Neuroscience Letters 577 (2014) 56–60
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Decline of umami preference in aged rats Hirohito Miura ∗ , Makoto Ooki, Norikazu Kanemaru, Shuitsu Harada Department of Oral Physiology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
h i g h l i g h t s • • • •
Neural responses to both sweet and umami taste did not differ between young and aged rats. Preference for umami taste markedly declined in aged rats. Preference for sweet taste was maintained in aged rats. Aging affects central mechanisms of taste preferences for umami.
a r t i c l e
i n f o
Article history: Received 25 March 2014 Received in revised form 25 May 2014 Accepted 6 June 2014 Available online 14 June 2014 Keywords: Aging Taste preference Taste nerve responses Umami Sweet
a b s t r a c t The effects of aging on the umami sensation were compared between the preference and neural responses from the greater superficial petrosal nerve (GSP innervating the soft palate) and the chorda tympani nerve (CT innervating the fungiform papillae) in the Sprague Dawley rat. A two-bottle preference test revealed that younger rats (5–12 weeks) preferred significantly 0.001 M 5 -inosine monophosphate (IMP), 0.01 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP + 0.01 M MSG than deionized water. However, aged rats (21–22 months) showed no significant preference to these umami solutions compared to deionized water. Among the other four basic taste stimuli, there were no significant differences in preference between young and aged rats. Regardless of the age of the rat, neural responses from the GSP and CT produced robust integrated responses to all three umami solutions used in the two-bottle tests. These results indicate that the lack of preference to umami in aged rats is a central nervous system phenomenon and suggests that the loss of preference to umami taste in aged rats is caused by homeostatic changes in the brain incurred by aging. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A number of studies in humans reported that taste thresholds for classical four basic tastes, sweet, bitter, sour and salty, generally increased with age [1]. However, in some reports, elderly individuals were shown to have a decrease in taste intensity only for bitter and sour taste stimuli [2–5] or solely for just bitter stimuli [6–8]. Further, taste thresholds to sweet and salty stimuli did not change with age [4,5,7,9–11]. It is pointed out that discrepancies among individual studies may be caused by the differences in age-group and/or unawareness of health condition or drug use of aged human subjects [1]. In contrast to numerous reports on the effects of classical four basic tastes, data on the aging effects on umami taste are very limited, and also still controversial. The detection thresholds for glutamate salts in humans were 5.04 times higher in elderly
subjects [12], while the concentration of pleasant umami taste was not significantly different between the young and old [13]. A few reports exist on the change with age in taste threshold or preference to taste stimuli in animals. Behavioral experiment in the rat revealed that taste thresholds increased with age in sucrose (Suc) and NaCl but not to quinine hydrochloride (QHCl) [14]. However, other experiments in C57BL/6J and 129X1/SvJ mice showed that age contributed little to the variation in taste preferences [15], while only IMP was used as an umami stimulus in the experiment. Changes with age in umami taste are still unclear even in animals. The present study aims to elucidate the peripheral neural effects of aging on preference to umami taste by recording responses from the greater superficial petrosal (GSP) and the chorda tympani (CT) nerve that primarily mediate umami taste information [16]. 2. Materials and methods 2.1. Animals
∗ Corresponding author. Tel.: +81 99 275 6122; fax: +81 99 275 6128. E-mail address: [email protected] (H. Miura). http://dx.doi.org/10.1016/j.neulet.2014.06.018 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Male rats (Sprague Dawley, young: 5–12 weeks, aged 21–22 month) were used. Animals were used for a two-bottle preference
H. Miura et al. / Neuroscience Letters 577 (2014) 56–60
test and sacrificed for neural experiments of the GSP or CT. All animal experiments were conducted in Kagoshima University, and all experimental procedures were approved by the institutional animal care and use committees before the onset of the experiments. 2.2. Two-bottle preference test Young (5–12 weeks, n = 7) and aged (21–22 months, n = 4) male rats were used for a two-bottle preference test. Rats were housed individually in polycarbonate cages with free access to chow (CE7, Clea Japan, Inc.). Room temperature and test solutions were 22 ± 1 ◦ C and the light and dark cycle was 08:00–20:00. Paired solutions were presented in two glass bottles, each with a liquid tube made of stainless steel that controls liquid flow with a valve by spring (4.7 mm in diameter, Toyoriko, Japan). The horizontal distance between the two tips of the licking tubes was set 3 cm. During the first week of training, only deionized water (DW) was presented for 24 h. In the second training period of 4 days, two DW bottles were presented for 20 min at 8:00 am and 8:00 pm. The position of the two bottles was switched at each time to avoid placement effects. In the third training period of 2 days, 0.3 M Suc and DW were presented simultaneously for 20 min at 8 a.m. and 8 p.m. After the