Participation of the peripheral taste system in aging-dependent changes in taste sensitivity

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PARTICIPATION OF THE PERIPHERAL TASTE SYSTEM IN AGING-DEPENDENT CHANGES IN TASTE SENSITIVITY

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MASATAKA NARUKAWA, AZUSA KUROKAWA, RIE KOHTA AND TAKUMI MISAKA *

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Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

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Abstract—Previous studies have shown that aging modifies taste sensitivity. However, the factors affecting the changes in taste sensitivity remain unclear. To investigate the cause of the age-related changes in taste sensitivity, we compared the peripheral taste detection systems in young and old mice. First, we examined whether taste sensitivity varied according to age using behavioral assays. We confirmed that the taste sensitivities to salty and bitter tastes decreased with aging. In other assays, the gustatory nerve responses to salty and sweet tastes increased significantly with aging, while those to bitter taste did not change. Thus, the profile of the gustatory nerve responses was inconsistent with the profile of the behavioral responses. Next, we evaluated the expressions of taste-related molecules in the taste buds. Although no apparent differences in the expressions of representative taste receptors were observed between the two age groups, the mRNA expressions of signaling effectors were slightly, but significantly, decreased in old mice. No significant differences in the turnover rates of taste bud cells were observed between the two age groups. Thus, we did not observe any large decreases in the expressions of taste-related molecules and turnover rates of taste bud cells with aging. Based on these findings, we conclude that changes in taste sensitivity with aging were not caused by aging-related degradation of peripheral taste organs. Meanwhile, the concentrations of several serum components that modify taste responses changed with age. Thus, taste signal-modifying factors such as serum components may have a contributing role in aging-related changes in taste sensitivity. Ó 2017 The Author(s). Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

Key words: aging, taste, taste detection, peripheral taste system. 10

*Corresponding author. Address: Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Fax: +81 3 5841 8100. E-mail address: [email protected] (T. Misaka). Abbreviations: BrdU, 5-bromo-20 -deoxyuridine; CT, chorda tympani; CvP, circumvallate papillae; ENaC, epithelium sodium channel; FuP, fungiform papillae; GL, glossopharyngeal; IMP, inosine 50 monophosphate; MSG, monosodium glutamate; PKD, polycystic kidney disease; PLCb2, phospholipase Cb2.

INTRODUCTION

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In advanced countries, the general population is getting older. Taste is a vital sense during nutritional intake and consequently in the maintenance of health and longevity. In general, the pleasure derived from eating food diminishes with age, partly through deterioration of smell and taste sensations. Elucidation of the agedependent changes in taste cognition is important for better understanding of the factors that govern the changes in taste perception during an individual’s lifespan. Taste is classified into five basic categories: salty, sweet, bitter, sour, and umami. The taste of food is detected by taste cells that transmit information to the gustatory nerves and subsequently to the central nervous system (Yarmolinsky et al., 2009). Recent studies have identified taste receptors and taste-related molecules in taste bud cells. Tas1R2/Tas1R3, Tas1R1/ Tas1R3, and Tas2Rs play as the receptors for sweet, umami, and bitter tastes, respectively. Transient receptor potential channel type M5 and phospholipase Cb2 (PLCb2) are known as the downstream signaling effectors of these taste receptors. Polycystic kidney disease (PKD) 2L1/PKD1L3 and epithelial sodium channel (ENaC) are candidates for the sour and salty taste receptors, respectively (Yarmolinsky et al., 2009). Several studies have investigated the correlation between aging and taste sensation in rodents. Thaw (1996) reported that 28-month-old Sprague–Dawley rats showed higher thresholds for detection of sucrose and NaCl than rats younger than 23 months (Thaw, 1996). Tordoff (2007) described that old C57BL/6J mice aged 125 weeks showed a higher preference for NaCl than young mice aged 8 weeks, but found no age-related changes in the preferences for saccharin, quinine hydrochloride, and citric acid (Tordoff, 2007). Shin et al. (2012) reported that although 18-month-old B6C3F1/J mice showed a lower sweet response than 10-month-old mice, there were no changes in other taste qualities with aging (Shin et al., 2012). Although these reports produced inconsistent results, they all suggested that taste sensitivity is modified by aging. However, the factors affecting the changes in taste sensitivity remain poorly understood. To clarify the participation of the peripheral taste system in the aging-dependent changes in taste sensitivity, in this study, we compared the peripheral taste detection systems in young and old mice. First, we investigated whether taste sensitivities changed with aging under our experimental conditions. Next, to

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http://dx.doi.org/10.1016/j.neuroscience.2017.06.054 0306-4522/Ó 2017 The Author(s). Published by Elsevier Ltd on behalf of IBRO. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1

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identify the cause of the changes in taste sensitivities, we analyzed the expressions of taste-associated molecules, turnover rates of taste bud cells, and levels of serum components known to modify taste responses. As differences in experimental conditions such as species, age, and concentration range of tested taste solutions were considered the main reasons for the inconsistent results among previous studies, we designed our study under the following conditions: (1) C57BL/6J (B6) mice aged >120 weeks were used as the old group; (2) mice were not exposed to strong taste stimuli until the behavioral experiments in both the young and old groups; and (3) a broad concentration range from weak to strong taste was used for taste stimulation in behavioral assays.

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MATERIALS AND METHODS

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Materials

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Citric acid, NaCl, and sucrose were purchased from Kanto Chemical (Tokyo, Japan). Denatonium benzoate (denatonium), monosodium glutamate (MSG), inosine 50 -monophosphate (IMP), and amiloride were purchased from Sigma (St. Louis, MO). All other reagents were of analytical grade and obtained from standard suppliers.

