Vitamin C deficiency in osteogenic disorder Shionogi/Shi Jcl-od/od rats: effects on sour taste preferences, lick rates, chorda tympani nerve responses, and taste transduction elements.

Animals use sour taste to avoid spoiled food and to choose foods containing vitamins and minerals. To investigate the response to sour taste substances during vitamin C (ascorbic acid; AA) deficiency, we conducted behavioral, neural, anatomical, and molecular biological experiments with osteogenic disorder Shionogi/Shi Jcl-od/od rats, which lack the ability to synthesize AA. Rats had higher 3 mM citric acid and 10 mM AA preference scores when AA-deficient than when replete. Licking rates for sour taste solutions [AA, citric acid, acetic acid, tartaric acid, and HCl] were significantly increased during AA deficiency relative to pre- and postdeficiency. Chorda tympani nerve recordings were conducted to evaluate organic acid taste responses in the AA-deficient and replete rats. Nerve responses to citric acid, acetic acid, and tartaric acid were significantly diminished in AA-deficient rats relative to replete controls. There was no significant difference in the number of fungiform papillae taste buds per unit area in the AA-deficient rats relative to the replete rats. However, mRNA expression levels of Gnat3 (NM_173139.1), Trpm5 (NM_001191896.1), Tas1r1 (NM_053305.1), Car4 (NM_019174.3), and Gad1 (NM_017007.1) in fungiform papillae taste bud cells from AA-deficient rats were significantly lower than those in replete rats. Our data suggest that AA deficiency decreases avoidance of acids and reduces chorda tympani nerve responses to acids. AA deficiency downregulates some taste-related genes in fungiform papillae taste bud cells. However, the results also reveal that the mRNA expression of some putative sour taste receptors in fungiform papillae taste bud cells is not affected by AA deficiency.

Novel approaches to the study of viscosity discrimination in rodents.

Texture has enormous effects on food preferences. The materials used to study texture discrimination also have tastes that experimental animal can detect; therefore, such studies must be designed to exclude taste differences. In this study, to minimize the effects of material tastes, we utilized high- and low-viscosity forms of carboxymethyl cellulose (CMC-H and CMC-L, respectively) at the same concentrations (0.1-3%) for viscosity discrimination tests in rats. In two-bottle preference tests of water and CMC, rats avoided CMC-H solutions above 1% (63 mPa·s) but did not avoid less viscous CMC-L solutions with equivalent taste magnitudes, suggesting that rats spontaneously avoided high viscosity. To evaluate low-viscosity discrimination, we performed conditioned aversion tests to 0.1% CMC, which initially showed a comparable preference ratio to water in the two-bottle preference tests. Conditioning with 0.1% CMC-L (1.5 mPa·s) did not induce aversion to 0.1% CMC-L or CMC-H. However, rats acquired a conditioned aversion to 0.1% CMC-H (3.6 mPa·s) even after latent inhibition to CMC taste by pre-exposure to 0.1% CMC-L. These results suggest that rats can discriminate considerably low viscosity independent of CMC taste. This novel approach for viscosity discrimination can be used to investigate the mechanisms of texture perception in mammals.

Influence of dental education on eye gaze distribution when observing facial profiles with varying degrees of lip protrusion.

OBJECTIVES: The purpose of the present study was to investigate the influence of dental education on eye gaze distribution when perceiving facial profile images with varying degrees of lip protrusion. METHODS: Fifty-one subjects with dental education (dental-education group) and 47 without dental education (no-dental-education group) were included in this study. The eye gaze distribution of the subjects was recorded for 10 seconds using a non-contact eye tracker when determining whether or not the subjects judged each various facial profile image inappropriate. The facial profile images had varying degrees of upper-lip and lower-lip protrusion. The frequency of eye fixation (lasting >0.06 seconds) was counted. The duration was measured as the total time of eye fixation. The facial images were divided into 4 regions of interest (ROIs): the forehead (ROI-I); the orbit and nose root (ROI-II); the nose tip and upper lip (ROI-III); and the lower lip and chin (ROI-IV). The frequency and duration of eye fixation on each ROI was compared. RESULTS: The frequency of eye fixation on ROI-II was greater in comparison to ROI-I and ROI-IV. The frequency of eye fixation on ROI-III was also greater in comparison to ROI-I and ROI-IV. The duration of eye fixation on ROI-III in the dental-education group was longer in comparison to the no-dental-education group. CONCLUSIONS: Dental education influenced the eye fixation on the nose tip and upper lip when perceiving facial profile images.