training period, a test solution and DW were presented for 20 min at 8 a.m. and 8 p.m. for two days and the consumption of each solution was measured. The position of two bottles was switched at each time. Then, two bottles of DW were presented for 1 day as a resting day. Test solutions were 0.1 M NaCl, 0.3 M Suc, 0.01 M HCl, 0.001 M QHCl, 0.001 and 0.003 M 5 -inosine monophosphate (IMP), 0.01 and 0.03 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP + 0.01 M MSG, 0.003 M IMP + 0.03 M MSG. The degree of preference was calculated by a preference ratio = 2 × a/(a + b) − 1; a = intake volume of test solution, b = intake volume of DW. Student’s t-test was used to determine whether any difference was between a paired consumption of test solution and DW. 2.3. Neurophysiological recordings Young (5–12 weeks, n = 7) and aged (21–22 months, n = 9) male rats were used for the neurophysiological experiments. The
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procedure to dissect the GSP and CT were described previously [17,18]. Responses from the whole nerve were amplified, integrated and displayed on a thermal array recorder at a speed of 1 mm/s as described [17]. Taste stimuli were applied to a deionized water (DW) rinse (1 ml/s) through polyethylene tubing (2 mm id.). The constantly flowing rinse water was switched to the stimulus solution for 10 s. The stimulus solution reached its maximum concentration within 1 s. Test solutions were 0.1 M NaCl, 0.5 M Suc, 0.01 M HCl, 0.01 M QHCl, 0.001 and 0.003 M IMP, 0.01 and 0.03 M MSG, and binary mixtures of 0.001 M IMP + 0.01 M MSG, 0.003 M IMP + 0.03 M MSG. Stimuli and rinse solutions were presented at 20 ± 1 ◦ C. Na+ produced large integrated response from the CT which masked the umami responses to the Na salts of IMP and MSG. Umami substances were therefore applied after a 10 s preadaptation to the same concentration of NaCl; 0.001 M NaCl–0.001 M IMP, 0.01 M NaCl–0.01 M MSG. 0.01 M NaCl was used for the preadaptation for testing binary mixtures of MSG + IMP since 0.01 M NaCl instead of 0.011 M adaptation is sufficient. The peaks of the initial phasic integrated responses were measured. The phasic response to the standard stimuli (0.1 M NaCl) was used as the standard response magnitude. The response magnitude of each response was calculated relative to the magnitude of the standard response. For the CT responses to umami substances, the magnitude of the response after 10 s adaptation to NaCl was measured from the base line to the peak response (arrows in Fig. 3). 2.4. Blood exam The blood exam was performed in young (8-week, n = 5) and aged (19-month, n = 5) rats to measure levels of plasma totalprotein, albumin, blood urea nitrogen, creatinine, and glucose with SPOTCHEM EZ (SP-4430, ARKRAY USA, Edina, Minnesota). 2.5. Data analysis For the results of the behavioral experiment, Student’s t-test was used to determine if significant differences occurred between a preference to stimulation and DW. For the neurophysiological experiments, the response magnitudes to three umami
Fig. 1. Preference ratio obtained by a two-bottle test to four basic taste and umami stimuli in young (5–12 weeks, n = 7) and aged (21–22 months, n = 4) rats. A test solution and DW were presented simultaneously, and the preference ratio was calculated by the equation: preference ratio = 2 × a/(a + b) − 1; a = intake volume of test solution, b = intake volume of DW. Error bars shows SE of the mean. Asterisks indicate statistical significance between a test solution and DW by Paired t-test; *** p < 0.0005, ** p < 0.005, * p < 0.05.
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H. Miura et al. / Neuroscience Letters 577 (2014) 56–60
Fig. 2. Integrated responses of the GSP in a young (8-week) and an aged (24-month) rat to 0.003 M IMP, 0.03 M MSG, binary mixture of 0.003 M IMP + 0.03 M MSG, 0.5 M Suc, 0.1 M NaCl and DW. Stimuli were applied for 10 s.
stimulations were compared with a two-way ANOVA and post hoc Bonferroni/Dunn test. 3. Results 3.1. Two-bottle preference test The results of the two-bottle preference tests revealed that both young and aged rats significantly reject 0.001 M QHCl (p < 0.0005) and prefer 0.3 M Suc (young p < 0.0005, aged p < 0.005). Although umami solutions induced a strong preference in young rats (p < 0.0005), this was not observed in aged rats (Fig. 1). 3.2. Neural recordings from the GSP and CT in aged rats Four basic taste and umami substances produced robust GSP responses in the aged rats (Figs. 2 and 3), and binary mixtures of 0.001 M IMP + 0.01 M MSG produced significant synergistic effects (Fig. 3), as shown in young rats [16]. NaCl produced large responses in the CT at the start of the 10 s adaptation to NaCl (0.001 M, 0.01 M, 0.1 M NaCl). After the 10 s adaptation with NaCl, the Na salts of umami substances were stimulatory as indicated by arrows (Fig. 4A). Significant synergistic effects were observed to binary mixtures of 0.001 M IMP + 0.01 M MSG in aged rats (Fig. 4), as also shown in young rats [16]. 3.3. Blood exam To compare the physiological condition of aged rats to that of young rats, blood exam was conducted. The results showed no
Fig. 3. Mean relative phasic response magnitudes to 0.01 M HCl, 0.01 M QHCl, 0.5 M Suc, 0.001 M IMP, 0.01 M MSG, and binary mixtures of 0.001 M IMP + 0.01 M MSG from the GSP of 21–24 month old rats. Error bars show SE of the mean (n = 4). Asterisks indicate statistical significance by post hoc analysis; ** p < 0.005, * p < 0.05.