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Animals

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The study population comprised male B6 mice (CLEA Japan, Tokyo, Japan). The animals were housed at The University of Tokyo Animal Care Facility and had ad libitum access to a standard laboratory chow and distilled water. A total of 30 mice were used in this study. The surrounding temperature and humidity were maintained at 23 °C and 55%, respectively, with a 12h/12-h light/dark cycle (lights switched on at 0800 h). We divided the mice into two age groups: young group aged 8–24 weeks (body weight: 24.6 ± 0.6 g at 12 weeks) and old group aged 120–139 weeks (body weight: 35.2 ± 0.8 g at 120 weeks). Mice with normal shapes and feeding behaviors were used. All experiments were performed in accordance with protocols approved by The University of Tokyo Animal Care Committee (Approval Number: P10-457).

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Experiment schedule

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Initially, the mice performed a brief access test, followed by a 48-h two-bottle preference test. After the behavioral assays, the gustatory nerve activities were measured. Subsequently, taste bud and blood samples were collected. For immunohistochemistry, we used another group of mice which did not perform the behavioral assays.

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BEHAVIORAL EXPERIMENTS

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Brief access test

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Ten mice from each age group were used for the brief access test, which was performed for 2 weeks. The numbers of licks to aversive and attractive taste

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substances were measured in the first and second weeks, respectively.

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Evaluation of aversive taste substances in the first week

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Each animal with 23-h water deprivation was placed in a test cage on day 1 of training and given free access to distilled water during a 1-h session. The number of licks per 5 s was measured by a custom-built gustometer (Neuroscience, Tokyo, Japan). Days 2–3 were the training session. During this period, the animal was trained to drink distilled water on an interval schedule, consisting of 5-s periods of presentation of distilled water with 30-s intervals. Days 4–5 were the test session. The numbers of licks for denatonium, citric acid, and distilled water by each animal were counted during the first 5 s after the animal’s first lick. After the test session, the mice were rested.

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Evaluation of attractive taste substances in the second week

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Each animal’s water intake was limited to the average water intake recorded just before the brief access test. Days 8–10 were the training session. On days 11–13, the numbers of licks for sucrose, MSG + IMP, and NaCl by each animal were counted during the first 5 s after the animal’s first lick. As young and old mice had different motivations for water (average lick numbers for water after 23-h water deprivation: young mice, 32.4 ± 0.9; old mice, 27.8 ± 1.2), the taste sensitivities to tastants were expressed as lick ratios. The lick ratios of the tastants were calculated as follows: number of licks of tastant/ number of licks of water. When the lick ratios to attractive taste substances were calculated, the average lick numbers for water during aversive taste test sessions were utilized. To avoid restriction effects, data for mice whose body weight fell below 80% of the ad libitum normal free-feeding value were excluded from the analyses. Tastant solutions for the brief access test were (in mM): 0.3–100 citric acid, 0.1–10 denatonium, 10–1000 NaCl, 1–300 sucrose, and 1–300 MSG + 0.5 IMP.

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Forty-eight-hour two-bottle preference test

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Young mice (n = 10) and old mice (n = 6–10) were caged individually and given 48 h of access to two bottles, one containing deionized water and the other containing a tastant solution. After 24 h the bottle positions were switched to avoid positional effects. The ratio of tastant volume to total liquid consumed was recorded. The preference ratios of the tastants were calculated as follows: tastant intake/total fluid intake (tastant intake + water intake). The tastant solutions for the two-bottle preference test were (in mM): 1–10 citric acid, 0.03–3 denatonium, 10–500 NaCl, 1–100 sucrose, and 0.1–10 MSG + 0.5 IMP.

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Gustatory nerve recordings

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Whole-nerve responses to lingual application of tastants were recorded from the chorda tympani (CT) nerve. After behavioral experiments, the CT nerve responses were measured in young mice (n = 5) and old mice (n = 4–6) under anesthesia by intraperitoneal injection of sodium pentobarbital (50 mg/kg) and urethane (500 mg/kg). A tracheal cannula was implanted in each animal, and the animal was secured with a headholder. The CT nerve was exposed at its exit from the lingual nerve by removal of the internal pterygoid muscle, dissected free from surrounding tissues, and cut at the point of its entry into the bulla. The nerve was desheathed, and then placed on a platinum wire electrode. An indifferent electrode was positioned nearby in the wound. Whole-nerve activities were amplified, displayed on an oscilloscope, and monitored with an amplifier (DAM50; World Precision Instruments Inc., Sarasota, FL). The amplified signal was passed through an integrator with a time constant of 1 s. The magnitude of the whole-nerve response was measured as the height of the integrated response from baseline (before stimulation) at approximately 5 s after the onset of stimulation to avoid any tactile effect of the stimuli. The taste solution was applied for 30 s, followed by a >30-s rinse with deionized water. Application of each taste solution was repeated at least twice, and the mean response was calculated. Tastant solutions for the CT nerve responses were (in mM): 10 and 30 citric acid, 10 and 30 denatonium, 100 and 300 NaCl, 100 and 300 NaCl + 30 mM amiloride, 100 and 300 sucrose, and 30 and 100 MSG + 0.5 IMP. The relative response magnitude to each tastant was calculated by comparison with the response magnitude to 100 mM NH4Cl (control).

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Real-time RT-PCR

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The tongues of the mice (young mice (n = 6) and old mice (n = 6)) were removed after the CT nerve recordings. The epithelium was peeled using an enzymatic treatment (Narukawa et al., 2006, 2008, 2009). The fungiform (FuP) and circumvallate papillae (CvP) regions were separated from the epithelium. Total RNAs from the FuP and CvP were extracted using RNeasy Mini Columns (Qiagen, Venlo, Netherlands). Genomic DNA digestion was performed with an RNasefree DNase Set (Qiagen). First-strand cDNA was generated from total RNA by reverse transcription (Superscript III Reverse Transcription Kit; Life Technologies, Gaithersburg, MD). The mRNA transcript levels were determined by real-time RT-PCR (ABI Prism 7000 Sequence Detection System; Applied Biosystems, Foster City, CA). PCR amplification was performed using TaqMan technology (TaqMan Gene Expression Assays; Applied Biosystems). The TaqMan probe IDs used were Kcnq1 (Mm00803386_m1), Tas2r105 (Mm00498502_s1), Tas1r1 (Mm00473433_m1), Tas1r2 (Mm00499716_m1), Tas1r3 (Mm00473459_g1), Pkd2l1 (Mm00619572_m1), Gustducin (Mm01165313_m1), and Plcb2 (Mm01338057_m1). The delta-delta method was used

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for relative quantification (Livak and Schmittgen, 2001). As the epithelial tissue surrounding the taste buds was present in the papillae samples, the mRNA expression of KCNQ1, a typical taste bud cell marker, was used as an internal control.

