Neurophysiological and biophysical evidence on the mechanism of electric taste

The phenomenon of electric taste was investigated by recording from the chorda tympani nerve of the rat in response to both electrical and chemical stimulations of the tongue with electrolytes in order to gain some insight into its mechanism on both a neurophysiological and biophysical basis. The maximum neural response levels were identical for an individual salt (LiCl, NaCl, KCl, or CaCl2), whether it was presented as a chemical solution or as an anodal stimulus through a subthreshold solution. These observations support the idea that stimulation occurs by iontophoresis of ions to the receptors at these current densities (less than 100 microA/cm2). Electric responses through dilute HCl were smaller than the chemically applied stimulations, but the integrated anodal responses appeared similar to chemical acid responses, as evidenced by an OFF response to both forms of stimuli. Hydrogen may be more permeant to the lingual epithelium and would thus be shunted away from the taste receptors during anodal stimulation. When the anion of electric taste was varied via subthreshold salt solutions, the response magnitude increased as the mobility of the anion decreased. The transport numbers of the salts involved adequately explains these differences. The physical aspects of ion migration occurring within the adapting fluid on the tongue are also discussed. Direct neural stimulation by the current appears to occur only at higher current densities (greater than 300 microA/cm2). If the taste cells of the tongue were inactivated with either iodoacetic acid (IAA) or N-ethyl maleimide (NEM), or removed with collagenase, then responses from the chorda tympani could be obtained only at these higher current densities. Latency measurements before and after IAA or NEM treatment corroborated these findings. The results are discussed in terms of several proposed mechanisms of electric taste and it is concluded that an ion accumulation mechanism can adequately explain the data.     

Developmental changing of rat taste responses from chorda tympani nerve

In order to clarify developmental changing of gustatory system, histological and electrophysiological experiments were performed in the rat. Histological examination on the anterior tongue innervated by chorda tympani nerve showed that the ratio of matured taste buds which possess a taste pore were only 9% of all taste buds observed at 1 week of postnatal age, and 81.3% at 3 weeks of age. Recording integrated responses from the chorda tympani nerve reveals that taste buds with a pore at 1 week of age responded to NaCl, HCl, and quinine-HCl as well as in adult rats, which suggests that these relatively young taste buds are matured functionally for these three stimuli. However, the response magnitudes for various sugars at 1 week of age were smaller compared to those in the adult rat, reached to the maximum at 3 weeks of age, then decreased gradually with age. Also, results from the experiment of cross-adaptation among different sugars, effects of pronase-E treatment of the tongue, analysis of correlation between on- and off-responses to sugars, showed that qualitative changes for sugar responses continues after 3 weeks of age. These results suggest that functional changes occur in the gustatory processing of sugars during postnatal development in the rat chorda tympani nerve.     

Sour taste responses in mice lacking PKD channels

Background

The polycystic kidney disease-like ion channel PKD2L1 and its associated partner PKD1L3 are potential candidates for sour taste receptors. PKD2L1 is expressed in type III taste cells that respond to sour stimuli and genetic elimination of cells expressing PKD2L1 substantially reduces chorda tympani nerve responses to sour taste stimuli. However, the contribution of PKD2L1 and PKD1L3 to sour taste responses remains unclear.

Methodology/Principal Findings

We made mice lacking PKD2L1 and/or PKD1L3 gene and investigated whole nerve responses to taste stimuli in the chorda tympani or the glossopharyngeal nerve and taste responses in type III taste cells. In mice lacking PKD2L1 gene, chorda tympani nerve responses to sour, but not sweet, salty, bitter, and umami tastants were reduced by 25–45% compared with those in wild type mice. In contrast, chorda tympani nerve responses in PKD1L3 knock-out mice and glossopharyngeal nerve responses in single- and double-knock-out mice were similar to those in wild type mice. Sour taste responses of type III fungiform taste cells (GAD67-expressing taste cells) were also reduced by 25–45% by elimination of PKD2L1.

Conclusions/Significance

These findings suggest that PKD2L1 partly contributes to sour taste responses in mice and that receptors other than PKDs would be involved in sour detection.     