significant differences in the total plasma protein and creatinine levels between young and aged rats, while the levels were significantly lower for plasma albumin (p < 0.05), BUN (p < 0.05) and glucose (p < 0.005) in the aged compared to younger rats (Fig. 5).
Fig. 4. (A) Integrated responses of the CT in a aged (24-month) rat to 0.001 M IMP subsequent to adaptation to 0.001 M NaCl, 0.01 M MSG subsequent to adaptation to 0.01 M NaCl, binary mixture of 0.001 M IMP + 0.01 M MSG subsequent to adaptation to 0.01 M NaCl, and 0.1 M NaCl. Response magnitude was measured from base line to the peak response (arrow). (B) Mean relative response magnitudes to 0.001 M IMP, 0.01 M MSG, and the binary mixture of 0.001 M IMP + 0.01 M MSG from the GSP of 21–24 month old rats. Error bars show SE of the mean (n = 5). Asterisks indicate statistical significance by post hoc analysis; ** p < 0.005, * p < 0.05.
H. Miura et al. / Neuroscience Letters 577 (2014) 56–60
Fig. 5. Results of the blood exam in young (8-week) and aged (19-month) male rats. T-Pro: total plasma protein, Alb: albumin, BUN: blood urea nitrogen, Cre: creatinine, Glu: glucose. n = 5; ** p < 0.005, * p < 0.05.
4. Discussion Umami taste provides an important clue to evoke ingestive behavior since birth. Brest milk is rich in free glutamate [19,20], and human neonates show a strong preference to MSG [21]. The present experiments showed that young rats, as in humans, showed strong preferences to IMP, MSG and to the binary mixture of IMP and MSG. However, the preference to umami substances disappeared in aged rats. Gustatory information of umami was previously reported to be conveyed dominantly via the GSP and CT but not glossopharyngeal (GL) among three major taste nerves, based on the behavioral analysis of the rat with taste nerves transected [16]. In aged rats, in spite of the disappearance of umami preference, both GSP and CT produced robust umami responses to MSG, which was characterized by synergistic effects with IMP [22]. In contrast to umami, sweet taste induced strong preference behavior in not only young but also old rats. The aging effects on umami and sweet taste responses are expected to be similar in both the neurophysiological and behavioral experiments, since they share the same taste receptor subunit for these substances in taste buds. Specific combinations of Tas1r family members serve as sweet or umami taste receptor: Tas1r1 and Tas1r2 for umami, and Tas1r2 and Tas1r3 for sweet [23]. Both in young and aged rats, similarly to the sweet taste responses to Suc, the robust neural umami responses were produced by IMP, MSG and binary mixture of IMP and MSG in the GSP and CT, showing that umami information is conveyed to the brain regardless of age. There results indicate a robustness of peripheral taste system for sweet and umami. However, the behavioral experiments showed that a preference behavior to umami in young rats disappeared in aged rats, although aged rats still significantly preferred Suc. The preference for umami was previously reported to be altered depending on nutritional conditions. Rats fed diets deficient in protein or an essential l-amino acid, l-lysine (Lys), increased the consumption of Lys, glycine and NaCl, but not umami substances; however, a preference for umami substances appeared when the rats were recovered from their previous deficiency [22]. Our results of the blood exam between young and aged rats showed no significant difference in the total plasma protein and no morbid condition of starvation, therefore, the reduced preference to umami substances in aged rats must not be associated with the plasma level of protein. The homeostatic control of plasma glucose directly links to the regulation of food intake [24]. When glucose level lowered,
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ingestive behavior is evoked. In the present report, plasma glucose level was lower in aged rats than in young rats. A similar decline of plasma glucose with age was previously reported in aged male rats [25]. On the other hand, food intake and energy consumption were shown to be decreased with age [26]. This inverse relationship between plasma glucose and food intake implies the aging effect on appetite regulated by a variety of neuropeptides and hormones [27]. Taste-evoked ingestive behavior is assumed to be controlled by some of neuropeptides regulating appetite [28]. Orexin-A and neuropeptide Y (NPY) are involved in sweetener-induced overconsumption in adult rats [29]: Drinking a sweet solution, saccharin, elevated mRNA level of orexin-A and NPY in the hypothalamus, and, on