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Immunohistochemistry

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At 7 days after intraperitoneal injection of 5-bromo-20 deoxyuridine (BrdU; Sigma) at a dose of 25 mg/kg body weight for 3 days in young mice (n = 5) and old mice (n = 5), the FuP and CvP were dissected from the tongue and embedded in OCT compound (Sakura Finetek, Tokyo, Japan). BrdU is a thymidine analog that labels proliferating cells passing through S phase of the cell cycle and is used to detect DNA synthesis (Gratzner, 1982). The embedded samples were snapfrozen in liquid nitrogen and stored at 80 °C until use. Fixed papillae samples were cut into 6-mm-thick sections using a cryostat (Cryostar NX70; Thermo Scientific, Waltham, MA), and then attached to silanized slides (Matsunami Glass, Osaka, Japan). The sections were fixed in 4% paraformaldehyde for 10 min, washed twice with phosphate-buffered saline (PBS), blocked in MOM diluent (Vector MOM Immunodetection Kit; Vector Laboratories, Burlingame, CA) for 5 min at room temperature, and incubated overnight at 4 °C with rabbit anti-KCNQ1 primary antibody (Millipore, Billerica, MA) diluted 1:1000 in MOM diluent. The sections were then washed three times with PBS and incubated at room temperature for 1 h with the secondary antibody, Alexa Fluor 488-conjugated goat anti-rabbit IgG (Life Technologies), diluted 1:500 in MOM diluent. Next, the sections were washed three times with PBS, re-fixed in 4% paraformaldehyde for 10 min, washed twice with PBS, and incubated in 2 N HCl for 15 min at 37 °C. After two washes with PBS, the sections were incubated in MOM Mouse Ig Blocking Reagent for 1 h at room temperature. The sections were washed twice with PBS, blocked in MOM diluent for 5 min at room temperature, and incubated at room temperature with mouse anti-BrdU primary antibody (BrdU Labeling and Detection Kit II; Roche Diagnostics, Basel, Switzerland) diluted 1:100 in MOM diluent. After three washes with PBS, the sections were incubated with the secondary antibody, Alexa Fluor 555-conjugated goat anti-mouse IgG (Life Technologies), diluted 1:500 in MOM diluent for 1 h. After washing with PBS, the sections were mounted with Fluoromount (Diagnostic Biosystems, Pleasanton, CA). The cells with KCNQ1 and BrdU signals were counted in five sections (every 30 mm in CvP sections or different taste buds in FuP sections). We calculated the percentages of BrdU-positive cells in the KCNQ1-positive cells. The FuP sections were also used to examine ENaCa expression. The specific primary antibodies used were rabbit anti-ENaCa (1:500; Abcam ab77385, Cambridge, UK) and goat anti-KCNQ1 (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit IgG (1:500; Life Technologies) and Alexa Fluor 555-conjugated donkey anti-goat IgG (1:500; Life Technologies). Fluorescent images were obtained with an FV10i confocal laser-scanning

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microscope (Olympus, Tokyo, Japan) and a BX51 microscope equipped with a DP73 CCD digital camera (Olympus). Measurement of serum components

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After the gustatory nerve recordings, blood was drawn from the inferior vena cava for young mice (n = 4–6) and old mice (n = 5–6). The collected blood was stored overnight A 1.2 at 4 °C and the serum was separated by centrifugation. The serum levels of calcium, iron, magnesium, and zinc were 0.8 respectively measured with Metalloassay Calcium, Iron, Magnesium, and Zinc Kits (MG Metallogenics, Chiba, Japan). The 0.4 serum levels of sodium, angiotensin II, and leptin were measured with a Sodium Enzymatic Assay Kit (Diazyme Laboratories Poway, CA), 0 Angiotensin II EIA Kit (RayBiotech, 0.1 1 Norcross, GA), and Mouse/Rat Citric Leptin ELISA Kit (Morinaga Institute C 0.4 of Biological Science, Yokohama, Japan), respectively. Absorbances were measured in a microplate reader (Flexstation III; Molecular 0.3 Devices, Sunnyvale, CA).

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Statistical analysis

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The results were expressed as mean ± standard error of mean (SEM). All statistical analyses were conducted with GraphPad Prism 6 software (GraphPad Software, CA, USA). Behavioral assays were compared between young and old mice with a two-way repeated-measures analysis of variance (ANOVA). The other assays were compared between young and old mice with Welch’s t-test. For all analyses, differences with p-values lower than 0.05 were considered significant.

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RESULTS

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Changes in taste sensitivities with aging

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First, we confirmed whether aging modified taste sensitivities under our experimental conditions. For this, we tested the taste sensitivities to the five basic tastes, sour (citric acid), bitter (denatonium), salty (NaCl), sweet (sucrose), and umami (MSG + IMP), at various concentrations using two behavioral tests, a brief

Lick ratio

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access test (Fig. 1) and a 48-h two-bottle preference test (Fig. 2). Initially, we measured the taste sensitivities in the brief access test. The lick ratio for bitter taste in the old mice showed lower avoidance compared with that in the young mice (Fig. 1B; denatonium: F(1,90) = 8.09, p < 0.01). The lick ratio for salty taste in the old mice