Effects of isoproterenol treatment on gustatory neural responses in three inbred strains of mice

1. Treatment of a beta-agonist, isoproterenol, for 5 days reduced chorda tympani responses to sucrose by about 40% of the control without affecting responses to other taste stimuli, such as NaCl, HCl and quinine HCl, in balb CrSlc mice whereas such reduction of sucrose responses was not observed in C57BL/6-CrSlc and C3H/HeSlc mice, although in the latter two strains long-lasting off-responses to quinine HCl appeared after the treatment. 2. In BALB mice, the magnitude of reduction of sucrose responses by isoproterenol increased with prolonging the treatment from 1 to 5 days, although it reached almost its maximum level by the 3 days treatment. 3. BALB mice with the removal of the submandibular glands showed slightly greater control responses of the chorda tympani nerve to sucrose than BALB mice with the sham-operation or the removal of the sublingual glands, and showed no significant reduction of sucrose responses by isoproterenol treatment. 4. These results suggest that isoproterenol probably did not act directly on sweetener receptors of taste cell membranes but affect them through the submandibular salivary system.     

A study on the gustatory effects of tetrodotoxin in rat (author's transl)]

Effects of tetrodotoxin (TTX) on neural responses of the chorda tympani to four basic taste stimuli were investigated electrophysiologically in rats. When the TTX (10 mg/ml) was applied directly to the tongue surface for 3 minutes, magnitude of the integrated responses of the chorda tympani was diminished to about 60% of that of the control response. This diminution of response was recovered within 30 minutes by degrees and the effect of the TTX was antagonized by guanylate. This result gives a suggestion that guanidyl group in the TTX may play an important role for the inhibitory actions to the responses of the chorda tympani. On the other hand, when the TTX (0.25 mg/100 g b. wt.) was applied intravenously, magnitude of the responses of the chorda tympani to four basic taste stimuli decreased gradually to 20 approximately 30% of that of the control responses within 60 minutes and did not recover more than 10 hours. This is assumed due to the blocking of the sodium pump of nerve fibers in the chorda tympani by the TTX.     

Gustatory neural responses to umami taste stimuli in C57BL/6ByJ and 129P3/J mice

In long-term two-bottle tests, mice from the C57BL/6ByJ (B6) strain drink more monosodium L-glutamate (MSG) and inosine-5'-monophosphate (IMP) compared with mice from the 129P3/J (129) strain. The goal of this study was to assess the role of afferent gustatory input in these strain differences. We measured integrated responses of the mouse chorda tympani and glossopharyngeal nerves to lingual application of compounds that evoke umami taste in humans: MSG, monoammonium L-glutamate (NH(4) glutamate), IMP and guanosine-5'-monophosphate (GMP) and also to other taste stimuli. Chorda tympani responses to MSG and NH(4) glutamate were similar in B6 and 129 mice. Chorda tympani responses to IMP and GMP were lower in B6 than in 129 mice. Responses to umami stimuli in the glossopharyngeal nerve did not differ between the B6 and 129 strains. Responses to MSG, IMP and GMP were not affected by sodium present in these compounds because B6 and 129 mice had similar neural taste responses to NaCl. This study has demonstrated that the increased ingestive responses to the umami stimuli in B6 mice are accompanied by either unchanged or decreased neural responses to these stimuli. Lack of support for the role of the chorda tympani or glossopharyngeal nerves in the enhanced consumption of MSG and IMP by B6 mice suggests that it is due to some other factors. Although results of our previous study suggest that postingestive effects of MSG can affect its intake, contribution of other gustatory components (e.g. greater superficial petrosal nerve or central gustatory processing) to the strain differences in consumption of umami compounds also cannot be excluded. Strain differences in gustatory neural responses to nucleotides but not glutamate suggest that these compounds may activate distinct taste transduction mechanisms.     

