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  • Age-related changes in taste preference using a 48-h two-bottle test: A 48-h two-bottle test, a standard behavioral test for taste preference, was conducted on 46 male Sprague-Dawley rats (CLEA Japan, Inc., Japan) between 3 and 72 weeks of age and weighing 95–1050 g. Rats generally have a mean lifespan of 2–3 years that includes two critical time-points: the end of weaning and reproduction. Therefore, we divided the rats into five groups: juvenile (3–6 weeks, just after weaning, n = 9), young adult (8–11 weeks, early reproductive phase, n = 8), adult (17–20 weeks, late reproductive phase, n = 9), middle-aged (34–37 weeks, end of reproduction, n = 10), and old-aged (69–72 weeks, n = 10). We did not use rats over 74 weeks of age because they carry a high risk of spontaneous disease. The experience of consuming taste solution is likely to have an effect on subsequent ingestive behaviors. To avoid this, a different set of rats was used for each age group. The juvenile group was purchased at 3 weeks. The young adult and the adult groups were purchased one week before the behavioral experiments. As rats of more than 30 weeks old were not available for purchase, the middle-aged and the old-aged groups were purchased at 30 weeks and raised until the appropriate age for testing in the animal-breeding facilities of the faculty. All rats were allowed food pellets (MF, Oriental Yeast, Osaka, Japan) and distilled water (DW) ad libitum, and handled by the experimenters every day before attaining the appropriate age. Animals were housed individually in plastic cages suitable for their body mass: 225 × 338 × 140 mm for rats 3–11 weeks old, and 345 × 403 × 177 mm for rats 17–72 weeks old. Cages were changed once a week. Since environmental changes could alter animals’ consumption behavior, the taste stimulus was presented after at least 60 hours of acclimation in the new plastic cage. The ambient temperature was maintained at 23°C in a 12:12 h light/dark cycle (lights on between 8:00 and 20:00). All animal care and experimental guidelines conformed to those established by the National Institutes of Health and were approved by “Guide for the Care and Use of Laboratory Animals” in the Osaka Dental University Animal Care and Use Committee (Permit Number: 12–02045). After the acclimation, all rats were presented with two bottles in their home cages: one containing DW and the other containing a taste solution. The bottle consisted of a 100-ml plastic syringe (JS-S00S, JMS Co., Ltd, Tokyo, Japan) and a stainless steel spout (TV-25, CLEA, Tokyo, Japan). The rats could freely access both bottles and chow for 48 h. To avoid positional preference, the positions of the bottles were switched 24 h after the start of the presentation. We recorded 48-h fluid consumption by measuring the weight of the bottles. The taste solutions were sucrose (0.3 and 0.5 M), sodium saccharin (saccharin, 5 mM), NaCl (0.1 and 0.3 M), QHCl (0.03 and 0.3 mM), MSG (0.1 M), and HCl (10 and 50 mM). To exclude the possibility of order effects, the taste solutions were presented in pseudorandom order, without grouping similar solutions by concentration. In addition, the presentation order was different among different rats. The order was one of the following: 1) 0.3 M sucrose, 0.1 M NaCl, 0.3 mM QHCl, 5 mM saccharin, 50 mM HCl, 0.1 M MSG, 0.03 mM QHCl, 0.5 M sucrose, 0.3 M NaCl and 10 mM HCl; 2) reverse order of 1); 3) 0.1 M MSG, 50 mM HCl, 5 mM saccharin, 0.3 mM QHCl, 0.1 M NaCl, 0.3 M sucrose, 0.03 mM QHCl, 0.5 M sucrose, 0.3 M NaCl and 10 mM HCl. We spent 4 weeks (e.g., 3–6 weeks of age in the juvenile group) completing the presentation of all 10 taste solutions. Age-related changes in preference for low and high concentrations of taste solutions using a 48-h two-bottle test: It was possible that the differences in the consumption of the taste solutions and water across the life stages were due to the changes in the taste thresholds. In order to answer to this question, we investigated the intake of low and high concentrations of taste solutions in the second experiment. It