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MSG + 0.5 mM IMP (mM) Fig. 1. Lick ratios for basic tastes in young and old mice. The lick ratios for (A) citric acid, (B) denatonium, (C) NaCl, (D) sucrose, and (E) MSG + 0.5 mM IMP are shown. Gray and red circles indicate young and old mice, respectively. * and ** indicate p < 0.05 and p < 0.01, respectively (two-way repeated-measures ANOVA, n = 10). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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showed a higher preference compared with that in the young mice (Fig. 1C; NaCl: F(1,108) = 5.84, p < 0.03). However, there were no significant differences between the young and old mice in the lick ratios for the other basic tastes (Fig. 1A, D, and E; citric acid: F(1,108) = 0.01, p = 0.92; sucrose: F(1,108) = 0.29, p = 0.59; MSG + IMP: F(1,108) = 3.35, p = 0.07). When the preference ratios were compared between the young and old mice, the preference ratio for bitter taste in the old mice showed decreased avoidance compared with that in the young mice (Fig. 2B; denatonium: F(1,80) = 23.25, p < 0.0001). Meanwhile, the preference ratio for salty taste in the old mice increased, while that in the young mice decreased (Fig. 2C; NaCl: F(1,91) = 12.21, p < 0.001). There were no significant differences in the preference ratios for sweet, sour, and umami tastes between the young and old mice (Fig. 2A, D, and E; citric acid: F(1,43) = 2.03, p = 0.16; sucrose: F(1,39) = 3.96, p > 0.05; MSG + IMP: F(1,39) = 1.11, p = 0.30). Thus, the behavioral assays revealed changes in the sensitivities for bitter and salty tastes. The changes in fluid intake are shown in Fig. 2. The total fluid intakes in the old mice were lower than those in the young mice (Fig. 2A–E; citric acid: F(1,43) = 54.1, p < 0.0001; denatonium: F(1, p < 0.0001; NaCl: 80) = 19.0, F(1,91) = 3.56, p = 0.06; sucrose: F(1, 39) = 8.67, p < 0.01; MSG

3 Fig. 2. Preference ratios for basic tastes in young and old mice. The preference ratios (left) relative to water and fluid intakes (right) for (A) citric acid, (B) denatonium, (C) NaCl, (D) sucrose, and (E) MSG + 0.5 mM IMP are shown. Gray and red symbols indicate young and old mice, respectively (For young mice, preference ratio = Y_Pref, total fluid intake = Y_To, and taste solution intake = Y_Ta; For old mice, preference ratio = O_Pref, total fluid intake = O_To, and taste solution intake = O_Ta). For preference ratios, *** and **** indicate p < 0.001 and p < 0.0001, respectively. For total fluid intakes, yy, yyy, and yyyy indicate p < 0.01, p < 0.001, and p < 0.0001, respectively. For tastant solutions, àà indicates p < 0.01. All statistical analyses were performed by twoway repeated-measures ANOVA (n = 5–10).

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Comparison of gustatory nerve activities between young and old mice

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As the sensitivities to some of the basic tastes changed with aging, we measured the activities of the gustatory

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old mice, while those of sucrose and MSG + IMP decreased.

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+ IMP: F(1,39) = 15.3, p < 0.0005). Regarding the tastant solutions, there were significant differences in tastant intakes between the young and old mice except for citric acid (Fig. 2A–E; citric acid: F(1,43) = 0.80, p = 0.38; denatonium: F(1,80) = 9.56, p < 0.003; NaCl: F(1,91) = 7.17, p < 0.01; sucrose: F(1,39) = 8.12, p = 0.01; MSG + IMP: F(1,39) = 12.5, p = 0.003). The tastant intakes of denatonium and NaCl increased in the

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(NaCl –(NaCl + 30 µM amiloride)) Fig. 3. Gustatory nerve responses to basic tastes in young and old mice. (A) Representative traces of integrated chorda tympani nerve responses for 100 mM NH4Cl, 30 mM citric acid, 30 mM denatonium, 300 mM NaCl, 300 mM NaCl + 30 lM amiloride, 300 mM sucrose, and 100 mM MSG + 0.5 mM IMP are shown. The nerve responses to (B) citric acid, (C) denatonium, (D) NaCl, (E) amilorid- insensitive NaCl component (NaCl + 30 mM amiloride), (F) amiloride-sensitive NaCl component (NaCl – (NaCl + 30 mM amiloride)), (G) sucrose, and (H) MSG + 0.5 mM IMP are shown. Gray and red squares indicate young and old mice, respectively. * indicates p < 0.05 (t-test, n = 4–6). Please cite this article in press as: Narukawa M et al. Participation of the peripheral taste system in aging-dependent changes in taste sensitivity. Neuroscience (2017), http://dx.doi.org/10.1016/j.neuroscience.2017.06.054

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nerves that transmit taste information received on the tongue to central nerves (Fig. 3A). There were no significant differences in the CT responses to sour, bitter, or umami tastes between the young and old mice (Fig. 3B, C, and H), while the responses to salty and sweet tastes in the old mice were stronger than those in the young mice (Fig. 3D, G). Thus, we observed that the gustatory responses to salty and sweet tastes were increased. The profile of the CT responses showed little correspondence with the profile of the behavioral assay responses. The taste response to NaCl has two components: amiloride-sensitive and amiloride-insensitive responses (Ninomiya and Funakoshi, 1988; Chandrashekar et al., 2010; Oka et al., 2013). The amiloride-sensitive and amiloride-insensitive components induce attractive and aversive salty taste responses, respectively (Chandrashekar et al., 2010; Oka et al., 2013). To elucidate the component contributing to the increased NaCl response, we measured the CT responses to a mixture of NaCl and amiloride. While the amiloride-insensitive responses were very similar in both age groups (Fig. 3E), amiloride-sensitive components in old mice were larger than those in young mice (Fig. 3F). These findings suggest that an increase in the amiloridesensitive component may underlie the increase in the NaCl response. Expression of taste-related molecules in taste bud cells To identify the cause of the changes in taste sensitivities, we measured the expressions of representative tasterelated molecules in the FuP and CvP. Using quantitative RT-PCR, we investigated the mRNA expressions of the following taste receptor molecules: bitter taste receptor Tas2R105; umami taste receptor subunit Tas1r1; sweet taste receptor subunit Tas1r2; sweet and umami taste receptor subunit Tas1r3; and sour taste receptor candidate Pkd2l1. In both the FuP and the CvP, there were no significant differences in the expression levels of the taste receptor mRNAs between the two age groups (Fig. 4A, B). We also investigated the expression of salty taste receptor channel subunit ENaCa in the FuP by immunostaining. Since the expression of ENaCa mRNA is observed in tongue epithelium surrounding taste buds (Li et al., 1994), we investigated protein expression of ENaCa. Immunoreactivity to the anti-ENaCa antibody was observed in both age groups and no apparent differences in the expression patterns were observed (Fig. 4C). Thus, there were no significant differences in the expressions of taste receptor molecules between the young and old mice. Next, we measured the mRNA expressions of two signaling molecules, the G protein coupled with the bitter taste receptor, Gustducin (Clapp et al., 2001) and the second messenger in bitter, umami, and sweet signaling, Plcb2 (Zhang et al., 2003). The mRNA levels of these signaling molecules in the old mice were significantly lower than those in the young mice (Fig. 4A, B). The mRNA level of Plcb2 was decreased in the CvP, while that of gustducin was decreased in both

