Stimulation of bitterness by capsaicin and menthol: differences between lingual areas innervated by the glossopharyngeal and chorda tympani nerves

Capsaicin is viewed as a purely chemesthetic stimulus that selectively stimulates the somatosensory system. Here we show that when applied to small areas of the tongue, capsaicin can produce a bitter taste as well as sensory irritation. In experiment 1, individuals were screened for the ability to perceive bitterness from capsaicin on the circumvallate papillae. Fifteen of 25 subjects who reported at least weak bitterness rated the intensity of taste, irritation and coolness produced by 100-320 microM capsaicin and 100-320 mM menthol applied via cotton swabs to the tip (fungiform region), the posterior edge (foliate region), and the dorsal posterior surface (circumvallate region) of the tongue. Sucrose, citric acid, sodium chloride and quinine hydrochloride were applied to the same areas to assess tastes responsiveness. On average, capsaicin and menthol produced "moderate" bitterness (and no other significant taste qualities) in the circumvallate region, and weaker bitterness on the side and tip of the tongue. Sensory irritation from capsaicin was rated significantly higher at the tongue tip, whereas menthol coolness was rated higher in the circumvallate region. In experiment 2 we applied sucrose and quinine hydrochloride together with capsaicin to investigate the effects other taste stimuli might have on capsaicin's reported bitterness. As expected, adding quinine produced stronger bitterness in the circumvallate and fungiform regions, and adding sucrose significantly reduced the bitterness of capsaicin in the circumvallate region. Overall, the results suggest that capsaicin and menthol are capable of stimulating a subset of taste neurons that respond to bitter substances, perhaps via receptor-gated ion channels like those recently found in capsaicin- and menthol-sensitive trigeminal ganglion neurons, and that the glossopharyngeal nerve may contain more such neurons than the chorda tympani nerve. That some people fail to perceive bitterness from capsaicin further implies that the incidence of capsaicin-sensitive taste neurons varies across people as well as between gustatory nerves.     

Intranasal localizability of odorants: influence of stimulus volume

When an odorant is presented to one side of the nose and air to the other, the ability to localize which side received the odorant depends upon trigeminal nerve stimulation. It has been shown that performance on this lateralization task increases as stimulus concentration increases. In this study, we determined the influences of stimulus volume and sex on the ability to localize each of 8 odorants presented at neat concentrations: anethole, geraniol, limonene, linalool, menthol, methyl salicylate, phenyl ethanol, and vanillin. At a low stimulus volume (11 mL), only menthol was localized at an above-chance level. At a high stimulus volume (21 mL), above-chance localization occurred for all odorants except vanillin. Women were significantly better than men in localizing menthol. Stimuli rated as most intense were those that were most readily localized. The detection performance measures, as well as rated intensity values, significantly correlated with earlier findings of the trigeminal detectability of odorants presented to anosmic and normosmic subjects. This study suggests that differences in stimulus volume may explain some discrepant findings within the trigeminal chemosensory literature and supports the concept that vanillin may be a "relatively pure" olfactory stimulus.     