Peripheral neural basis for behavioural discrimination between glutamate and the four basic taste substances in mice

1. Single chorda tympani fibres sensitive to monosodium L-glutamate (MSG), elicit a unique taste in humans and gave a greater response to NaCl and/or sucrose than to MSG whereas several MSG-sensitive glossopharyngeal fibres responded only slightly if at all to NaCl and sucrose. 2. The across-fibre correlations showed that MSG and NaCl produced similar response patterns in the chorda tympani fibres but different response patterns in the glossopharyngeal fibres. 3. These results suggest that taste information of glossopharyngeal fibres plays a relatively more important role in the qualitative discrimination between MSG and the four basic taste substances than that of chorda tympani fibres.     

Taste responses of the gerbil IXth nerve

Summated taste responses to 12 taste solutions were recordedfrom the IXth (glossopharyngeal) nerve of 38 Mongolian gerbils(Meriones unguiculatus). 0.3 M NH₄Cl was the most effectivestimulant. The relative magnitude of the peak summated responsewas a positively accelerated function of log molar concentration.Absolute thresholds were determined for three chemicals: 0.002M NaCl, 0.0003 M HCl, and 0.002 M sucrose. The relative magnitudesof the responses to quinine, NH₄Cl, and KCl were greater forthe IXth nerve than for the chorda tympani nerve, whereas NaClwas more effective for the chorda tympani. A similar patternis seen in the rat. Acetic and citric acid may bind to commonreceptor sites. NH₄Cl, KCl, and HCl may also have receptor sitesin common.     

Lack of functional and morphological susceptibility of the greater superficial petrosal nerve to developmental dietary sodium restriction

Restriction of dietary sodium during gestation has major effects on taste function and anatomy in the offspring. The chorda tympani nerve of offspring that are maintained on sodium-reduced chow throughout life (NaDep) has reduced neurophysiological responses to sodium and altered morphology of its terminal field in the nucleus of the solitary tract. There are many anatomical and physiological similarities between the chorda tympani nerve that innervates taste buds on the anterior tongue and the greater superficial petrosal nerve (GSP) that innervates taste buds on the palate. To determine if the GSP is similarly susceptible to the effects of dietary sodium restriction, the present study examined neurophysiological responses and the terminal field of the GSP in NaDep and control rats. Neurophysiological responses of the GSP to a variety of sodium and non-sodium stimuli did not differ between NaDep and control rats. Furthermore, the volume and shape of the GSP terminal field in the nucleus of the solitary tract did not differ between the groups. Therefore, despite the high degree of functional and anatomical correspondence between the chorda tympani nerve and the GSP, the GSP does not appear to be susceptible to the effects of lifelong dietary sodium restriction.     

Effects of chorda tympani nerve anesthesia on taste responses in the NST

Human clinical and psychophysical observations suggest that the taste system is able to compensate for losses in peripheral nerve input, since patients do not commonly report decrements in whole mouth taste following chorda tympani nerve damage or anesthesia. Indeed, neurophysiological data from the rat nucleus of the solitary tract (NST) suggests that a release of inhibition (disinhibition) may occur centrally following chorda tympani nerve anesthesia. Our purpose was to study this possibility further. We recorded from 59 multi- and single- unit taste-responsive sites in the rat NST before, during and after recovery from chorda tympani nerve anesthesia. During anesthesia, average anterior tongue responses were eliminated but no compensatory increases in palatal or posterior tongue responses were observed. However, six individual sites displayed increased taste responsiveness during anesthesia. The average increase was 32.9%. Therefore, disinhibition of taste responses was observed, but infrequently and to a small degree in the NST At a subset of sites, chorda tympani-mediated responses decreased while greater superficial petrosal-mediated responses remained the same during anesthesia. Since this effect was accompanied by a decrease in spontaneous activity, we propose that taste compensation may result in part by a change in signal-to-noise ratio at a subset of sites.      