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papillae. Thus, the age-dependent mRNA expressions of signaling effectors were decreased significantly, but the degrees of the decreases were slight.

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To investigate the effect of aging on the cell turnover rates in the FuP and CvP, we stained the cells in the taste buds with BrdU. The ratios of BrdU-positive cells in the taste bud cells of the FuP in the young and old mice were 0.06 ± 0.02 (0.80 ± 0.23 BrdU-positive cells in 13.5 ± 0.9 taste bud cells/section) and 0.05 ± 0.03 (0.54 ± 0.33 BrdU-positive cells in 12.8 ± 0.7 taste bud cells/section), respectively (Fig. 5A). Thus, the numbers of BrdU-positive cells in the FuP were similar between the two groups. In the taste bud cells of the CvP, the ratios of BrdU-positive cells in the young and old mice were 0.05 ± 0.02 (0.58 ± 0.21 BrdU-positive cells in 12.6 ± 0.5 taste bud cells/section) and 0.01 ± 0.00 (0.11 ± 0.04 BrdU-positive cells in 11.3 ± 0.5 taste bud cells/section), respectively (Fig. 5B). Thus, the number of BrdU-positive cells in the CvP tended to be lower in the old mice compared with the young mice. However, we did not observe any significant effects of aging on the turnover rates of taste bud cells.

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Measurement of serum components affecting taste responses

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Several studies have reported that serum components, such as mineral and hormone levels, modify taste responses (Liu et al., 1991; Tordoff, 1992; Kawai et al., 2000; Curtis et al., 2001; Goto et al., 2001; Okada et al., 2012; Shigemura et al., 2013). Therefore, we measured the concentrations of various serum components to investigate their effects on the change in palatability with aging. Because aging dramatically altered the taste responses to NaCl among the basic tastes (Figs. 2C and 3D), we focused on the concentrations of minerals and angiotensin II, a major mediator of body fluid and sodium homeostasis, which are known to affect responses to salty taste in young and old mice (Shigemura et al., 2013). The serum concentrations of the components are shown in Table 1. The serum concentrations of calcium, iron, and magnesium were significantly higher in the old mice compared with the young mice, while the serum concentration of sodium tended to be higher in the old mice. The serum concentration of zinc did not change with aging. The serum concentration of angiotensin II in the old mice was significantly lower than that in the young mice. Thus, the young and old mice exhibited different concentrations of serum components. This might mean that changes in the levels of serum components may affect the changes in taste sensitivities with aging. As aging altered the CT responses to sweet taste, the serum concentration of leptin, which is known to affect the sweet taste response (Kawai et al., 2000), was measured. Although the serum concentration of leptin tended to be higher in the old mice, there was no significant difference between the young and old mice.

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among the experimental conditions, mice older than 120 weeks, which is close to the average mouse lifespan, 10 were used as the old mice in this study. To confirm whether aging leads to 1 alterations in taste sensitivity, we employed two behavioral assays, a 48-h two-bottle preference test and a 0.1 brief access test. The two-bottle Tas2r105 Tas1r1 Tas1r2 Tas1r3 Pkd2l1 Gustducin Plcβ2 preference test is a conventional method for determining preference. 100 The mice had no restrictions for eating and drinking during the 10 measurements. However, because they could access the taste solution for a long time, we must consider 1 that the taste preference may reflect orosensory stimulation as well as post-ingestive effects (Manabe et al., 0.1 2010). The brief access test is based Tas2r105 Tas1r1 Tas1r2 Tas1r3 Pkd2l1 Gustducin Plcβ2 on behavior that mice lick the taste 䕔 Young, 䕔 Old solution. The intensity of taste sensitivity is shown by the initial licking rate. As the initial licking rate is usuKCNQ1 Merge ENaC ally measured within 1 min, postingestive effects are completely excluded (Manabe et al., 2010). Therefore, the initial lick number is considered to reflect preference for the solution with oral sensation. However, the mice usually have restrictions on eating and drinking, which KCNQ1 Merge ENaC induce stress. Therefore, we performed both tests in the present study. Aging changed the sensitivities for bitter and salty tastes, but did not significantly change the sensitivities for sour, sweet, and umami tastes. Thus, aging induced taste sensitivity changes in a subset of taste qualities, Fig. 4. Molecular expression of taste-related genes in taste bud cells. Relative mRNA expression levels of Tas2r105, Tas1r1, Tas1r2, Tas1r3, Pkd2l1, gustducin, and Plcb2 in the fungiform (A) and rather than all taste qualities. circumvallate papillae (B). Kcnq1 mRNA was used as an internal control. Gray and red bars To investigate whether aging * indicate young and old mice, respectively. Indicates p < 0.05 (t-test, n = 6). (C) Immunostaining affects taste responses at the of ENaCa (red) and KCNQ1 (green) in fungiform papillae from young and old mice. Scale nervous level, we measured the bar=50 lm. (For interpretation of the references to colour in this figure legend, the reader is gustatory nerve responses to taste referred to the web version of this article.) stimuli. The activity of the gustatory system at the periphery was DISCUSSION monitored, as a reliable measure of taste receptor cell function (Dahl et al., 1997). The CT In this study, to investigate the cause of the age-related and glossopharyngeal (GL) nerves relay gustatory inforchanges in taste sensitivity, we compared the peripheral mation from the anterior and posterior parts of the tongue, taste detection systems between young and old mice. respectively. The FuP and CvP innervate the CT and GL Although previous studies that investigated the potential nerves, respectively. It is known that these nerves have relationship between aging and taste sensation in distinct response profiles to taste substances (Shingai rodents produced inconsistent results, they all and Beidler, 1985). While the CT nerves are purely gustasuggested that aging may change taste sensitivity tory, the GL nerves contain both gustatory and (Thaw, 1996; Tordoff, 2007; Shin et al., 2012). Differsomatosensory components (Arai et al., 2010). Thereences in experimental conditions such as species, age, fore, we measured the CT nerve responses to taste suband concentration range of tested taste substances were stances in this study. While the CT responses to NaCl and considered potential reasons for the inconsistent results. sucrose were dramatically increased in the old mice, there As age of mice was thought to be of primary importance