Selectivity of lingual nerve fibers to chemical stimuli

The cell bodies of the lingual branch of the trigeminal nerve were localized in the trigeminal ganglion using extracellular recordings together with horseradish peroxidase labeling from the tongue. Individual lingual nerve fibers were characterized with regard to their conduction velocities, receptive fields, and response to thermal, mechanical, and chemical stimuli. Fibers were classified as C, A delta, A beta, cold, and warm. The chemical stimuli included NaCl, KCl, NH4Cl, CaCl2, menthol, nicotine, hexanol, and capsaicin. With increasing salt concentration the latency of the response decreased and the activity increased. The responses elicited by salts (to 2.5 M), but not nonpolar stimuli such as menthol, were reversibly inhibited by 3.5 mM of the tight junction blocker, LaCl3. These data suggest that salts diffuse into stratified squamous epithelia through tight junctions in the stratum corneum and stratum granulosum, whereupon they enter the extracellular space. 11 C fibers were identified and 5 were characterized as polymodal nociceptors. All of the C fibers were activated by one or more of the salts NaCl, KCl, or NH4Cl. Three C fibers were activated by nicotine (1 mM), but none were affected by CaCl2 (1 M), menthol (1 mM), or hexanol (50 mM). However, not all C fibers or even the subpopulation of polymodals were activated by the same salts or by nicotine. Thus, it appears that C fibers display differential responsiveness to chemical stimuli. A delta fibers also showed differential sensitivity to chemicals. Of the 35 characterized A delta mechanoreceptors, 8 responded to NaCl, 9 to KCl, 9 to NH4Cl, 0 to CaCl2, menthol, or hexanol, and 2 to nicotine. 8 of 9 of the cold fibers (characterized as A delta''s) responded to menthol, none responded to nicotine, 8 of 16 were inhibited by hexanol, 9 of 19 responded to 2.5 M NH4Cl, 5 of 19 responded to 2.5 M KCl, and 1 of 19 responded to 2.5 M NaCl. In summary, lingual nerve fibers exhibit responsiveness to chemicals introduced onto the tongue. The differential responses of these fibers are potentially capable of transmitting information regarding the quality and quantity of chemical stimuli from the tongue to the central nervous system.     

Olfactory discrimination ability of human subjects for ten pairs of enantiomers.

We tested the ability of human subjects to distinguish between enantiomers, i.e. odorants which are identical except for chirality. In a forced-choice triangular test procedure 20 subjects were repeatedly presented with 10 enantiomeric odor pairs and asked to identify the bottle containing the odd stimulus. We found (i) that as a group, the subjects were only able to significantly discriminate the optical isomers of alpha-pinene, carvone and limonene, whereas they failed to distinguish between the (+)- and (-)-forms of menthol, fenchone, rose oxide, camphor, alpha-terpineol, beta-citronellol and 2-butanol; (ii) marked individual differences in discrimination performance, ranging from subjects who were able to significantly discriminate between 6 of the 10 odor pairs to subjects who failed to do so with 9 of the 10 tasks; (iii) that with none of the 10 odor pairs were the antipodes reported to differ significantly in subjective intensity when presented at equal concentrations; and (iv) that error rates were quite stable and did not differ significantly between sessions, and thus, we observed a lack of learning or training effects. Additional tests of the degree of trigeminality and threshold measurements of the optical isomers of alpha-pinene, carvone and limonene suggest that the discriminability of these three enantiomeric odor pairs is indeed due to differences in odor quality. These findings support the assumption that enantioselective molecular odor receptors may only exist for some but not all volatile enantiomers and thus that chiral recognition of odorants may not be a general phenomenon but is restricted to some substances.     

Novel menthol-derived cooling compounds activate primary and second-order trigeminal sensory neurons and modulate lingual thermosensitivity

We presently investigated 2 novel menthol derivatives GIV1 and GIV2, which exhibit strong cooling effects. In previous human psychophysical studies, GIV1 delivered in a toothpaste medium elicited a cooling sensation that was longer lasting compared with GIV2 and menthol carboxamide (WS-3). In the current study, we investigated the molecular and cellular effects of these cooling agents. In calcium flux studies of TRPM8 expressed in HEK cells, both GIV1 and GIV2 were approximately 40- to 200-fold more potent than menthol and WS-3. GIV1 and GIV2 also activated TRPA1 but at levels that were 400 times greater than those required for TRPM8 activation. In calcium imaging studies, subpopulations of cultured rat trigeminal ganglion and dorsal root ganglion cells responded to GIV1 and/or GIV2; the majority of these were also activated by menthol and some were additionally activated by the TRPA1 agonist cinnamaldehyde and/or the TRPV1 agonist capsaicin. We also made in vivo single-unit recordings from cold-sensitive neurons in rat trigeminal subnucleus caudalis (Vc). GIV 1 and GIV2 directly excited some Vc neurons, GIV1 significantly enhanced their responses to cooling, and both GIV1 and GIV2 reduced responses to noxious heat. These novel cooling compounds provide additional molecular tools to investigate the neural processes of cold sensation.     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).     