Sugar-activated ion transport in canine lingual epithelium. Implications for sugar taste transduction

There is good evidence indicating that ion-transport pathways in the apical regions of lingual epithelial cells, including taste bud cells, may play a role in salt taste reception. In this article, we present evidence that, in the case of the dog, there also exists a sugar-activated ion-transport pathway that is linked to sugar taste transduction. Evidence was drawn from two parallel lines of experiments: (a) ion-transport studies on the isolated canine lingual epithelium, and (b) recordings from the canine chorda tympani. The results in vitro showed that both mono- and disaccharides in the mucosal bath stimulate a dose-dependent increase in the short-circuit current over the concentration range coincident with mammalian sugar taste responses. Transepithelial current evoked by glucose, fructose, or sucrose in either 30 mM NaCl or in Krebs-Henseleit buffer (K-H) was partially blocked by amiloride. Among current carriers activated by saccharides, the current response was greater with Na than with K. Ion flux measurements in K-H during stimulation with 3-O-methylglucose showed that the sugar-evoked current was due to an increase in the Na influx. Ouabain or amiloride reduced the sugar-evoked Na influx without effect on sugar transport as measured with tritiated 3-O-methylglucose. Amiloride inhibited the canine chorda tympani response to 0.5 M NaCl by 70-80% and the response to 0.5 M KCl by approximately 40%. This agreed with the percent inhibition by amiloride of the short-circuit current supported in vitro by NaCl and KCl. Amiloride also partially inhibited the chorda tympani responses to sucrose and to fructose. The results indicate that in the dog: (a) the ion transporter subserving Na taste also subserves part of the response to K, and (b) a sugar-activated, Na-preferring ion-transport system is one mechanism mediating sugar taste transduction. Results in the literature indicate a similar sweet taste mechanism for humans.     

Taste transduction mechanism: similar effects of various modifications of gustatory receptors on neural responses to chemical and electrical stimulation in the frog

Responses in the frog glossopharyngeal nerve induced by electrical stimulation of the tongue were compared with those induced by chemical stimuli under various conditions. (a) Anodal stimulation induced much larger responses than cathodal stimulation, and anodal stimulation of the tongue adapted to 5 mM MgCl2 produced much larger responses than stimulation with the tongue adapted to 10 mM NaCl at equal current intensities, as chemical stimulation with MgCl2 produced much larger responses than stimulation with NaCl at equal concentration. (b) The enhansive and suppressive effects of 8-anilino-1-naphthalenesulfonate, NiCl2, and uranyl acetate on the responses to anodal current were similar to those on the responses to chemical stimulation. (c) Anodal stimulation of the tongue adapted to 50 mM CaCl2 resulted in a large response, whereas application of 1 M CaCl2 to the tongue adapted to 50 mM CaCl2 produced only a small response. This, together with theoretical considerations, suggested that the accumulation of salts on the tongue surface is not the cause of the generation of the response to anodal current. (d) Cathodal current suppressed the responses induced by 1 mM CaCl2, 0.3 M ethanol, and distilled water. (e) The addition of EGTA or Ca-channel blockers (CdCl2 and verapamil) to the perfusing solution of the lingual artery reversibly suppressed both the responses to chemical stimulus (NaCl) and to anodal current with 10 mM NaCl. (f) We assume from the results obtained that electrical current from the microvillus membrane of a taste cell to the synaptic area supplied by anodal stimulation or induced by chemical stimulation activates the voltage-dependent Ca channel at the synaptic area.     

Response properties of macaque monkey chorda tympani fibers

Many of the chorda tympani fibers of crab-eating monkeys respond to more than one of the four basic stimuli (NaCl, sucrose, HCl, and quinine hydrochloride) as well as cooling or warming of the tongue. Fibers could be classified into four categories depending on their best sensitivity to any one of the four basic stimuli. Sucrose-best and quinine-best fibers are rather specifically sensitive to sucrose and quinine, respectively, while salt-best and acid-best fibers respond relatively well to HCl and NaCl, respectively. Saccharin, dulcin, and Pb acetate produce a good response in sucrose-best fibers, but quinine- best and salt-best fibers also respond to saccharin. Highly significant positive correlations exist between amounts of responses to sucrose and those to saccharin, dulcin, and Pb acetate, indicating that these substances produce in the monkey a taste quality similar to that produced by sucrose. Compared with chroda tympani fibers of rats, hamsters, and squirrel monkeys, macaque monkey taste fibers are more narrowly tuned to one of the four basic taste stimuli and more highly developed in sensitivity to various sweet-tasting substances. Also LiCl and NaCl are more effective stimuli for gustatory receptors in macaque monkeys than NH4Cl and KCl. This contrasts with a higher sensitivity to KCl and NH4Cl than to NaCl in chorda tympani fibers of squirrel monkeys.     