Effect of the duration of a conditioned stimulus on component recognition in binary taste mixtures in rats.

OBJECTIVES: The aim of this behavioral study was to investigate the duration of a conditioned stimulus (CS-duration) necessary for rats to recognize the components of a binary taste mixture in a conditioned taste aversion (CTA) paradigm as well as the relationship between CS-duration and their spontaneous recovery. METHODS: The experimental rats were categorized under conditioned and control groups and further divided into three groups according to the CS-duration: 10, 30, and 60 s. As the test stimuli, a mixture of 100 mM sucrose (S) + 30 µM quinine hydrochloride (Q) and its components were used. RESULTS: On day 1 of the CTA test, the number of licks (NL) for S + Q and S in all conditioned groups was significantly lower than that of the control group presented with CS for 60 s (CON-60), which was the representative control group determined by the initial CTA test. For Q, there was no significant difference between NL of the CTA group presented with CS for 10 s and that of CON-60; however, NL in the other two CTA groups, i.e., CTA-30 and CTA-60, was significantly lower than that of CON-60. When the rats were presented with a shorter CS-duration, they showed spontaneous recovery earlier depending on the CS-duration. CONCLUSIONS: These results suggest that rats can recognize a binary taste mixture and its components using a CS-duration of more than 30 s and that spontaneous recovery from CTA learning depends on the CS- duration.

Behavioral and Neural Responses to Vitamin C Solution in Vitamin C-deficient Osteogenic Disorder Shionogi/Shi Jcl-od/od Rats.

To investigate the appetite for vitamin C (VC), we conducted behavioral and neural experiments using osteogenic disorder Shionogi/Shi Jcl-od/od (od/od) rats, which lack the ability to synthesize VC, and their wild-type controls osteogenic disorder Shionogi/Shi Jcl- +/+ (+/+) rats. In the behavioral study, rats were deprived of VC for 25 days and then received two-bottle preference tests with a choice between water and 10 mM VC. The preference for 10 mM VC solution of od/od rats was significantly greater than that of +/+ rats. In the neural study, the relative magnitudes of the whole chorda tympani nerve (CTN) responses to 100-1000 mM VC, 3-10 mM HCl, 100-1000 mM NaCl, and 20 mM quinine▪HCl in the VC-deficient rats were significantly smaller than those in the nondeficient ones. Further, we conducted additional behavioral experiments to investigate the appetite for sour and salty taste solutions of VC-deficient od/od rats. Preference scores for 3 mM citric acid increased in od/od rats after VC removal, compared with before, whereas preference scores for 100 and 150 mM NaCl were decreased in VC-deficient od/od rats. The preference for 300 mM NaCl was not changed. Hence, our results suggest that the reduction of the aversive taste of VC during VC deficiency may have involved the reduction of CTN responses to acids. Overall, our results indicate that VC-deficient rats ingest sufficient VC to relieve their deficiency and that VC deficiency causes changes in peripheral sensitivity to acids, but nongustatory factors may also affect VC intake and choice.

Recognition by Rats of Binary Taste Solutions and Their Components.

This behavioral study investigated how rats conditioned to binary mixtures of preferred and aversive taste stimuli, respectively, responded to the individual components in a conditioned taste aversion (CTA) paradigm. The preference of stimuli was determined based on the initial results of 2 bottle preference test. The preferred stimuli included 5mM sodium saccharin (Sacc), 0.03M NaCl (Na), 0.1M Na, 5mM Sacc + 0.03M Na, and 5mM Sacc + 0.2mM quinine hydrochloride (Q), whereas the aversive stimuli tested were 1.0M Na, 0.2mM Q, 0.3mM Q, 5mM Sacc + 1.0M Na, and 5mM Sacc + 0.3mM Q. In CTA tests where LiCl was the unconditioned stimulus, the number of licks to the preferred binary mixtures and to all tested preferred components were significantly less than in control rats. No significant difference resulted between the number of licks to the aversive binary mixtures or to all tested aversive components. However, when rats pre-exposed to the aversive components contained of the aversive binary mixtures were conditioned to these mixtures, the number of licks to all the tested stimuli was significantly less than in controls. Rats conditioned to components of the aversive binary mixtures generalized to the binary mixtures containing those components. These results suggest that rats recognize and remember preferred and aversive taste mixtures as well as the preferred and aversive components of the binary mixtures, and that pre-exposure before CTA is an available method to study the recognition of aversive taste stimuli.