Central Processing of Odor Concentration is a Temporal Phenomenon as Revealed by Chemosensory Event-Related Potentials (CSERP)

Chemosensory event-related potentials (CSERP) can be used toexamine central nervous odor processing. An important questionfor understanding odor perception is how different concentrationsare processed. In the present study two odors were chosen whichactivate either the olfactory (linalool) or the trigeminal (menthol)system. Both odors were presented to 11 subjects in four differentconcentrations. Four subjects had to attend actively to theodors while the others perceived the odors under passive attention.The results showed that increased concentrations of the olfactorystimulus resulted in shorter latencies of the N1 component butdid not affect the amplitudes of the CSERP. However, the amplitudesof the stimulus dependent, exogenous components (N1, P2) increasedwith higher concentrations of the trigeminal stimulus. The amplitudeof the late positive complex, which reflects endogenous processes,was usually larger when the odorous stimuli had to be attendedto actively. It is concluded that olfactory intensity codingresults in a qualitatively different but not in a stronger neuronalresponse of the human brain. Chem. Senses 22: 9–26, 1997.     

The influence of training on chemosensory event-related potentials and interactions between the olfactory and trigeminal systems

It is not possible to accurately predict the perceptual response to odorants and odorant mixtures without understanding patterns of suppression and facilitation that result from interactions between the olfactory and trigeminal systems. The current study extends previous findings by exploring the effect of intensive training on the interaction between these systems and also by using a different mixed chemosensory stimulus to examine whether the principles established in earlier studies generalize to different odorants. Stimuli were chosen so as to selectively activate the olfactory (H2S) and trigeminal (CO2) nerves. In addition, linalool was included as a stimulus that activated both systems. Thirty-five participants (19 men, 16 women) rated the intensity of each stimulus when presented both alone and in binary mixtures (linalool + H2S, and linalool + CO2). Chemosensory event-related potentials were obtained from three recording positions. Analysis of intensity ratings showed that linalool was significantly less intense than the other stimuli when presented alone. In binary mixtures, H2S was strongly suppressed by linalool. One week of intensive odor training produced significant and specific reductions in the intensity of linalool and H2S, both alone and in their mixture. Training with a different odor (champignol) had no effect. Chemosensory event-related potential data confirmed previous findings showing changes in topographical distribution that reflected the degree of trigeminal activity. Binary mixtures generally produced larger amplitudes than single stimuli. Latencies clearly differentiated between the three single stimuli and the binary mixtures. Changes were observed in event-related potentials that reflected those obtained for intensity ratings in that they were observed for linalool and H2S in the linalool trained group only. The amplitude of the late 'endogenous' component (P3) was significantly decreased for these odors at frontal recording sites. In summary, strong and specific training effects were observed in intensity ratings for participants trained with the test odor (linalool), but not for those trained with a different odor. This was supported by a significant decrease of amplitudes of the event-related potentials at frontal recording sites following training with the test odor only     

Responses of the ciliates tetrahymena and paramecium to vertebrate odorants and tastants

The ciliates Tetrahymena and Paramecium respond to strong depolarizing stimuli with Ca(2+)-based action potentials, ciliary reversals, and consequent bouts of backward and forward swimming called "avoidance reactions" (ARs). We found that several representative tastants and odorants cause repetitive ARs in Tetrahymena and Paramecium at low (nM to microM) concentrations. Tetrahymena responded well to capsaicin, quinine, quinacrine, denatonium benzoate, eugenol, piperine, chloroquine, carvacrol, allyl isothiocyanate (AITC), and menthol. Chemosensory adaptation was seen with carvacrol, eugenol, quinacrine, and capsaicin. Cross-adaptation was seen between some of these compounds, suggesting possible similarities in their chemosensory transduction or adaptation pathways. Paramecium only responded well to AITC, quinacrine, piperine, and eugenol (with the effective concentration for 50% response [EC(50)] values in the microM range) while chemosensory adaptation was only seen to eugenol in Paramecium, suggesting possible species differences. Tetrahymena and Paramecium may have primitive receptors that can recognize these and other compounds or some of these compounds can act independently of specific receptors.     