Behavioral discrimination between glutamate and the four basic taste substances in mice

1. Behavioural studies using the conditioned taste aversion (CTA) paradigm in mice showed that aversion conditioned to monosodium L-glutamate (MSG), which elicits a unique taste in humans, did not strongly generalize to any of the four basic taste stimuli, suggesting that mice could behaviourally discriminate between MSG and the four basic taste stimuli. 2. Denervation of bilateral glossopharyngeal nerve significantly increased behavioural similarities (the strength of generalization in the CTA paradigm) between MSG and sodium salts. This was not the case after destruction of the bilateral chorda tympani nerve. 3. These results suggest that taste information of glossopharyngeal nerve plays a more important role in the behavioural discrimination between MSG and the four basic tastes than does that of the chorda tympani nerve.     

Anion modulation of taste responses in sodium-sensitive neurons of the hamster chorda tympani nerve

Beidler's work in the 1950s showed that anions can strongly influence gustatory responses to sodium salts. We have demonstrated "anion inhibition" in the hamster by showing that the chorda tympani nerve responds more strongly to NaCl than to Na acetate over a wide range of concentrations. Iontophoretic presentation of Cl- and acetate to the anterior tongue elicited no response in the chorda tympani, suggesting that these anions are not directly stimulatory. Drugs (0.01, 1.0, and 100 microM anthracene-9-carboxylate, diphenylamine-2-carboxylate, 4- acetamido-4'-isothiocyanatostilbene-2,2'-disulfonate, and furosemide) that interfere with movements of Cl- across epithelial cells were ineffective in altering chorda tympani responses to 0.03 M of either NaCl or Na acetate. Anion inhibition related to movements of anions across epithelial membranes therefore seems unlikely. The chorda tympani contains a population of nerve fibers highly selective for Na+ (N fibers) and another population sensitive to Na+ as well as other salts and acids (H fibers). We found that N fibers respond similarly to NaCl and Na acetate, with spiking activity increasing with increasing stimulus concentration (0.01-1.0 M). H fibers, however, respond more strongly to NaCl than to Na acetate. Furthermore, H fibers increase spiking with increases in NaCl concentration, but generally decrease their responses to increasing concentrations of Na acetate. It appears that anion inhibition applies to taste cells innervated by H fibers but not by N fibers. Taste cells innervated by N fibers use an apical Na+ channel, whereas those innervated by H fibers may use a paracellularly mediated, basolateral site of excitation.     

Whole nerve chorda tympani responses to sweeteners in C57BL/6ByJ and 129P3/J mice

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.     

Strain differences in amiloride inhibition of NaCl responses in mice,Mus musculus

Summary The effects of lingual treatment with amiloride, an inhibitor of salt taste responses in several mammalian species, on NaCl responses of the chorda tympani nerve were compared between four inbred strains of mouse (BALB/cCrSlc, DBA/2CrSlc, C57BL/6CrSlc and C3H/HeSlc). In C57BL and C3H mice amiloride significantly suppressed responses of the chorda tympani nerve to NaCl at a concentration 0.1 M or more whereas in BALB and DBA mice the drug did not significantly affect the responses to NaCl at any concentration, suggesting a lack of the amiloride-sensitive receptor component for NaCl in the latter two strains.A two-bottle preference test demonstrated that all strains of mouse usually showed no preference for NaCl at any concentration and avoided NaCl at 0.3 M or more, although some differences were observed in that C57BL and C3H mice showed aversive responses to 0.1 and 0.15 M NaCl, whereas BALB and DBA mice were indifferent to these solutions.The results suggest that there exist prominent differences between mouse strains in the amiloride-sensitive component of their salt receptor systems. However, in mice the taste information derived from the amiloride-sensitive receptor component probably has no remarkable effect on behavioral responses to NaCl except for a possible contribution to decreasing aversion thresholds for NaCl by increasing overall taste information about NaCl.     