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Fig. 5. BrdU-positive cells in the fungiform (FuP) and circumvallate papillae (CvP). Immunostaining with antibodies against BrdU (red) and KCNQ1 (green) in the FuP (A) and CvP (B). Arrowheads show BrdU signals. Scale bar=50 lm. The ratios of BrdU-positive cells in the taste bud cells of the FuP and CvP are shown on the right column (n = 5). In the FuP, a total of 18 and 20 taste buds were analyzed for young and old mice, respectively. In the CvP, a total of 350 and 310 taste buds were analyzed for young and old mice, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Table 1. Comparison of serum components between young and old mice

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Serum components

Young

Old

Calcium (mg/dL) Iron (lg/dL) Magnesium (mg/dL) Sodium (mg/dL) Zinc (lg/dL) Angiotensin II (pg/mL) Leptin (ng/mL)

10.4 ± 0.2 169 ± 6 3.4 ± 0.1 342 ± 8 104 ± 7 63.0 ± 4.9 3.7 ± 0.5

13.3 ± 0.6** 205 ± 12* 6.2 ± 0.2*** 394 ± 20 101 ± 8 49.3 ± 1.6* 5.9 ± 1.5

**

, , and n = 4–6).

***

indicate p < 0.05, p < 0.01 and p < 0.001, respectively (t-test,

were no significant differences between the two age groups in the CT responses to bitter, sour, and umami tastes. The profile of the CT responses showed little correspondence with the profile of the responses in the behavioral assays. The CT responses to NaCl were markedly increased in the old mice. To investigate this further, we employed amiloride, an inhibitor of ENaC, which has been implicated as a sensor of sodium salt taste (Heck et al., 1984; Chandrashekar et al., 2010). As there was no significant difference in the amiloride-insensitive component, it was considered that the increased NaCl response arose

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from the amiloride-sensitive component in the old mice. As the amiloride-sensitive component induces an attractive salty taste (Chandrashekar et al., 2010; Oka et al., 2013), an increase in the amiloride-sensitive component may have caused the increase in NaCl sensitivity in the old mice. Although changes in sensitivity to bitter taste were observed in the behavioral assays, there was no significant difference between the two age groups in the CT responses to bitter taste. As the gustatory nerve responses to bitter substances are stronger in GL nerves than in CT nerves (Danilova and Hellekant, 2003), a difference in the CT responses may not be observed. To identify the cause of the changes in taste sensitivities, we analyzed the expressions of representative taste-related molecules and the cell turnover rates. No significant effects of aging on the mRNA expressions of the representative taste receptor molecules were observed. We examined ENaCa expression in the FuP by immunostaining. ENaC is known to consist of at least three subunits (a, b, and c) (Canessa et al., 1994). The ENaCa has an amiloridesensitive sodium current (Canessa et al., 1994). By studying ENaCa KO, ENaCa-, and ENaCb-GFP mice, Chandrashekar et al. (2010) reported that functional ENaC is found in the FuP, but not in the CvP (Chandrashekar et al., 2010). Therefore, we investigated ENaCa expression in the FuP. Immunoreactivity for the anti-ENaCa antibody was observed in both age groups, with no apparent difference in the expression patterns observed macroscopically. Thus, apparent differences were not observed in the expression levels of taste receptors. Meanwhile, slight, but significant, decreases were observed in the mRNA expressions of signaling effectors such as Gustducin and Plcb2. Thus, the protein level of the signaling effectors may be also decreased in old mice. As gustducin is a known G protein coupled with the bitter taste receptor (Clapp et al., 2001), the sensitivities of bitter-responding taste cells may underlie the decrease in bitter taste avoidance. Taste cells turn over throughout life. The average turnover rate for taste cells was reported to be approximately 8–12 days (Beidler and Smallman, 1965; Farbman, 1980). It was further reported that significant reductions in taste bud size and number of taste cells per taste bud, but not in number of taste buds, were observed with aging (Shin et al., 2012). The number of taste cells per taste bud tended to decrease in both papillae of the old mice. Although the turnover rate of the taste bud cells tended to be slower in the CvP of the old mice, the difference was not significant. In the FuP, we did not observe alterations in the turnover rate between the young and old mice. Thus, under our experimental conditions, no apparent changes in the peripheral taste organs were observed with aging. Therefore, we conclude that the decreased sensitivities to bitter and salty tastes were not induced by a decline in function of the peripheral taste detection ability. Although aging induced changes in taste sensitivity, it did not affect all taste qualities in a similar manner, indicating the existence of certain taste quality-specific changes. In addition, apparent differences were not