Proportion of odorants impacts the configural versus elemental perception of a binary blending mixture in newborn rabbits

Processing of odor mixtures by neonates is weakly understood. Previous studies showed that a binary mixture of ethyl isobutyrate/ethyl maltol (odorants A/B) blends in newborn rabbits at the 30/70 ratio: Pups would perceive a configural odor in addition to the components' odors. Here, we investigated whether the emergence of this additional odor in AB is determined by specific ratio(s) of A and B. To that goal, we tested whether pups discriminated between AB mixtures with lower (A(-)B, 8/92 ratio) or higher (A(+)B, 68/32) proportion of A. In Experiment 1, pups conditioned to A (or B) responded to A(-)B and A(+)B but not to AB. In Experiment 2, pups responded to A(-)B after learning of A(-) (and to A(+)B after learning of A(+)) but not to AB. In Experiment 3, after conditioning to A(-)B pups responded to A(-) and B (and to A(+) and B after learning of A(+)B) but not or less to AB. In Experiment 4, pups responded to A(-)B and A(+)B after conditioning to AB. These results confirm the configural perception of certain odor mixtures by young organisms and reveal that the proportion of components is a key factor influencing their coding, recognition, and discrimination of complex stimuli.     

Phosphoinositide 3-Kinase Dependent Inhibition as a Broad Basis for Opponent Coding in Mammalian Olfactory Receptor Neurons

Phosphoinositide 3-kinase (PI3K) signaling has been implicated in mediating inhibitory odorant input to mammalian olfactory receptor neurons (ORNs). To better understand the breadth of such inhibition in odor coding, we screened a panel of odorants representing different chemical classes, as well as odorants known to occur in a natural odor object (tomato), for their ability to rapidly activate PI3K-dependent inhibitory signaling. Odorants were screened on dissociated native rat ORNs before and after pre-incubation with the PI3K-isoform specific blockers AS252424 and TGX221. Many different odorants increased their excitatory strength for particular ORNs following PI3K blockade in a manner consistent with activating PI3K-dependent inhibitory signaling in those cells. The PI3K-dependent inhibitory odorants overlapped with conventional excitatory odorants, but did not share the same bias, indicating partial partitioning of the odor space. Finding that PI3K-dependent inhibition can be activated by a wide range of otherwise conventional excitatory odorants strongly implies PI3K-dependent inhibition provides a broad basis for opponent coding in mammalian ORNs.     

Functional consequences following infection of the olfactory system by intranasal infusion of the olfactory bulb line variant (OBLV) of mouse hepatitis strain JHM

The present study assessed the functional consequences of viral infection with a neurotropic coronavirus, designated MHV OBLV, that specifically targets central olfactory structures. Using standard operant techniques and a 'go, no-go' successive discrimination paradigm, six BALB/c mice were trained to discriminate between the presentation of an air or odor stimulus (three mice for each of the odorants propanol and propyl acetate). Two additional BALB/c mice were trained to discriminate between the presentation of air and the presentation of either vanillin or propionic acid. Following criterion performance, each mouse received an additional 2000 trials of overtraining. At completion of overtraining one mouse from the propanol and propyl acetate groups were allocated as untreated. The remaining six mice were inoculated with 300 microl of the OBLV stock per nostril for a total of 1.5 x 10(6) p.f.u. in 600 microl. Following a 1 month rest, untreated and inoculated animals were again tested on their respective air versus odor discrimination task. Untreated animals immediately performed at criterion levels. In contrast, inoculated animals varied in their capacity to discriminate between air and odorant. Five of the six inoculated mice showed massive disruption of the olfactory bulb, including death of mitral cells; the other was more modestly affected. In addition, the density of innervation of the olfactory mucosa by substance P-containing trigeminal fibers is also affected by inoculation. Those mice that remained anosmic to the training odorants had the most severe reduction in mitral cell number and substance P fiber density among the inoculated animals.     