the other hand, intracerebroventricular (ICV) administrations of orexin-A, NPY and melanin-concentrating hormone (MCH) increased the intake of saccharin in adult rats. Also, ICV injection of orexin-A, NPY and ghrelin is showed to dose-dependently increase food intake in both young (4 month old) and adult (11 month old) rats [26]. However, in aged (25–27 month old) rats, orexin-A and NPY did not stimulate food intake at any dose, and ghrelin still induced food intake but with reduced efficacy. Based on these studies, the difference in aging effect on the taste preference between sweet and umami in the present report may be due to the difference in contributing neuropeptides. Preference behavior to umami substances depends on neuropeptides such as orexin-A and NPY, which may not be effective in aged rats, but not ghrelin. Hence, preference to umami was not evoked, despite umami information conveyed to the brain via CT and GSP nerves. In contrast, preference to Suc may be mediated by such as ghrelin or some other neuropeptide that is effective in aged rats. 5. Conclusion In conclusion, this is the first report indicating the existence of aging effects on the modification of umami preference in the CNS. Further experiments are necessary to clarify the central mechanisms that caused the depletion of umami preference in aged rats. Acknowledgments We thank Dr. John Caprio for valuable comments on the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (C) (Nos. 06671865, 23593036) from the Ministry of Education, Science and Culture in Japan. References [1] J. Mojet, E. Christ-Hazenlhof, J. Heidema, Taste perception with age: generic or specific losses in threshold sensitivity to the five basic tastes, Chem. Senses 26 (2001) 845–860. [2] L.M. Bartoshuk, B. Rifkin, L.E. Marks, P. Bars, Taste and aging, J. Gerontol. 41 (1986) 51–57. [3] B.J. Cowart, Relationships between taste and smell across the adult life span, Ann. N.Y. Acad. Sci. 561 (1989) 39–55. [4] R.J. Hyde, R.P. Feller, Age and sex effects on taste of sucrose, NaCl, citric acid and caffeine, Neurobiol. Aging 2 (1981) 315–318. [5] C. Murphy, M.M. Gilmore, Quality-specific effects of aging on the human taste system, Percept. Psychophys. 45 (1989) 121–128. [6] J. Mojet, J. Heidema, E. Christ-Hazelhof, Taste perception with age: generic or specific losses in supra-threshold intensities of five taste qualities, Chem. Senses 28 (2003) 397–413. [7] D.A. Stevens, H.T. Lawless, Age-related changes in flavor perception, Appetite 2 (1981) 127–136. [8] S.S. Schiffman, E.A. Sattely-Miller, I.A. Zimmerman, B.G. Graham, R.P. Erickson, Taste perception of monosodium glutamate (MSG) in foods in young and elderly subjects, Physiol. Behav. 56 (1994) 265–275. [9] Z.S. Warwick, S.S. Schiffman, Sensory evaluations of fat-sucrose and fat-salt mixtures: relationship to age and weight status, Physiol. Behav. 48 (1990) 633–636.
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[10] J.M. Weiffenbach, B.J. Cowart, B.J. Baum, Taste intensity perception in aging, J. Gerontol. 41 (1986) 460–468. [11] J.M. Weiffenbach, C.A. Tylenda, B.J. Baum, Oral sensory changes in aging, J. Gerontol. 45 (1990) M121–M125. [12] S.S. Schiffman, A.E. Frey, J.A. Luboski, M.A. Foster, R.P. Erickson, Taste of glutamate salts in young and elderly subjects: role of inosine 5 -monophosphate and ions, Physiol. Behav. 49 (1991) 843–854. [13] J. Mojet, E. Christ-Hazelhof, J. Heidema, Taste perception with age: pleasantness and its relationships with threshold sensitivity and suprathreshold intensity of five taste qualities, Food Qual. Preference 16 (2005) 413–423. [14] A.K. Thaw, Changes in taste threshold over the life span of the Sprague-Dawley rat, Chem. Senses 21 (1996) 189–193. [15] M.G. Tordoff, Taste solution preferences of C57BL/6J and 129X1/SvJ mice: influence of age, sex, and diet, Chem. Senses 32 (2007) 655–671. [16] N. Sako, S. Harada, T. Yamamoto, Gustatory information of umami substances in three major taste nerves, Physiol. Behav. 71 (2000) 193–198. [17] S. Harada, D.V. Smith, Gustatory sensitivities of the hamster’s soft palate, Chem. Senses 17 (1992) 37–51. [18] S. Harada, T. Yamamoto, K. Yamaguchi, Y. Kasahara, Different characteristics of gustatory responses between the greater superficial petrosal and chorda tympani nerves in the rat, Chem. Senses 22 (1997) 133–140. [19] K. Kurihara, Glutamate: from discovery as a food flavor to role as a basic taste (umami), Am. J. Clin. Nutr. 90 (2009) 719S–722S. [20] D.K. Rassin, J.A. Sturman, G.E. Guall, Taurine and other free amino acids in milk of man and other mammals, Early Hum. Dev. 2 (1978) 1–13.
