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

Experience-dependent changes in affective valence of taste in male mice Taste plays an essential role in the evaluation of food quality by detecting potential harm and benefit in what ani‑ mals are about to eat and drink. While the affective valence of taste signals is supposed to be innately determined, taste preference can also be drastically modified by previous taste experiences of the animals. However, how the experience‑ dependent taste preference is developed and the neuronal mechanisms involved in this process are poorly understood. Here, we investigate the effects of prolonged exposure to umami and bitter tastants on taste preference using two‑bottle tests in male mice. Prolonged umami exposure significantly enhanced umami preference with no changes in bitter preference, while prolonged bitter exposure significantly decreased bitter avoidance with no changes in umami preference. Because the central amygdala (CeA) is postulated as a critical node for the valence processing of sensory information including taste, we examined the responses of cells in the CeA to sweet, umami, and bitter tastants using in vivo calcium imaging. Interestingly, both protein kinase C delta (Prkcd)-positive and Somatostatin (Sst)‑positive neurons in the CeA showed an umami response comparable to the bitter response, and no difference in cell type ‑specific activity patterns to different tastants was observed. Meanwhile, fluorescence in situ hybridization with c-Fos antisense probe revealed that a single umami experience significantly activates the CeA and several other gustatory‑related nuclei, and especially CeA Sst‑positive neurons were strongly activated. Intriguingly, after prolonged umami experience, umami tastant also significantly activates the CeA neurons, but the Prkcd ‑positive neurons instead of Sst‑positive neurons were highly activated. These results suggest a relationship between amygdala activity and experience‑ dependent plasticity developed in taste preference and the involvement of the genetically defined neural populations in this process. Keywords Gustatory circuit, Umami, Bitter, Amygdala, Taste preference, Plasticity, Calcium imaging † 3 Shun Hamada, Kaori Mikami, Shuhei Ueda and Masashi Nagase contributed Department of Neuroscience I, Research Institute of Environmental equally to this work Medicine, Nagoya University, Nagoya 464‑8601, Japan Department of Molecular/Cellular Neuroscience, Nagoya University *Correspondence: Graduate School of Medicine, Nagoya 466‑8550, Japan Ayako M. Watabe Department of Neurochemistry, Graduate School of Medicine, The awatabe@jikei.ac.jp University of Tokyo, Tokyo 113‑0033, Japan Department of Biochemistry, Faculty of Medicine, University of Yamanashi, Yamanashi 409‑3898, Japan Institute of Clinical Medicine and Research, Research Center for Medical Sciences, The Jikei University School of Medicine, 163‑1 Kashiwashita, Kashiwa, Chiba 277‑8567, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hamada et al. Molecular Brain (2023) 16:28 Page 2 of 16 (CeA) directly or indirectly [3, 15, 16]. Recent studies Introduction have demonstrated that taste information is processed by The taste system is crucial for animals to detect poten - a labeled-line system, such that information about each tial benefits, e.g., nutrients, and potential harm, e.g., taste quality has a discrete pathway from its taste recep- toxins, in what they are about to eat and drink [1, 2]. tors to the corresponding neuronal taste circuits [17, 18]. There are five basic taste qualities: sweet, sour, salty, bit - While some neurons in the central taste pathway are ter, and umami. The taste system has an emotional and tuned to particular taste qualities, other neurons respond motivational aspect, and affective taste, such as sweet more broadly to multiple tastes [19]. Furthermore, some and umami, drives approaching and appetitive behav- neurons change in their responsive profiles according to iors, while aversive taste, such as bitter and sour, drives experience and time [20], which suggests some plastic- avoidance behaviors [3–5]. The attractive and aversive ity in the taste coding rule. Therefore, neural plasticity valence of taste signals is innately determined. For exam- within the described taste circuitry can regulate expe- ple, human newborn infants exhibit affective behav - rience-dependent changes in the affective and aversive iors such as lip sucking, elevation of the corners of the valence of particular taste signals. mouth, and rhythmic tongue protrusions when exposed In the present study, we have addressed this issue to sweet or umami solutions [6, 7]. They also show aver - by establishing a behavioral paradigm for experience- sive responses such as nose wrinkling and grimacing dependent plasticity in the taste preference for attractive when exposed to bitter or sour solutions. Rats and mice (umami) and aversive (bitter) taste quality, and examined also exhibit affective behaviors such as rhythmic and lat - the neuronal correlates in mice using in  vivo calcium eral tongue protrusions when administered with sweet or imaging and fluorescence in  situ hybridization in multi - umami solutions [7–9]. They also show aversive behav - ple brain regions. iors, such as gasping, chin rubbing, and handshaking, when exposed to quinine solution. Materials and methods The attractive and aversive valence of taste can also be Animals acquired so that experiences can modify the preference Adult male C57BL/6J mice (Japan SLC, Inc., Shizuoka, for certain tastants. For instance, many people may expe- Japan) were group housed (3–4 mice per cage) on a rience the development of an acquired taste for