observed in the expression levels of taste receptors. Although the mRNA expressions of signaling effectors were significantly decreased in the old mice, the degrees of the decreases were slight. PLCb2 is a common second messenger in bitter, umami, and sweet taste signaling (Zhang et al., 2003). Thus, although we expected that the sensitivities to umami and sweet tastes may be affected by aging as well as the sensitivity to bitter taste, the behavioral responses to umami and sweet tastes were free of the influence of aging. Furthermore, although we observed an increase in the CT response to sucrose in the old mice, there was no significant difference in the mRNA expression levels of Tas1rs between the two age groups. These findings suggest the existence of some taste quality-specific factors. Several studies have reported that changes in the levels of serum components are associated with changes in the physiological state that modifies taste responses (Liu et al., 1991; Tordoff, 1992; Kawai et al., 2000; Curtis et al., 2001; Goto et al., 2001; Okada et al., 2012; Shigemura et al., 2013). For example, it was reported that certain mineral deficiencies lead to increased preference for NaCl (Liu et al., 1991; Tordoff, 1992; Curtis et al., 2001; Goto et al., 2001; Okada et al., 2012). Additionally, intracellular signaling effectors such as intracellular pH (Lyall et al., 2002), intracellular calcium (DeSimone et al., 2012), osmolarity (Lyall et al., 1999), and cAMP (Mummalaneni et al., 2014) have been reported to alter CT response to NaCl. It is also known that angiotensin II modifies the NaCl and sucrose responses (Shigemura et al., 2013). As sensitivity to salty taste was most affected by aging among the basic tastes, we measured the concentrations of serum components that are known to modify NaCl responses. A study that investigated the change of the mineral status in various tissues with age showed that although mineral concentrations such as those of iron, zinc, calcium, magnesium, and copper did not change significantly in blood with age, iron, copper, and calcium concentrations were increased significantly with age in some tissues. In contrast, zinc and magnesium concentrations decreased significantly with age in brain and kidney, respectively. This suggests that each mineral show a different metabolic status (Morita et al., 1994). In this study, while the serum zinc levels did not change with aging, the levels of the other tested minerals increased (Table 1). These findings imply that the old mice were not in a mineral-deficient state, and suggest that mineral deficiency did not induce the change in taste sensitivity to NaCl. However, the serum angiotensin II concentration decreased significantly with aging. Shigemura et al. (2013) reported that an increase in the blood angiotensin II level decreased the amiloride-sensitive NaCl response. Therefore, the decreased serum angiotensin II concentration may be one of the reasons for the increase in taste responses to NaCl in the old mice. Although the sensitivity to sucrose did not differ between the young and old mice, the CT response to sucrose increased with aging. To investigate this discrepancy, we measured the serum level of leptin, a known sweet response-modifying hormone (Kawai et al., 2000), in the young and old mice. An increase in the blood leptin level was reported to

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decrease the gustatory response to sweet compounds (Kawai et al., 2000). However, there was no significant difference in the serum leptin concentrations between the two groups. Some hormones, such as glucagon-like peptide-1 and cannabinoids, are known as sweet response-modifying hormones (Shin et al., 2008; Yoshida et al., 2010). Therefore, such factors may participate in the sweet response. In addition, aging generally reduces metabolic expenditure. As sweet taste signals the existence of energy (Beauchamp, 2016), a decrease in metabolic expenditure may participate in the change in response to sweet taste. The intake of sucrose solution in the old mice was much lower than that in the young mice. As old mice can sense sweetness strongly, they may be satisfied with weak sweetness. In previous studies performed under different experimental conditions, taste sensitivity to NaCl increased with aging (Thaw, 1996; Tordoff, 2007). As a similar result was obtained in the present study, we consider that NaCl perception is susceptible to changes with aging.

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CONCLUSION

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In this study, we investigated the factors affecting changes in taste sensitivity with aging. There were no apparent differences in peripheral taste organs between young and old mice. Therefore, we conclude that the changes in taste sensitivity with aging were not caused by aging-related degradation of peripheral taste organs. Meanwhile, the concentrations of several serum components that modify taste responses changed with age. One of the reasons as to why taste sensitivity decreases with aging may be indirect effects of taste signal-modifying factors such as serum components.

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Acknowledgments—We thank Ms. Eri Saita, Ms. Yuki Yoshizumi, and Mr. Yuga Watanabe (The University of Tokyo, Japan) for their support with animal care.

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This work was supported in part by a Grant-in-Aid for Challenging Exploratory Research 26660106 to MN in the Priority Area Food Science from the Japan Society for the Promotion of Science, by The Salt Science Research Foundation No. 16D3, and by the Council for Science, Technology and Innovation (CSTI), Crossministerial Strategic Innovation Promotion Program (SIP) ‘‘Technologies for creating next-generation agriculture, forestry and fisheries.”

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REFERENCES

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Arai T, Ohkuri T, Yasumatsu K, Kaga T, Ninomiya Y (2010) The role of transient receptor potential vanilloid-1 on neural responses to acids by the chorda tympani, glossopharyngeal and superior laryngeal nerves in mice. Neuroscience 165:1476–1489. Beauchamp GK (2016) Why do we like sweet taste: A bitter tale? Physiol Behav 164:432–437. Beidler LM, Smallman RL (1965) Renewal of cells within taste buds. J Cell Biol 27:263–272. Canessa CM, Schild L, Buell G, Thorens B, Gautschi I, Horisberger JD, Rossier BC (1994) Amiloride-sensitive epithelial Na+ channel is made of three homologous subunits. Nature 367:463–467. Chandrashekar J, Kuhn C, Oka Y, Yarmolinsky DA, Hummler E, Ryba NJP, Zuker CS (2010) The cells and peripheral representation of sodium taste in mice. Nature 464:297–301.