[21] J. Steiner, What the neonate can tell us about umami, in: Y. Kawamura, M.R. Kare (Eds.), Umami: A Basic Taste, Marcel Dekker, New York, NY, 1987, pp. 97–123. [22] K. Torii, T. Mimura, Y. Yugari, Biochemical mechanism of umami taste perception and effect of dietary protein on the taste preference for amino acids and sodium chloride in rats, in: Y. Kawamura, M.R. Kare (Eds.), Umami: A Basic Taste, Marcel Dekker, New York, NY, 1987, pp. 513–563. [23] S. Damak, M. Rong, K. Yasumatsu, Z. Kokrashvili, V. Varadarajan, S. Zou, et al., Detection of sweet and umami taste in the absence of taste receptor T1r3, Science 301 (2003) 850–853. [24] M.W. Schwartz, S.C. Woods, D. Porte, R.J. Seeley, D.G. Baskin, Central nervous system control of food intake, Nature 404 (2000) 661–671. [25] J.B. Van Liew, P.J. Davis, F.B. Davis, L.L. Bernardis, M.R. Deziel, L.N. Marinucci, et al., Effects of aging, diet, and sex on plasma glucose, fructosamine, and lipid concentrations in barrier-raised Fischer 344 rats, J. Gerontol. 48 (1993) B184–B190. [26] S. Akimoto, K. Miyasaka, Age-associated changes of appetite-regulating peptides, Geriatr. Gerontol. Int. 10 (Suppl. 1) (2010) S107–S119. [27] G.J. Morton, D.E. Cummings, D.G. Baskin, G.S. Barsh, M.W. Schwartz, Central nervous system control of food intake and body weight, Nature 443 (2006) 289–295. [28] Y. Yamamoto, Central mechanisms of taste: cognition, emotion and tasteelicited behaviors, Jpn. Dent. Sci. Rev. 44 (2008) 91–99. [29] Y. Furudono, C. Ando, C. Yamamoto, M. Kobashi, T. Yamamoto, Involvement of specific orexigenic neuropeptides in sweetener-induced overconsumption in rats, Behav. Brain Res. 175 (2006) 241–248.
Contents lists available at ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Decline of umami preference in aged rats Hirohito Miura ∗ , Makoto Ooki, Norikazu Kanemaru, Shuitsu Harada Department of Oral Physiology, Kagoshima University Graduate School of Medical and Dental Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan
h i g h l i g h t s • • • •
Neural responses to both sweet and umami taste did not differ between young and aged rats. Preference for umami taste markedly declined in aged rats. Preference for sweet taste was maintained in aged rats. Aging affects central mechanisms of taste preferences for umami.
a r t i c l e
i n f o
Article history: Received 25 March 2014 Received in revised form 25 May 2014 Accepted 6 June 2014 Available online 14 June 2014 Keywords: Aging Taste preference Taste nerve responses Umami Sweet
a b s t r a c t The effects of aging on the umami sensation were compared between the preference and neural responses from the greater superficial petrosal nerve (GSP innervating the soft palate) and the chorda tympani nerve (CT innervating the fungiform papillae) in the Sprague Dawley rat. A two-bottle preference test revealed that younger rats (5–12 weeks) preferred significantly 0.001 M 5 -inosine monophosphate (IMP), 0.01 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP + 0.01 M MSG than deionized water. However, aged rats (21–22 months) showed no significant preference to these umami solutions compared to deionized water. Among the other four basic taste stimuli, there were no significant differences in preference between young and aged rats. Regardless of the age of the rat, neural responses from the GSP and CT produced robust integrated responses to all three umami solutions used in the two-bottle tests. These results indicate that the lack of preference to umami in aged rats is a central nervous system phenomenon and suggests that the loss of preference to umami taste in aged rats is caused by homeostatic changes in the brain incurred by aging. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction A number of studies in humans reported that taste thresholds for classical four basic tastes, sweet, bitter, sour and salty, generally increased with age [1]. However, in some reports, elderly individuals were shown to have a decrease in taste intensity only for bitter and sour taste stimuli [2–5] or solely for just bitter stimuli [6–8]. Further, taste thresholds to sweet and salty stimuli did not change with age [4,5,7,9–11]. It is pointed out that discrepancies among individual studies may be caused by the differences in age-group and/or unawareness of health condition or drug use of aged human subjects [1]. In contrast to numerous reports on the effects of classical four basic tastes, data on the aging effects on umami taste are very limited, and also still controversial. The detection thresholds for glutamate salts in humans were 5.04 times higher in elderly
subjects [12], while the concentration of pleasant umami taste was not significantly different between the young and old [13]. A few reports exist on the change with age in taste threshold or preference to taste stimuli in animals. Behavioral experiment in the rat revealed that taste thresholds increased with age in sucrose (Suc) and NaCl but not to quinine hydrochloride (QHCl) [14]. However, other experiments in C57BL/6J and 129X1/SvJ mice showed that age contributed little to the variation in taste preferences [15], while only IMP was used as an umami stimulus in the experiment. Changes with age in umami taste are still unclear even in animals. The present study aims to elucidate the peripheral neural effects of aging on preference to umami taste by recording responses from the greater superficial petrosal (GSP) and the chorda tympani (CT) nerve that primarily mediate umami taste information [16]. 2. Materials and methods 2.1. Animals