Self- and cross-desensitization of oral irritation by menthol and cinnamaldehyde (CA) via peripheral interactions at trigeminal sensory neurons

Menthol and cinnamaldehyde (CA) are plant-derived spices commonly used in oral hygiene products, chewing gum, and many other applications. However, little is known regarding their sensory interactions in the oral cavity. We used a human psychophysics approach to investigate the temporal dynamics of oral irritation elicited by sequential application of menthol and/or CA, and ratiometric calcium imaging methods to investigate activation of rat trigeminal ganglion (TG) cells by these agents. Irritancy decreased significantly with sequential oral application of menthol and CA (self-desensitization). Menthol cross-desensitized irritation elicited by CA, and vice versa, over a time course of at least 60 min. Seventeen and 19% of TG cells were activated by menthol and CA, respectively, with ～50% responding to both. TG cells exhibited significant self-desensitization to menthol applied at a 5, but not 10, min interval. They also exhibited significant self-desensitization to CA at 400 but not 200 μM. Menthol cross-desensitized TG cell responses to CA. CA at a concentration of 400 but not 200 μM also cross-desensitized menthol-evoked responses. The results support the argument that the perceived reductions in oral irritancy and cross-interactions between menthol and CA and menthol observed (at least at short interstimulus intervals) can be largely accounted for by the properties of trigeminal sensory neurons innervating the tongue.     

Learning to smell the roses: experience-dependent neural plasticity in human piriform and orbitofrontal cortices

It is widely presumed that odor quality is a direct outcome of odorant structure, but human studies indicate that molecular knowledge of an odorant is not always sufficient to predict odor quality. Indeed, the same olfactory input may generate different odor percepts depending on prior learning and experience. Combining functional magnetic resonance imaging with an olfactory paradigm of perceptual learning, we examined how sensory experience modifies odor perception and odor quality coding in the human brain. Prolonged exposure to a target odorant enhanced perceptual differentiation for odorants related in odor quality or functional group, an effect that was paralleled by learning-induced response increases in piriform cortex and orbitofrontal cortex (OFC). Critically, the magnitude of OFC activation predicted subsequent improvement in behavioral differentiation. Our findings suggest that neural representations of odor quality can be rapidly updated through mere perceptual experience, a mechanism that may underlie the development of odor perception.     

Release of calcitonin gene-related peptide in the pig nasal mucosa by antidromic nerve stimulation and capsaicin

The overflow of calcitonin gene-related peptide like-immunoreactivity (CGRP-LI) in the nasal venous effluent upon antidromic stimulation of the maxillary division of the trigeminal nerve with 6.9 Hz for 3 min or upon capsaicin (0.3 mumol bolus injection) were analysed in the nasal mucosa of sympathectomized pentobarbital anaesthetized pigs. The overflow of CGRP-LI upon antidromic stimulation displayed a slower appearance in the venous effluent than the overflow upon bolus injection of capsaicin. The vascular effects as revealed by the arterial blood flow, the venous blood flow, the blood volume of the nasal mucosa, i.e., the filling of the capacitance vessels and the superficial mucosal blood flow as revealed by the laser-Doppler signal were also studied. Antidromic stimulation of the trigeminal nerve as well as capsaicin bolus injection induced a marked vasodilation which was parallel to the overflow of CGRP. However, capsaicin bolus injection also resulted in a marked increase in the mean arterial blood pressure which may be due to reflex activation of sympathetic fibers. In conclusion, we have demonstrated that chemical stimulation with capsaicin as well as antidromic stimulation of nasal sensory nerves in sympathectomized animals induces both vasodilation and overflow of CGRP-LI in vivo. This indicates that CGRP may contribute to the sensory regulation of the microcirculation in the nasal mucosa.     