766 767 768 769 770 771 772

11

Clapp TR, Stone LM, Margolskee RF, Kinnamon SC (2001) Immunocytochemical evidence for co-expression of Type III IP3 receptor with signaling components of bitter taste transduction. BMC Neurosci 2:6. Curtis KS, Krause EG, Contreras RJ (2001) Altered NaCl taste responses precede increased NaCl ingestion during Na+ deprivation. Physiol Behav 72:743–749. Dahl M, Erickson RP, Simon SA (1997) Neural responses to bitter compounds in rats. Brain Res 756:22–34. Danilova V, Hellekant G (2003) Comparison of the responses of the chorda tympani and glossopharyngeal nerves to taste stimuli in C57BL/6J mice. BMC Neurosci 4:5. DeSimone JA, Ren ZJ, Phan HT, Heck GL, Mummalaneni S, Lyall V (2012) Changes in taste receptor cell [Ca2+]i modulate chorda tympani responses to salty and sour taste stimuli. J Neurophysiol 108:3206–3220. Farbman AI (1980) Renewal of taste bud cells in rat circumvallate papillae. Cell Tissue Kinet 13:349–357. Goto T, Komai M, Suzuki H, Furukawa Y (2001) Long-term zinc deficiency decreases taste sensitivity in rats. J Nutr 131:305–310. Gratzner HG (1982) Monoclonal antibody to 5-bromo- and 5iododeoxyuridine: A new reagent for detection of DNA replication. Science 218:474–475. Heck GL, Mierson S, Desimone JA (1984) Salt taste transduction occurs through an amiloride-sensitive sodium transport pathway. Science 223:403–405. Kawai K, Sugimoto K, Nakashima K, Miura H, Ninomiya Y (2000) Leptin as a modulator of sweet taste sensitivities in mice. Proc Natl Acad Sci U S A 97:11044–11049. Liu XW, Dejima Y, Suzuki T, Himeno S, Okazaki Y (1991) Marginal zinc deficiency and changes in behavioral salt taste threshold and salt preference in mice. J Nutr Sci Vitaminol 37:185–199. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDC method. T Methods 25:402–408. Lyall V, Alam RI, Phan THT, Russell OF, Malik SA, Heck GL, DeSimone JA (2002) Modulation of rat chorda tympani NaCl responses and intracellular Na+ activity in polarized taste receptor cells by pH. J Gen Physiol 120:793–815. Lyall V, Heck GL, DeSimone JA, Feldman GM (1999) Effects of osmolarity on taste receptor cell size and function. Am J Physiol 277:C800–C813. Manabe Y, Matsumura S, Fushiki T (2010) Chapter 10 Preference for high-fat food in animals. In: Montmayeur JP, le Coutre J, editors. Fat detection: taste, texture, and post ingestive effects. Boca Raton, Florida: CRC Press. Morita A, Kimura M, Itokawa Y (1994) The effect of aging on the mineral status of female mice. Biol Trace Elem Res 42:165–177. Mummalaneni S, Qian J, Phan THT, Rhyu MR, Heck GL, DeSimone JA, Lyall V (2014) Effect of ENaC modulators on rat neural responses to NaCl. PLoS One 9:e98049. Narukawa M, Kitagawa-Iseki K, Oike H, Abe K, Mori T, Hayashi Y (2008) Characterization of umami receptor and coupling G protein in mouse taste cells. Neuroreport 19:1169–1173. Narukawa M, Minamisawa E, Hayashi Y (2009) Signalling mechanisms in mouse bitter responsive taste cells. Neuroreport 20:936–940. Narukawa M, Mori T, Hayashi Y (2006) Umami changes intracellular Ca2+ levels using intracellular and extracellular sources in mouse taste receptor cells. Biosci Biotechnol Biochem 70:2613–2619. Ninomiya Y, Funakoshi M (1988) Amiloride inhibition of responses of rat single chorda tympani fibers to chemical and electrical tongue stimulations. Brain Res 451:319–325. Oka Y, Butnaru M, von Buchholtz L, Ryba NJP, Zuker CS (2013) High salt recruits aversive taste pathways. Nature 494:472–475. Okada S, Abuyama M, Yamamoto R, Kondo T, Narukawa M, Misaka T (2012) Dietary zinc status reversibly alters both the feeding behaviors of the rats and gene expression patterns in diencephalon. BioFactors 38:203–218. Shigemura N, Iwata S, Yasumatsu K, Ohkuri T, Horio N, Sanematsu K, Yoshida R, Margolskee RF, Ninomiya Y (2013) Angiotensin II

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M. Narukawa et al. / Neuroscience xxx (2017) xxx–xxx

modulates salty and sweet taste sensitivities. J Neurosci 33:6267–6277. Shin YK, Cong WN, Cai H, Kim W, Maudsley S, Egan JM, Martin B (2012) Age-related changes in mouse taste bud morphology, hormone expression, and taste responsivity. J Gerontol A Biol Sci Med Sci 67:336–344. Shin YK, Martin B, Golden E, Dotson CD, Maudsley S, Kim W, Jang HJ, Mattson MP, Drucker DJ, Egan JM, Munger SD (2008) Modulation of taste sensitivity by GLP-1 signaling. J Neurochem 106:455–463. Shingai T, Beidler LM (1985) Response characteristics of three taste nerves in mice. Brain Res 335:245–249. Thaw AK (1996) Changes in taste threshold over the life span of the Sprague–Dawley rat. Chem Senses 21:189–193.

Tordoff MG (1992) Salt intake of rats fed diets deficient in calcium, iron, magnesium, phosphorus, potassium, or all minerals. Appetite 18:29–41. Tordoff MG (2007) Taste solution preferences of C57BL/6J and 129X1/SvJ mice: Influence of age, sex, and diet. Chem Senses 32:655–671. Yarmolinsky DA, Zuker CS, Ryba NJP (2009) Common sense about taste: from mammals to insects. Cell 139:234–244. Yoshida R, Ohkuri T, Jyotaki M, Yasuo T, Horio N, Yasumatsu K, Sanematsu K, Shigemura N, Yamamoto T, Margolskee RF, Ninomiya Y (2010) Endocannabinoids selectively enhance sweet taste. Proc Natl Acad Sci U S A 107:935–939. Zhang YF, Hoon MA, Chandrashekar J, Mueller KL, Cook B, Wu DQ, Zuker CS, Ryba NJP (2003) Coding of sweet, bitter, and umami tastes: Different receptor cells sharing similar signaling pathways. Cell 112:293–301.

(Received 23 March 2017, Accepted 28 June 2017) (Available online xxxx)

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