Experience-dependent changes in affective valence of taste in male mice

Chronic umami or bitter exposure induced increased preference for umami or decreased aversion to bitter

To investigate the influence of prolonged experience of umami and bitter tastants on taste preference, mice were reared with ad libitum water (Water group), umami solution (Umami group), or bitter solution (Bitter group) for 3 weeks in the immediate post-weaning period (Fig. 1A), which did not affect body weight gain (Water group, 4.45 ± 0.32 g; Umami group, 3.96 ± 0.22 g; Bitter group, 4.10 ± 0.42 g). After prolonged taste exposure, we performed the two-bottle test between water and umami to assess umami preference. The Umami group exhibited a significant increase in intake of umami solution compared with water, whereas the Water and Bitter groups showed no difference in water and umami intake (Water group, p = 0.6588; Umami group, p = 0.0003; Bitter group, p = 1.0000; Fig. 1B). Total intake of both water and umami was comparable between the three groups (Water group, 0.76 ± 0.08 g; Umami group, 1.04 ± 0.13 g; Bitter group, 0.94 ± 0.15 g). We calculated the ratio of umami intake to total intake of water and umami as a preference ratio, so that a preference ratio higher than the 50% value indicated that umami was preferred over water. The preference ratio of umami in the Umami group was significantly higher than 50% (p < 0.0001; Fig. 1C). In addition, comparison of the preference ratios among the three groups revealed that the Umami group showed a significantly high preference ratio of umami (F2,21 = 30.09, p < 0.0001; Umami vs. Water group, p < 0.0001; Umami vs. Bitter group, p < 0.0001; Water vs. Bitter group, p = 0.9934; Fig. 1C), which indicated that prolonged exposure to umami increased its preference. We also analyzed access duration to the water and umami bottles to assess exploring behavior to each tastant. All groups showed significant increase in access duration to the umami bottle at several time points when analyzed every 5 min (Additional file 3: Fig. S1A–C). Therefore, potential neophobia to the unexperienced tastants, which may have been observed in the first 5 min, would have been canceled or at least negligible in our experimental condition. In total access duration during the whole test session, both the Water and Umami groups contacted the umami bottle longer duration than the water bottle (Water group, p = 0.0315; Umami group, p = 0.0008; Fig. 1D). The Bitter group showed a tendency of increased access duration to the umami bottle (p = 0.0676; Fig. 1D). The ratio of access duration to the umami bottle in the Water and Umami groups was significantly high compared with the chance rate (50%), and that of the Bitter group was slightly but not significantly higher than 50% (Water group, p = 0.0483; Umami group, p < 0.0001; Bitter group, p = 0.0676; Fig. 1E). These observations indicate that not only the Umami group but also the Water and Bitter groups showed interest in umami. The Umami group, however, had a significantly higher access ratio to the umami bottle compared with that of the other groups (F2,21 = 15.12, p < 0.0001; Umami vs. Water group, p = 0.0002; Umami vs. Bitter group, p = 0.0008; Water vs. Bitter group, p = 0.9063; Fig. 1E).

Fig. 1figure 1

Preference for umami or bitter in the two-bottle test in prolonged taste exposure mice. A Experimental paradigm of prolonged taste exposure and two-bottle test. B Intake of water and umami during 15-min two-bottle test. C Preference ratios of umami. Preference ratios were calculated as the ratio of the umami intake to the total intake. D Access duration to water or umami bottle. E Access ratio of umami bottle. F Intake of water and bitter during 15-min two-bottle test. G Preference ratios of bitter. H Access duration to bitter bottle. I Access ratio of water or bitter bottle. Each circle represents results from one mouse. Data are represented as mean ± SEM. Water group, n = 8; Umami group, n = 9; Bitter group, n = 7. *p < 0.05, ***p < 0.001 (paired t-test); #p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001 (Tukey’s post hoc test); †p < 0.05, ††††p < 0.0001 (one sample t-test)

To further confirm that the experiments with umami (100 mM monosodium glutamate, MSG) reflects umami effects rather than sodium effects, we also investigated the influence of the prolonged experience of umami (100 mM monopotassium glutamate, MPG). Mice were reared with ad libitum water (Water group), MPG-based umami solution (MPG group) for 3 weeks in the immediate post-weaning period (Additional file 3: Fig. S2A), which did not affect body weight gain (Water group, 5.32 ± 0.36 g; MPG group, 5.67 ± 0.35 g). We found that MPG experience for 3 weeks enhanced umami intake similar to MSG experience (Water group, p = 0.0261; Umami group, p = 0.0047; Additional file 3: Fig. S2B, Fig. 1B). Although both the Water and MPG groups showed attraction to umami, the MPG group exhibited higher preference ratio (MPG vs. Water group, p = 0.1336; Additional file 3: Fig. S2C). In addition, the MPG group exhibited increased access duration and access ratio compared to the Water group (Additional file 3: Fig. S2D, E). These results regarding access duration are similar to those obtained using MSG. Taken together, these experiments support the idea that our experiments with MSG also reflect the effect of umami. These results using MSG and MPG suggest that prolonged umami exposure increased the preference for umami. The following experiments were conducted with MSG-based umami solution that induced remarkable changes in umami preference in behavioral experiments.