The effect of hardness of food on amygdalar histamine release in rats.

When animals eat food, the oral cavity receives a variety of sensory information from food. The hardness of food, which elicits somatic sensation, is thought to affect feeding behavior, however, the details of neuronal mechanism are unclear. The histaminergic system is known to be involved in feeding behavior, and our previous studies indicated that gustatory information activates the histaminergic system, and that palatability of tastants influences its activity. From these findings, we hypothesized that the hardness of food may affect the histaminergic system. Thus, in the present study, we examined the effect of the hardness of food on histamine release in the central nucleus of amygdala when rats consumed either of two types of pellets composed of similar ingredients but having different degrees of hardness: hard and soft pellets. Histamine release was significantly increased in the rat fed with hard pellets. By contrast, histamine release was not enhanced in soft pellets-fed rats. There were no significant differences between the hard and soft pellet intakes during the experimental period. When rats acquired a conditioned aversion to soft pellets, histamine release was increased during feeding, in sharp contrast to no change of histamine release pattern seen during unconditioned soft pellet intake. These observations indicate that the amygdalar histaminergic system is modulated by oral somatic sensation from food, and by palatability of food texture.

Genetically-increased taste cell population with G(alpha)-gustducin-coupled sweet receptors is associated with increase of gurmarin-sensitive taste nerve fibers in mice.

BACKGROUND: The peptide gurmarin is a selective sweet response inhibitor for rodents. In mice, gurmarin sensitivity differs among strains with gurmarin-sensitive C57BL and gurmarin-poorly-sensitive BALB strains. In C57BL mice, sweet-responsive fibers of the chorda tympani (CT) nerve can be divided into two distinct populations, gurmarin-sensitive (GS) and gurmarin-insensitive (GI) types, suggesting the existence of two distinct reception pathways for sweet taste responses. By using the dpa congenic strain (dpa CG) whose genetic background is identical to BALB except that the gene(s) controlling gurmarin sensitivity are derived from C57BL, we previously found that genetically-elevated gurmarin sensitivity in dpa CG mice, confirmed by using behavioral response and whole CT nerve response analyses, was linked to a greater taste cell population co-expressing sweet taste receptors and a G(alpha)- protein, G(alpha)--gustducin. However, the formation of neural pathways from the increased taste cell population to nerve fibers has not yet been examined. RESULTS: Here, we investigated whether the increased taste cell population with G(alpha)--gustducin-coupled sweet receptors would be associated with selective increment of GS fiber population or nonselective shift of gurmarin sensitivities of overall sweet-responsive fibers by examining the classification of GS and GI fiber types in dpa CG and BALB mice. The results indicated that dpa CG, like C57BL, possess two distinct populations of GS and GI types of sweet-responsive fibers with almost identical sizes (dpa CG: 13 GS and 16 GI fibers; C57BL: 16 GS and 14 GI fibers). In contrast, BALB has only 3 GS fibers but 18 GI fibers. These data indicate a marked increase of the GS population in dpa CG. CONCLUSION: These results suggest that the increased cell population expressing T1r2/T1r3/G(alpha)--gustducin in dpa CG mice may be associated with an increase of their matched GS type fibers, and may form the distinct GS sweet reception pathway in mice. G(alpha)--gustducin may be involved in the GS sweet reception pathway and may be a key molecule for links between sweet taste receptors and cell type-specific-innervation by their matched fiber class.

Taste preference and nerve response to 5'-inosine monophosphate are enhanced by glutathione in mice.