Expression of amiloride-sensitive epithelial sodium channels in mouse taste cells after chorda tympani nerve crush

Our previous electrophysiological study demonstrated that amiloride-sensitive (AS) and -insensitive (AI) components of NaCl responses recovered differentially after the mouse chorda tympani (CT) was crushed. AI responses reappeared earlier (at 3 weeks after the nerve crush) than did AS ones (at 4 weeks). This and other results suggested that two salt-responsive systems were differentially and independently reformed after nerve crush. To investigate the molecular mechanisms of formation of the salt responsive systems, we examined expression patterns of three subunits (alpha, beta and gamma) of the amiloride-sensitive epithelial Na(+) channel (ENaC) in mouse taste cells after CT nerve crush by using in situ hybridization (ISH) analysis. The results showed that all three ENaC subunits, as well as alpha-gustducin, a marker of differentiated taste cells, were expressed in a subset of taste bud cells from an early stage (1-2 weeks) after nerve crush, although these taste buds were smaller and fewer in number than for control mice. At 3 weeks, the mean number of each ENaC subunit and alpha-gustducin mRNA-positive cells per taste bud reached the control level. Also, the size of taste buds became similar to those of the control mice at this time. Our previous electrophysiological study demonstrated that at 2 weeks no significant response of the nerve to chemical stimuli was observed. Thus ENaC subunits appear to be expressed prior to the reappearance of AI and AS neural responses after CT nerve crush. These results support the view that differentiation of taste cells into AS or AI cells is initiated prior to synapse formation.     

Sensitivities of single nerve fibers in the hamster chorda tympani to mixtures of taste stimuli

Responses of three groups of neural fibers from the chorda tympani of the hamster to binary mixtures of taste stimuli applied to the tongue were analyzed. The groups displayed different sensitivities to six chemicals at concentrations that had approximately equal effects on the whole nerve. Sucrose-best fibers responded strongly only to sucrose and D-phenylalanine. NaCl-best and HCl-best fibers, responded to four electrolytes: equally to CaCl2 and nearly equally to HCl, but the former responded more to NaCl, and the latter responded more to NH4Cl. The groups of fibers dealt differently with binary mixtures. Sucrose- best fibers responded to a mixture of sucrose and D-phenylalanine as if one of the chemicals had been appropriately increased in concentration, but they responded to a mixture of either one and an electrolyte as if the concentration of sucrose or D-phenylalanine had been reduced. NaCl- best fibers responded to a mixture as if it were a "mixture" of two appropriate concentrations of one chemical, or somewhat less. But, responses of HCl-best fibers to mixtures were greater than that, approaching a sum of responses to components. These results explain effects on the whole nerve, suggest that the sensitivity of a mammalian taste receptor to one chemical can be affected by a second, which may or may not be a stimulus for that receptor, and suggest that some effects of taste mixtures in humans may be the result of peripheral processes.     

Behavioural and electrophysiological experiments on the sense of taste in Saguinus midas tamarin (Callitrichidae) (author's transl)]

The activity in the gustatory nerve from the anterior part of the tongue, the chorda tympani proper nerve, has been recorded during stimulation of the tongue of a New World monkey, Saguinus midas tamarin. A series of 0.3 M sugars and 0.07 M NaCl, 0.07 M sucrose, 0.004 M acetic acid and 0.00005 M quinine hydrochloride were used as taste stimuli. The concentrations of the last four stimuli were the same as those earlier found to be the lowest concentrations at which this monkey in behavioural experiments discriminates between them and water. The records showed that these threshold concentrations all elicited a neural response. Further, the neural activities during stimulation with the series of 0.3 M sugars were recorded. Using the amplitudes of the responses as a measure, the order between them was found to be fructose greater than glucose greater than lactose greater than arabinose greater than sucrose = galactose greater than raffinose. This order was discussed and related with the order found in behavioural experiments. With both methods, fructose seemed to be the strongest stimulus, but then the order among the sugars differed.