∗ Corresponding author. Tel.: +81 99 275 6122; fax: +81 99 275 6128. E-mail address: [email protected] (H. Miura). http://dx.doi.org/10.1016/j.neulet.2014.06.018 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Male rats (Sprague Dawley, young: 5–12 weeks, aged 21–22 month) were used. Animals were used for a two-bottle preference
H. Miura et al. / Neuroscience Letters 577 (2014) 56–60
test and sacrificed for neural experiments of the GSP or CT. All animal experiments were conducted in Kagoshima University, and all experimental procedures were approved by the institutional animal care and use committees before the onset of the experiments. 2.2. Two-bottle preference test Young (5–12 weeks, n = 7) and aged (21–22 months, n = 4) male rats were used for a two-bottle preference test. Rats were housed individually in polycarbonate cages with free access to chow (CE7, Clea Japan, Inc.). Room temperature and test solutions were 22 ± 1 ◦ C and the light and dark cycle was 08:00–20:00. Paired solutions were presented in two glass bottles, each with a liquid tube made of stainless steel that controls liquid flow with a valve by spring (4.7 mm in diameter, Toyoriko, Japan). The horizontal distance between the two tips of the licking tubes was set 3 cm. During the first week of training, only deionized water (DW) was presented for 24 h. In the second training period of 4 days, two DW bottles were presented for 20 min at 8:00 am and 8:00 pm. The position of the two bottles was switched at each time to avoid placement effects. In the third training period of 2 days, 0.3 M Suc and DW were presented simultaneously for 20 min at 8 a.m. and 8 p.m. After the training period, a test solution and DW were presented for 20 min at 8 a.m. and 8 p.m. for two days and the consumption of each solution was measured. The position of two bottles was switched at each time. Then, two bottles of DW were presented for 1 day as a resting day. Test solutions were 0.1 M NaCl, 0.3 M Suc, 0.01 M HCl, 0.001 M QHCl, 0.001 and 0.003 M 5 -inosine monophosphate (IMP), 0.01 and 0.03 M mono sodium glutamate (MSG), and binary mixtures of 0.001 M IMP + 0.01 M MSG, 0.003 M IMP + 0.03 M MSG. The degree of preference was calculated by a preference ratio = 2 × a/(a + b) − 1; a = intake volume of test solution, b = intake volume of DW. Student’s t-test was used to determine whether any difference was between a paired consumption of test solution and DW. 2.3. Neurophysiological recordings Young (5–12 weeks, n = 7) and aged (21–22 months, n = 9) male rats were used for the neurophysiological experiments. The
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procedure to dissect the GSP and CT were described previously [17,18]. Responses from the whole nerve were amplified, integrated and displayed on a thermal array recorder at a speed of 1 mm/s as described [17]. Taste stimuli were applied to a deionized water (DW) rinse (1 ml/s) through polyethylene tubing (2 mm id.). The constantly flowing rinse water was switched to the stimulus solution for 10 s. The stimulus solution reached its maximum concentration within 1 s. Test solutions were 0.1 M NaCl, 0.5 M Suc, 0.01 M HCl, 0.01 M QHCl, 0.001 and 0.003 M IMP, 0.01 and 0.03 M MSG, and binary mixtures of 0.001 M IMP + 0.01 M MSG, 0.003 M IMP + 0.03 M MSG. Stimuli and rinse solutions were presented at 20 ± 1 ◦ C. Na+ produced large integrated response from the CT which masked the umami responses to the Na salts of IMP and MSG. Umami substances were therefore applied after a 10 s preadaptation to the same concentration of NaCl; 0.001 M NaCl–0.001 M IMP, 0.01 M NaCl–0.01 M MSG. 0.01 M NaCl was used for the preadaptation for testing binary mixtures of MSG + IMP since 0.01 M NaCl instead of 0.011 M adaptation is sufficient. The peaks of the initial phasic integrated responses were measured. The phasic response to the standard stimuli (0.1 M NaCl) was used as the standard response magnitude. The response magnitude of each response was calculated relative to the magnitude of the standard response. For the CT responses to umami substances, the magnitude of the response after 10 s adaptation to NaCl was measured from the base line to the peak response (arrows in Fig. 3). 2.4. Blood exam The blood exam was performed in young (8-week, n = 5) and aged (19-month, n = 5) rats to measure levels of plasma totalprotein, albumin, blood urea nitrogen, creatinine, and glucose with SPOTCHEM EZ (SP-4430, ARKRAY USA, Edina, Minnesota). 2.5. Data analysis For the results of the behavioral experiment, Student’s t-test was used to determine if significant differences occurred between a preference to stimulation and DW. For the neurophysiological experiments, the response magnitudes to three umami
Fig. 1. Preference ratio obtained by a two-bottle test to four basic taste and umami stimuli in young (5–12 weeks, n = 7) and aged (21–22 months, n = 4) rats. A test solution and DW were presented simultaneously, and the preference ratio was calculated by the equation: preference ratio = 2 × a/(a + b) − 1; a = intake volume of test solution, b = intake volume of DW. Error bars shows SE of the mean. Asterisks indicate statistical significance between a test solution and DW by Paired t-test; *** p < 0.0005, ** p < 0.005, * p < 0.05.