Previous human sensory evaluation studies have shown that glutathione (GSH) enhances deliciousness, accompanied by thickness, mouthfulness, and continuity feeling, which is known as "kokumi" in Japanese, in an umami solution containing monosodium glutamate and 5'-inosine monophosphate (IMP). We conducted behavioral and electrophysiological experiments to explore possible interactions of taste effectiveness between GSH and umami substances in mice. The 2-bottle preference test revealed that the mice preferred GSH at concentrations ranging from 1 to 10 mM. When GSH was added to IMP or a mixture of IMP and monopotassium glutamate (MPG), the mice showed increased preference for these solutions over the individual IMP or the binary mixture of IMP and MPG in both short-term and long-term tests. The addition of GSH to MPG, however, did not increase preference. Neural responses of the chorda tympani and glossopharyngeal nerves to the mixture of IMP and GSH showed synergism, whereas synergism was not observed in the mixture of MPG and GSH in either taste nerve. Another behavioral study with the use of the conditioned taste aversion paradigm showed that aversions to MPG generalized moderately to GSH, but aversions to GSH did not generalize to MPG. The present study suggests that GSH enhances preference for umami solutions containing 5'-ribonucleotide rather than glutamate. On the basis of these results, we discuss possible receptors involved for the action of GSH.

Gurmarin sensitivity of sweet taste responses is associated with co-expression patterns of T1r2, T1r3, and gustducin.

Gurmarin (Gur) is a peptide that selectively suppresses sweet taste responses in rodents. The inhibitory effect of Gur differs among tongue regions and mouse strains. Recent studies demonstrated that co-expression levels of genes controlling sweet receptors (T1r2/T1r3 heterodimer) versus Galpha-protein, gustducin, are much lower in Gur-insensitive posterior circumvallate papillae than in Gur-sensitive anterior fungiform papillae. Here, we investigated the potential link of Gur-sensitivity with the co-expression for T1r2/T1r3 receptors and gustducin by comparing those of taste tissues of Gur-sensitive (B6, dpa congenic strains) and Gur-weakly-sensitive (BALB) strains. The results indicated that co-expression ratios among T1r2, T1r3, and gustducin in the fungiform papillae were significantly lower in Gur-weakly-sensitive BALB mice than in Gur-sensitive B6 and dpa congenic mice. This linkage between Gur-sensitivity and co-expression for T1r2/T1r3 receptors versus gustducin suggests that gustducin may be a key molecule involved in the pathway for Gur-sensitive sweet responses.

Acute suppression, but not chronic genetic deficiency, of c-fos gene expression impairs long-term memory in aversive taste learning.

Several lines of evidence have indicated that the establishment of long-term memory requires protein synthesis, including the synthesis of immediate-early gene products. Although the anatomical expression patterns of the c-fos gene, a transcription factor-encoding immediate-early gene, in conditioned taste aversion (CTA) are well documented, the functional roles of c-fos gene expression and Fos-mediated transcription remain to be clarified. Using the antisense oligodeoxynucleotide (AS-ODN) method in rats and gene-targeting knockout techniques in mice (c-fos(-/-) mice), we examined the roles of c-fos gene expression in the acquisition, retrieval, and retention of CTA. Preconditioning microinfusion of AS-ODN directed against c-fos mRNA (c-fos AS-ODN) into the parabrachial nucleus (PBN) impaired the acquisition, whereas infusion of control ODNs consisting of a randomized or inverted base order had no effect. Microinfusion of c-fos AS-ODN into either the amygdala or insular cortex did not impair the acquisition, whereas it attenuated the retention. Retrieval and subsequent retention of an acquired CTA were not disrupted by c-fos AS-ODN infusion into the PBN or amygdala. Microinfusion of another AS-ODN directed against zif268 (egr-1, krox-24, NGFI-A) mRNA into the PBN or amygdala did not affect the acquisition and retention. The genetic deficiency in c-fos(-/-) mice caused normal acquisition and retention. The present results suggest that the Fos-mediated gene transcription in the PBN, amygdala, or insular cortex plays critical roles in the acquisition and/or consolidation, but not the retrieval, of long-term taste memory; nevertheless, some other factors could compensate CTA mechanism when Fos-mediated transcription is not available.

Conditioned food aversion elicited by the temperature of drinking water as a conditioned stimulus in rats.