Next, we performed the two-bottle test with water and bitter solution to assess whether prolonged umami or bitter exposure affected bitter aversiveness. The Water and Umami groups consumed significantly less bitter solution than water (Water group, p = 0.0004; Umami group, p = 0.0165; Fig. 1F) and showed small preference ratios of bitter compared to 50% (Water group, p < 0.0001; Umami group, p = 0.0113; Fig. 1G). In contrast, the Bitter group consumed as much bitter solution as water (p = 0.7539; Fig. 1F) and exhibited bitter preference ratio around 50% (p = 0.8361; Fig. 1G), which was significantly high compared with that of the other groups (F2,21 = 12.37, p = 0.0003; Umami vs. Water groups, p = 0.1372; Umami vs. Bitter groups, p = 0.0128; Water vs. Bitter groups, p = 0.0002; Fig. 1G), indicating that the Bitter group showed no aversion to bitter. The total intake of water and bitter was comparable between the three groups (Water group, 0.56 ± 0.07 g; Umami group, 0.49 ± 0.05 g; Bitter group, 0.66 ± 0.08 g). Although access duration to the bitter bottle was significantly less than that to the water bottle in the Water and Umami groups, the Bitter group accessed the bitter and water bottles for almost the same duration (Water group, p = 0.0005; Umami group, p = 0.0198; Bitter group, p = 0.8296; Fig. 1H; Additional file 3: Fig. S1D–F). The access duration ratio to the bitter bottle in the Bitter group was significantly higher than that of the other groups (F2,21 = 9.17, p = 0.0014; Umami vs. Water group, p = 0.2014; Umami vs. Bitter group, p = 0.0370; Water vs. Bitter group, p = 0.0010; Fig. 1I). These results suggest that prolonged bitter exposure decreased aversion to bitter, which was innately aversive.

The CeA is composed of neurons with heterogeneous response properties for various tastants

These changes in taste preference/avoidance due to prolonged taste exposure were considered as an adaptation accompanied by neuroplasticity. We next sought to determine the areas of the brain that display neuronal activity associated with these behavioral changes. Recent studies have reported that the CeA plays a pivotal role in emotional behavioral selection [28]. In addition, the CeA receives direct input from multiple nuclei of the gustatory circuit such as the NTS, PB, and IC [5, 29, 30]. Especially, it has been reported that Prkcd-positive neurons in the CeA are a population that responds to aversive tastant [2]. Therefore, one intriguing possibility is that there are cell-type specific responses to the negative and positive taste qualities within the CeA, and neuronal activity changes occur in this circuit may lead to the modification of outcome behavior toward the tastant. However, how each tastant, such as umami, regulates CeA activity, and the correspondence between cells encoding each taste qualities has not been fully elucidated, even under untreated naïve conditions. To investigate innate responses to various tastants in the CeA, we first performed in vivo calcium imaging for two major genetically identified CeA cell populations, Prkcd-positive and Sst-positive neurons. Mice were sequentially given water and bitter, sweet, and umami tastant solutions as shown in Fig. 2A. Some neurons showed responses prior to presentation of tastant solutions (Additional file 1: Movie S1 and Additional file 2: Movie S2). So we evaluated the difference in responses to water and each tastant solution to minimize the influences of physical stimuli such as oral insertion of a ball tip needle and non-taste-specific responses to drinking itself, and to extract taste-specific response neurons. In Prkcd-positive neurons, the largest populations (19.7%) responded to bitter tastant (Fig. 2B), as was reported in previous Fos-labeling studies [2, 31]. Notably, a comparative number of neurons (18.8%) also responded to umami, and a smaller number of neurons (7.2%) responded to sweet tastant (Fig. 2B, D, Additional file 3: Fig. S4A, B). Furthermore, we found that 17.8%, 11.0%, and 11.0% of Sst-positive neurons responded to umami, bitter, and sweet tastants, respectively (Fig. 2C, Additional file 3: Fig. S5A, B). Interestingly, one-third of sweet-response and one-fifth of umami-response Sst-positive neurons also responded to umami and sweet tastants, respectively, both of which are thought to be attractive tastants (Fig. 2C, E, Additional file 3: Fig. S5B). Taken together, both Prkcd-positive and Sst-positive neurons are not unique populations to respond to a particular tastant, but are composed of mixed cells that respond to negative and positive tastants, although there is a bias in the tendency of the responding tastant.