On the trigeminal percept of androstenone and its implications on the rate of specific anosmia

Specific anosmia is a term that describes an inability to perceive a particular odorant in the context of an otherwise normal olfactory acuity. The most common example, for the odor of androstenone, has been ascribed a prevalence ranging from 2 to 45%. In two experiments we sought to determine whether this wide range could be explained by the difference in steroid concentrations used, and by the degree to which the trigeminal system contributes to perception of androstenone. Experiment 1 demonstrated that high concentrations of androstenone stimulated the trigeminal system, as indicated by electrophysiological recordings. Experiment 2 demonstrated that conscious detection of androstenone is possible based solely on the trigeminal system. Interestingly, detection seems to interact with olfactory acuity in that subjects with a low olfactory sensitivity to androstenone were better able to detect its trigeminal component. The agreement between conscious experience and behavioral discrimination was not well calibrated, in that subjects demonstrated a clear overconfidence in their abilities. Altogether, the current study suggests that androstenone is an odorant that produces a concentration-dependent degree of trigeminal stimulation. This trigeminal component explains the diversity of the reported prevalence of specific anosmia for androstenone and might have implications on future use of specific anosmia as a tool to understand odor processing.     

Stimulus selection for intranasal sensory isolation: eugenol is an irritant

Both the olfactory and the trigeminal systems are able to respond to intranasal presentations of chemical vapor. Accordingly, when the nose detects a volatile chemical, it is often unclear whether we smell it, feel it, or both. The distinction may often be unimportant in our everyday perception of fragrances or aromas, but it can matter in experiments that purport to isolate olfactory processes or study the interaction between olfaction and chemesthesis. Researchers turn to a small pool of compounds that are believed to be "pure olfactory" stimuli with little or no trigeminal impact. The current report reexamines one such commonly used compound, namely eugenol, a flavor and fragrance ingredient that has anesthetic properties under some conditions. Using a standard method involving many trials during an experimental session (Experiment 1), subjects were unable to reliably lateralize eugenol, consistent with claims that this compound is detected primarily through olfaction. However, with more limited exposure (Experiments 2 and 3), subjects were able to lateralize eugenol. We speculate that anesthetic properties of eugenol could blunt its trigeminal impact in some paradigms. Regardless, the current experiments suggest that eugenol can in fact stimulate the trigeminal nerve but in a complex concentration-dependent manner. Implications and strategies for selection of model odorants are discussed.     

Membrane fluidity changes of liposomes in response to various odorants. Complexity of membrane composition and variety of adsorption sites for odorants.

Three kinds of liposomes prepared from phosphatidylcholine (PC), azolectin, and azolectin-containing membrane proteins of the canine erythrocytes were used as models for olfactory cells. To explore properties of the adsorption sites of odorants, membrane fluidity changes in response to various odorants were measured with various fluorescence dyes which monitor the fluidity at different depths and different regions of the membranes. (a) Application of various odorants changed the membrane fluidity of azolectin liposomes. The patterns of membrane fluidity changes in response to odorants having a similar odor were similar to each other and those in response to odorants having different odors were different from each other. These results suggested that odorants having a similar odor are adsorbed on a similar site and odorants having different odors are adsorbed on different sites. (b) Such variation of the pattern was not seen in liposomes of a simple composition (PC liposome). (c) In the proteoliposomes whose composition was more complex than that of azolectin liposomes, the patterns of membrane fluidity changes varied among odorants having a similar odor. It was concluded that liposomes of complex membrane composition have the variety of adsorption sites for odorants.