognition and discrimination.