Fig. 2figure 2

In vivo calcium imaging of central amygdala (CeA) neurons during taste stimulation. A Schematic of drinking experiment for calcium imaging of taste stimuli-evoked responses. B, C Pie charts showing the fraction of response cells for each taste in the total cell population (B 223 cells from four Prkcd-cre mice, C 191 cells from four Sst-cre mice). Venn diagrams showing the overlap of activated cells. D, E Average z-scored GCaMP6f signals of umami-activated (42 cells from Prkcd-cre mice and 34 cells from Sst-cre mice), bitter-activated (44 cells from Prkcd-cre mice and 21 cells from Sst-cre mice), and sweet-activated (16 cells from Prkcd-cre mice and 21 cells from Sst-cre mice) cells in response to umami (orange), bitter (green), sweet (magenta), and neutral (blue) tastant solution stimuli. Shading, ± s.e.m

Prkcd-positive neurons in the CeA were activated by umami after prolonged umami exposure

Prkcd-positive neurons of the CeA were thought to respond to bitter and suppress appetitive behavior [2, 31], but our calcium imaging results indicate that a part of Prkcd-positive neurons also respond to attractive taste umami. To elucidate the umami taste information processing in more detail, we investigated the responses of neurons in the CeA and upstream nuclei of the gustatory circuit: the NTS, PB, VTA, and IC. To evaluate the neuronal activities of these nuclei with regard to umami tastant, we performed Fos counting studies by fluorescence in situ hybridization (FISH). For the identification of these nuclei, we also used molecular marker genes, including protein kinase Prkcd and peptide hormone Sst in the CeA, nitric oxide synthase Nos1 in the IC, tyrosine hydroxylase (Th) in the VTA and NTS, and peptide hormones Calca and Adcyap1 in the PB (Additional file 3: Fig. S6A). The Fos antisense probe detected Fos-positive neurons in the pentylenetetrazole-treated mouse hippocampus, but not in the vehicle-treated mouse hippocampus. Sense probes did not detect the signal in mice hippocampi from both treatment groups (Additional file 3: Fig. S6B).

Initially, to investigate the immediate neuronal activity of the tastant, mice were individually housed with restricted feeding for over an hour and restricted drinking for 19–21 h before the taste experiment. To assess the innate taste response, naïve mice were exposed the water, umami, or bitter solutions (Fig. 3). However, the mice provided with bitter solution did not drink it (water, 0.37 ± 1.10 g; umami, 0.58 ± 0.11 g; bitter, 0.03 ± 0.01 g; F2,22 = 11.29, p = 0.0004, one-way ANOVA; Umami vs. Water group, p = 0.2188; Umami vs. Bitter group, p = 0.0004; Bitter vs. Water group, p = 0.0137, Tukey’s post hoc test). Therefore, we did not perform Fos FISH experiments in mice provided with bitter solution (Fig. 4). The Fos-positive neurons were increased in the CeA in umami-stimulated mice compared with water-stimulated mice (p < 0.0001; Fig. 4A). In addition, we investigated cell type-specific neuronal activity in the CeA by analyzing Fos and Prkcd- or Sst-double-positive neurons. The ratios of Fos and Sst double-positive neurons per Sst-positive neurons in the Umami group was significantly higher than those in the Water group, while Fos-positive neurons in the Prkcd-positive neurons was comparable between these groups (Sst, p = 0.016398; Prkcd, p = 0.4373; Fig. 4B, C). Next, we performed the Fos FISH assay in the CeA upstream gustatory nuclei (PB, NTS, VTA, and IC). In the IC, we focused on the area between Bregma + 1.1 mm and + 0.6 mm as the umami field, because the umami field is between the bitter and sweet hot fields [5, 17]. In the PB, we also calculated the ratio of Fos-positive neurons in the Calca- or Adcyap1-positive neurons, because these neurons are known to innervate the CeA [32, 33]. The number of Fos-positive neurons in the PB was not significant between the Water and Umami groups (PB, p = 0.2181; Fig. 4D). On the other hand, while Fos-positive neurons in the Calca-positive neurons in the PB was comparable between two groups, Fos-positive neurons in the Adcyap-positive neurons was increased in the Umami-tastant provided group (Calca, p = 0.9617; Adcyap, p = 0.0495; Fig. 4E, F). The NTS showed no difference in Fos-positive neurons, but Fos-positive neurons in the VTA and IC increased in the Umami group (NTS, p = 0.5137; VTA, p = 0.0174; IC, p = 0.0476; Fig. 4G–I). These results suggest that the nuclei in higher gustatory circuit, such as CeA, VTA, and IC are more activated by the umami administration than NTS and PB, which are the primary nuclei.