It is known that taste can act as a conditioned stimulus (CS) for conditioned food aversion. In the present study, in order to examine whether or not the temperature of drinking water can be a CS, we conducted behavioral experiments in Wistar rats. The following results were obtained: (1) The rats subjected to aversive conditioning to 5 or 40 degrees C distilled water could learn to avoid these CSs, but they did not avoid any taste stimuli. (2) The rats subjected to aversive conditioning to 5 or 40 degrees C 0.1 M sucrose developed a generalized avoidance to sucrose at any temperature. (3) When rats familiarized to 25 degrees C 5 mM saccharin-Na (Sacc) were subjected to aversive conditioning to 5 or 40 degrees C Sacc, they avoided the respective CS, but they did not generalize it to any other stimuli even if having the same temperature as the CS. (4) The rats which had undergone transection of the taste nerves (chorda tympani and glossopharyngeal nerves) could acquire the conditioned response to the temperature of the CS. These results suggest that rats can be conditioned to temperature aversion and that the taste nerves are not needed in the formation of this conditioning.

Synergistic responses of the chorda tympani to mixtures of umami and sweet substances in rats.

It has been known that umami substances such as monosodium L-glutamate (MSG) and 5'-inosine monophosphate (IMP) elicit a unique taste called 'umami' in humans. One of the characteristics of the umami taste is synergism: the synergistic enhancement of the magnitude of response produced by the addition of 5'-ribonucleotides to MSG. In addition to this well-documented synergism, we report here for the first time on another type of synergism between a glutamate receptor agonist, L-AP4, and sweet substances, by analyzing the chorda tympani responses in rats. The results are as follows: (i) when L-AP4 was mixed with one of the sweet substances, such as sucrose, glucose, fructose and maltose, large synergistic responses were observed. (ii) These synergistic responses, except to L-AP4 + sucrose, were not suppressed by sweet taste suppressants, gurmarin and pronase E. (iii) These synergistic responses were not suppressed by either metabotropic or ionotropic glutamate receptor antagonists. (iv) Fibers that responded well to the binary mixtures of L-AP4 and sweet substances also responded well to NaCl and HCl, but very weakly to sucrose. These findings are different from the characteristics of synergism between glutamate and IMP. The multiple transduction mechanisms for the umami taste in rat taste cells are discussed.

A comparison of voluntary salt-intake behavior in Nax-gene deficient and wild-type mice with reference to peripheral taste inputs.

The Na(x) channel, a subfamily of voltage-gated sodium channels, is thought to be a specific sodium receptor in the central nervous system. Our previous study revealed that Na(x)-gene-deficient mice consumed excessive amounts of NaCl even under water-deprived conditions. In the present study, to investigate whether the peripheral taste inputs are involved in the abnormal intake of salt in Na(x)-deficient mice (homo), voluntary intake of various taste solutions in homo and wild-type mice (wild) was examined under non-deprived conditions. Homo showed a higher preference for 0.15 M NaCl solution than wild. Preference ratios for other basic tastants were identical between groups. Transection of the chorda tympani (CT) or the glossopharyngeal (GP) nerve had little effect on salt-intake behavior in homo and wild. Although combined transection of the superior laryngeal (SL) and GP nerves decreased NaCl intake in homo but not in wild, there were no differences in preference ratios for NaCl in homo before and after SL+GP transection. On the other hand, preference ratios for NaCl in wild tended to increase after combined SL and GP transection. Consequently, preference ratios for NaCl after SL+GP transection were no different between homo and wild. While electrophysiological responses of the CT and the GP to various taste solutions were indistinguishable between homo and wild, those of the SL to NaCl in homo were smaller than those in wild only at lower concentrations (0.01 and 0.03 M). Thus, chemosensory inputs from the oro-pharyngeal regions had little effect on abnormal salt intake in homo, if any. From these results, it is suggested that the higher preference for NaCl in homo is mainly due to the lack of Na(x) channels in the central nervous system.

The hardness of food plays an important role in food selection behavior in rats.

To examine the importance of the hardness of foods, we conducted behavioral and electromyographical experiments in Wistar male rats using three kinds of pellets, a hard commercially made pellet (MF), a hard privately produced pellet (H) and a soft privately produced pellet (S). MF and H had the same hardness but contained different ingredients, and S and H had the same ingredients but different degrees of hardness (S