included a new series of 38 male Sprague-Dawley rats (CLEA Japan, Inc., Japan) aged 3–72 weeks and weighing 125–980 g. We divided the rats into five groups as in the first experiment: juvenile (n = 8), young adult (n = 8), adult (n = 7), middle-aged (n = 7), and old-aged (n = 8). The housing conditions were the same as described above. All rats were presented with two bottles containing the same taste solution for 48 h, one at a high concentration and the other a low concentration. The taste solutions were 0.3 M vs. 0.5 M sucrose, 5 mM vs. 50 mM saccharin, 0.03 mM vs. 0.3 mM QHCl, 0.1 M vs. 0.3 M NaCl, 0.1 M vs. 0.3 M MSG, and 10 mM vs. 50 mM HCl. The taste solutions were presented in pseudorandom order. The order was one of the following: 1) 0.3 and 0.5 M sucrose, 0.1 and 0.3 M NaCl, 5 mM and 50 mM saccharin, 0.1 M and 0.3 M MSG, 0.03 mM and 0.3 mM QHCl, and 10 mM and 50 mM HCl; 2) reverse order of 1); 3) 5 mM and 50 mM saccharin, 0.1 and 0.3 M NaCl, 0.3 and 0.5 M sucrose, 0.1 M and 0.3 M MSG, 0.03 mM and 0.3 mM QHCl, and 10 mM and 50 mM HCl. Rats from this test were subsequently used in the electrophysiological experiments. Electrophysiological measurements of the responses of the chorda tympani nerve to taste solutions: The rats were anesthetized with an intraperitoneal injection of 60 mg/kg sodium pentobarbital (Somnopentyl®; Kyoritsu Seiyaku, Tokyo, Japan). Supplementary injections of 0.3 g/kg urethane were administered as needed to maintain a surgical level of anesthesia. A tracheal cannula was implanted and the animal properly secured within a head holder. The chorda tympani nerve was cut near its entrance into the tympanic bulla and dissected free from the underlying tissues. An indifferent electrode was positioned nearby in the wound. The whole-nerve activity was amplified, displayed on an oscilloscope, and monitored using an audio amplifier. The amplified signals were passed through an integrator with a time constant of 0.3 s and displayed on a slip chart recorder. After confirmation of stable recording, we applied 5 ml of taste solution to the rat’s tongue for 30 s. The rat’s tongue was rinsed with DW after completion of taste stimulation. We measured the entire integrated response during the simulation as the whole nerve response. In electrophysiological experiments it is possible that any endogenous or exogenous factors may produce individual differences in the recording of neural activities. Therefore, we normalized the taste responses by dividing the magnitudes of the responses to each taste stimuli by the response to 0.1 M NH4Cl, which is generally used as a standard stimulus in electrophysiological recordings of peripheral taste nerves. Normalized food intake was calculated by dividing the 24-h food intake by BW (per 100 g) and analyzed using one-way analysis of variance (ANOVA) and post-hoc Tukey HSD tests. The data from the electrophysiological experiments were also analyzed using one-way ANOVA. The previous studies investigating drinking behavior generally used 24-h intake as the behavioral index. To enable a comparison of our results with the prior studies, we calculated the 24-h intake volume as half of the 48-h intake volume. The taste solution preference ratios in the first experiment were calculated by dividing the volume of taste solution ingested by the total intake of DW + taste solution, and analyzed using one-way ANOVA with Tukey's HSD post-hoc tests. The preference ratios for higher concentration taste solutions were calculated by dividing the intake of the higher concentration solution by the total intake of higher + lower concentration solutions, and analyzed using one-way ANOVA with Duncan’s post-hoc tests. We also analyzed whether the preference ratios were significantly different from chance level (0.5) using an independent t-test. The difference between net intake of DW and each taste solution for each age group was analyzed by paired t-test. All statistical analyses were performed using Statistica software (StatSoft, Inc., Tulsa, OK, USA). A P value < 0.05 was considered significant.
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