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Fig. 2. Integrated responses of the GSP in a young (8-week) and an aged (24-month) rat to 0.003 M IMP, 0.03 M MSG, binary mixture of 0.003 M IMP + 0.03 M MSG, 0.5 M Suc, 0.1 M NaCl and DW. Stimuli were applied for 10 s.
stimulations were compared with a two-way ANOVA and post hoc Bonferroni/Dunn test. 3. Results 3.1. Two-bottle preference test The results of the two-bottle preference tests revealed that both young and aged rats significantly reject 0.001 M QHCl (p < 0.0005) and prefer 0.3 M Suc (young p < 0.0005, aged p < 0.005). Although umami solutions induced a strong preference in young rats (p < 0.0005), this was not observed in aged rats (Fig. 1). 3.2. Neural recordings from the GSP and CT in aged rats Four basic taste and umami substances produced robust GSP responses in the aged rats (Figs. 2 and 3), and binary mixtures of 0.001 M IMP + 0.01 M MSG produced significant synergistic effects (Fig. 3), as shown in young rats [16]. NaCl produced large responses in the CT at the start of the 10 s adaptation to NaCl (0.001 M, 0.01 M, 0.1 M NaCl). After the 10 s adaptation with NaCl, the Na salts of umami substances were stimulatory as indicated by arrows (Fig. 4A). Significant synergistic effects were observed to binary mixtures of 0.001 M IMP + 0.01 M MSG in aged rats (Fig. 4), as also shown in young rats [16]. 3.3. Blood exam To compare the physiological condition of aged rats to that of young rats, blood exam was conducted. The results showed no
Fig. 3. Mean relative phasic response magnitudes to 0.01 M HCl, 0.01 M QHCl, 0.5 M Suc, 0.001 M IMP, 0.01 M MSG, and binary mixtures of 0.001 M IMP + 0.01 M MSG from the GSP of 21–24 month old rats. Error bars show SE of the mean (n = 4). Asterisks indicate statistical significance by post hoc analysis; ** p < 0.005, * p < 0.05.
significant differences in the total plasma protein and creatinine levels between young and aged rats, while the levels were significantly lower for plasma albumin (p < 0.05), BUN (p < 0.05) and glucose (p < 0.005) in the aged compared to younger rats (Fig. 5).
Fig. 4. (A) Integrated responses of the CT in a aged (24-month) rat to 0.001 M IMP subsequent to adaptation to 0.001 M NaCl, 0.01 M MSG subsequent to adaptation to 0.01 M NaCl, binary mixture of 0.001 M IMP + 0.01 M MSG subsequent to adaptation to 0.01 M NaCl, and 0.1 M NaCl. Response magnitude was measured from base line to the peak response (arrow). (B) Mean relative response magnitudes to 0.001 M IMP, 0.01 M MSG, and the binary mixture of 0.001 M IMP + 0.01 M MSG from the GSP of 21–24 month old rats. Error bars show SE of the mean (n = 5). Asterisks indicate statistical significance by post hoc analysis; ** p < 0.005, * p < 0.05.
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Fig. 5. Results of the blood exam in young (8-week) and aged (19-month) male rats. T-Pro: total plasma protein, Alb: albumin, BUN: blood urea nitrogen, Cre: creatinine, Glu: glucose. n = 5; ** p < 0.005, * p < 0.05.