Fig. 3figure 3

Experimental design of the Fos fluorescent in situ hybridization (FISH) assay. A Time course of mice brain sampling. B Circuit model of afferent projections of the CeA. C Representative images of the Fos FISH assay. Blue: DAPI, Green: c-Fos, Magenta: brain region- or cell type-specific markers. Each scale bar represents 300 μm. Central amygdala (CeA), nucleus tractus solitarius (NTS), lateral parabrachial nucleus (lPB), ventral tegmental area (VTA), insular cortex (IC)

Fig. 4figure 4

Fos FISH assay of single tastant treatment. A Fos FISH assay at the CeA. (Left) Representative images of the CeA after single water or umami treatment. Fos-positive cell counts/1 mm2 were not significantly different. Water, n = 32 slices from N = 8 mice; umami, n = 28 from N = 7. B, C Double Fos FISH assay with Sst or Prkcd markers. The ratios of Fos-positive neurons per each marker were not significant. Open and filled triangles indicate single- and double-positive cells, respectively. Sst: water, n = 16 from N = 4; umami, n = 16 from N = 4; Prkcd: water, n = 20 from N = 5; umami, n = 28 from N = 7. D, GI Fos FISH assay in the PB, NTS, VTA, and IC, which are upstream regions of the CeA. Fos-positive cell counts/1 mm2 were not significantly different. PB: water, n = 24 from N = 6; umami, n = 24 from N = 6; NTS: water, n = 20 from N = 5; umami, n = 24 from N = 6; VTA: water, n = 16 from N = 4; umami, n = 16 from N = 4; IC: water, n = 16 from N = 4; umami, n = 16 from N = 4. E, F Double Fos FISH assay with Calca or Adcyap1 markers in the PB. The ratios of Fos-positive neurons per each marker were not significant. Filled triangles indicate double-positive cells. Calca: water, n = 24 from N = 6; umami, n = 24 from N = 6; Adcyap1: water, n = 24 from N = 6; umami, n = 24 from N = 6. Each scale bar represents 25 μm. *p < 0.05, ****p < 0.0001 (unpaired t-test)

Next, to determine changes in neuronal activity by prolonged taste exposure, mice received water or umami solution ad libitum for 3 weeks, and Fos FISH assay was performed after taste stimulation (Fig. 5). As observed in the single taste stimulation (Fig. 4), Fos expression in the CeA was markedly increased by umami stimulation in prolonged taste exposure mice (p = 0.0008; Fig. 5A). Interestingly, there was no difference in the ratio of Fos-positive neurons in the Sst-positive neurons, while the ratio of Fos-positive neurons in the Prkcd-positive neurons was significantly increased in the Umami group (Prkcd, p = 0.0001; Sst, p = 0.2778; Fig. 5B, C). Among the higher gustatory nuclei, no difference was observed except for the VTA, unlike the single taste administration (PB, p = 0.8423; Calca, p = 0.8279, Adcyap1, p = 0.1059; NTS, p = 0.2047; IC, p = 0.2740; Fig. 5D–G, H). Intriguingly, the VTA showed a decrease in the Fos-positive neurons in the prolonged umami administration (VTA, p = 0.0276; Fig. 5H). These results suggest that prolonged exposure to umami taste induces some plastic changes in the gustatory circuit, particularly in the CeA, in a cell type-specific manner.

Fig. 5figure 5

Fos FISH assay of prolonged taste exposure mice. A Fos FISH assay at the CeA. (Left) Representative images of the CeA of the prolonged taste exposure mice after water or umami stimulation. Fos-positive cell counts/1 mm2 were increased by umami stimulation in the umami-exposed mice. Water, n = 24 slices from N = 6 mice; umami, n = 24 from N = 6. B, C Double Fos FISH assay with Sst or Prkcd markers. The ratio of Fos-positive neurons per each marker was increased in the Prkcd-positive neurons. Sst: water, n = 20 from N = 5; umami, n = 24 from N = 6; Prkcd: water, n = 16 from N = 4; umami, n = 16 from N = 4. D, GI Fos FISH assay in the PB, NTS, VTA, and IC, which are upstream regions of the CeA. Fos-positive cell counts/1 mm2 were not significantly different. PB: water, n = 24 from N = 6; umami, n = 24 from N = 6; NTS: water, n = 12 from N = 3; umami, n = 12 from N = 3; VTA: water, n = 20 from N = 5; umami, n = 24 from N = 6; IC: water, n = 16 from N = 4; umami, n = 16 from N = 4. E, F Double Fos FISH assay with Calca or Adcyap1 markers in the PB. The ratios of Fos-positive neurons per each marker were not significant. Calca: water, n = 24 from N = 6; umami, n = 24 from N = 6; Adcyap1: water, n = 20 from N = 5; umami, n = 20 from N = 5. Each scale bar represents 25 μm. Arrows indicate Fos-positive cells. Open and filled triangles indicate single- and double-positive cells, respectively. *p < 0.05, ***p < 0.001 (unpaired t-test)

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