4. Discussion Umami taste provides an important clue to evoke ingestive behavior since birth. Brest milk is rich in free glutamate [19,20], and human neonates show a strong preference to MSG [21]. The present experiments showed that young rats, as in humans, showed strong preferences to IMP, MSG and to the binary mixture of IMP and MSG. However, the preference to umami substances disappeared in aged rats. Gustatory information of umami was previously reported to be conveyed dominantly via the GSP and CT but not glossopharyngeal (GL) among three major taste nerves, based on the behavioral analysis of the rat with taste nerves transected [16]. In aged rats, in spite of the disappearance of umami preference, both GSP and CT produced robust umami responses to MSG, which was characterized by synergistic effects with IMP [22]. In contrast to umami, sweet taste induced strong preference behavior in not only young but also old rats. The aging effects on umami and sweet taste responses are expected to be similar in both the neurophysiological and behavioral experiments, since they share the same taste receptor subunit for these substances in taste buds. Specific combinations of Tas1r family members serve as sweet or umami taste receptor: Tas1r1 and Tas1r2 for umami, and Tas1r2 and Tas1r3 for sweet [23]. Both in young and aged rats, similarly to the sweet taste responses to Suc, the robust neural umami responses were produced by IMP, MSG and binary mixture of IMP and MSG in the GSP and CT, showing that umami information is conveyed to the brain regardless of age. There results indicate a robustness of peripheral taste system for sweet and umami. However, the behavioral experiments showed that a preference behavior to umami in young rats disappeared in aged rats, although aged rats still significantly preferred Suc. The preference for umami was previously reported to be altered depending on nutritional conditions. Rats fed diets deficient in protein or an essential l-amino acid, l-lysine (Lys), increased the consumption of Lys, glycine and NaCl, but not umami substances; however, a preference for umami substances appeared when the rats were recovered from their previous deficiency [22]. Our results of the blood exam between young and aged rats showed no significant difference in the total plasma protein and no morbid condition of starvation, therefore, the reduced preference to umami substances in aged rats must not be associated with the plasma level of protein. The homeostatic control of plasma glucose directly links to the regulation of food intake [24]. When glucose level lowered,
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ingestive behavior is evoked. In the present report, plasma glucose level was lower in aged rats than in young rats. A similar decline of plasma glucose with age was previously reported in aged male rats [25]. On the other hand, food intake and energy consumption were shown to be decreased with age [26]. This inverse relationship between plasma glucose and food intake implies the aging effect on appetite regulated by a variety of neuropeptides and hormones [27]. Taste-evoked ingestive behavior is assumed to be controlled by some of neuropeptides regulating appetite [28]. Orexin-A and neuropeptide Y (NPY) are involved in sweetener-induced overconsumption in adult rats [29]: Drinking a sweet solution, saccharin, elevated mRNA level of orexin-A and NPY in the hypothalamus, and, on the other hand, intracerebroventricular (ICV) administrations of orexin-A, NPY and melanin-concentrating hormone (MCH) increased the intake of saccharin in adult rats. Also, ICV injection of orexin-A, NPY and ghrelin is showed to dose-dependently increase food intake in both young (4 month old) and adult (11 month old) rats [26]. However, in aged (25–27 month old) rats, orexin-A and NPY did not stimulate food intake at any dose, and ghrelin still induced food intake but with reduced efficacy. Based on these studies, the difference in aging effect on the taste preference between sweet and umami in the present report may be due to the difference in contributing neuropeptides. Preference behavior to umami substances depends on neuropeptides such as orexin-A and NPY, which may not be effective in aged rats, but not ghrelin. Hence, preference to umami was not evoked, despite umami information conveyed to the brain via CT and GSP nerves. In contrast, preference to Suc may be mediated by such as ghrelin or some other neuropeptide that is effective in aged rats. 5. Conclusion In conclusion, this is the first report indicating the existence of aging effects on the modification of umami preference in the CNS. Further experiments are necessary to clarify the central mechanisms that caused the depletion of umami preference in aged rats. Acknowledgments We thank Dr. John Caprio for valuable comments on the manuscript. This work was supported in part by Grants-in-Aid for Scientific Research (C) (Nos. 06671865, 23593036) from the Ministry of Education, Science and Culture in Japan. References [1] J. Mojet, E. Christ-Hazenlhof, J. Heidema, Taste perception with age: generic or specific losses in threshold sensitivity to the five basic tastes, Chem. Senses 26 (2001) 845–860. [2] L.M. Bartoshuk, B. Rifkin, L.E. Marks, P. Bars, Taste and aging, J. Gerontol. 41 (1986) 51–57. [3] B.J. Cowart, Relationships between taste and smell across the adult life span, Ann. N.Y. Acad. Sci. 561 (1989) 39–55. [4] R.J. Hyde, R.P. Feller, Age and sex effects on taste of sucrose, NaCl, citric acid and caffeine, Neurobiol. Aging 2 (1981) 315–318. [5] C. Murphy, M.M. Gilmore, Quality-specific effects of aging on the human taste system, Percept. Psychophys. 45 (1989) 121–128. [6] J. Mojet, J. Heidema, E. Christ-Hazelhof, Taste perception with age: generic or specific losses in supra-threshold intensities of five taste qualities, Chem. Senses 28 (2003) 397–413. [7] D.A. Stevens, H.T. Lawless, Age-related changes in flavor perception, Appetite 2 (1981) 127–136. [8] S.S. Schiffman, E.A. Sattely-Miller, I.A. Zimmerman, B.G. Graham, R.P. Erickson, Taste perception of monosodium glutamate (MSG) in foods in young and elderly subjects, Physiol. Behav. 56 (1994) 265–275. [9] Z.S. Warwick, S.S. Schiffman, Sensory evaluations of fat-sucrose and fat-salt mixtures: relationship to age and weight status, Physiol. Behav. 48 (1990) 633–636.
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