coffee, 12  h light/12  h dark cycle and provided with food (CE- beer, or even quinine. Likewise, in rats and mice, expo- 2, CLEA Japan, Inc., Tokyo, Japan) and water ad libitum, sure to sour and bitter substances before and after wean- unless otherwise noted. Protein kinase C delta (Prkcd)- ing leads to a significant preference for those substances cre mice [Tg(Prkcd-glc-1/CFP,-cre)EH124Gsat; stock in adulthood [10–12]. Thus, the unconditioned avoidance #011559-UCD] and somatostatin (Sst)-cre mice [Sst of sour and bitter can be modulated by early-life experi- tm2.1(cre)Zjh/J; stock #013044] were obtained from the ences. Compared with that of unfavored tastes, litera- Mutant Mouse Resource & Research Center and the ture on the modification of hedonic valence of favored Jackson Laboratory, respectively, and were maintained tastes is limited. It has been shown that rats exposed to heterozygous on a C57BL/6J background. Adult male overconsumption of sucrose during adolescence display mice over 3  months old were used for in  vivo calcium reduced sweet consumption and hedonic perception in imaging studies. All experimental protocols in this study adulthood [13]. Furthermore, Ackroff et al. demonstrated that included the use of animals were approved by the that prior umami experience significantly enhances pref - Institutional Animal Care and Use Committee of The erence for umami solutions in mice [14]. These findings Jikei University (Kashiwa City, Japan) (Approval number suggest that both attractive and aversive valence of taste 2018-072, 2019-010) and Nagoya University (approval signals can be subject to influence from previous expe - number R210154). All experiments complied with the riences; however, the neuronal mechanisms underlying Guidelines for Proper Conduct of Animal Experiments these observations are not well understood. by the Science Council of Japan (2006) and those recom- Taste signals first arise via taste receptor cells in the mended by the International Association for the Study of taste buds, which detect tastant chemicals and activate Pain. All efforts were made to reduce the number of ani - matching ganglion neurons. These signals are transmit - mals used and the suffering of the animals. ted through gustatory nerves, including the chorda tym- pani and glossopharyngeal nerves, to the nucleus of the Prolonged taste exposure and two‑bottle tests solitary tract (NTS), which relays information to the pon- Mice in the prolonged taste exposure groups had ad libi- tine parabrachial nucleus (PB) in rodents. These neurons tum access to food and one of the following taste solu- then activate the ventral tegmental area (VTA), the ven- tions instead of water beginning at 4  weeks of age, tral posteromedial nucleus of the thalamus, and the insu- immediately after weaning, for 3  weeks. The umami lar cortex (IC), which all project to the central amygdala Hamada  et al. Molecular Brain (2023) 16:28 Page 3 of 16 solution contained 100  mM monosodium l-glutamate Norland Products, NJ, USA). Exposed skull was coated (Sigma-Aldrich, Darmstadt, Germany) or 100 mM mono- with super-bond (C&B Kit; Sun Medical, Shiga, Japan), potassium l-glutamate (Sigma-Aldrich, Darmstadt, Ger - additionally covered with dental cement (REPAIRSIN; many) and 10  mM disodium inosine-5′-monophosphate GC, Tokyo, Japan), and a stainless steel bar was attached (Sigma-Aldrich) mixture. The bitter solution contained for head fixation. GCaMP6f fluorescence was periodi - 0.3 mM quinine hydrochloride (FUJIFILM Wako, Osaka, cally evaluated using a miniature integrated microscope Japan). Mice that experienced prolonged taste exposure system (nVista HD 2.0; Inscopix), and when sufficient were subjected to a two-bottle test following 19–21  h of GCaMP6f expression was confirmed, the microscope water deprivation. Mice were acclimated to the stainless baseplate was mounted using blue light curing resin steel sipper tubes in the two-bottle test chamber (Drinko- (Flow-It ALC; Pentron, CT, USA). The sites of viral injec - measurer; DM-G1, trapezoid-shaped test chamber tion and lens probe implantation were confirmed histo - with 55  mm upper side × 205  mm lower side × 135  mm logically after imaging experiments (Additional file  3: Fig. depth × 200  mm height; TOP-3002WW, O’Hara & Co., S3). Ltd, Tokyo, Japan) within the sound-attenuating box (660 mm width × 460 mm depth × 690 mm height; TOP- In vivo calcium imaging during taste stimulation 4011, O’Hara & Co., Ltd) for 15  min per day for 4  days. More than 5  days after baseplate mounting, the mice The two-bottle test was performed with the bottle posi - were ready for imaging. After several days of imaging tions switched for 2 days to avoid side preference. On test studies for natural experiences, imaging experiments day 1 and day 2, an umami solution bottle and a water for taste stimuli-evoked responses were performed. A bottle were presented. On day 3 and day 4, a bitter solu- detailed procedure of the precedent imaging studies will tion bottle and a water bottle were presented. Access be described elsewhere. A day before the experiment, the duration to each bottle was measured as nose poking miniscope was attached to each mouse and each mouse time to the bottle and analyzed by Operant task Studio was head-fixed on a running disc wheel for 30  min to V2 (O’Hara & Co., Ltd). A preference ratio was calculated acclimatize, and then 100 µL of water was given six times as the ratio of the taste solution (umami or bitter) intake at 2-min intervals using an oral gavage ball tip needle. On to the total (taste solution and water) intake. An access the experimental day, mice were habituated for 30 min as ratio was determined as the ratio of the access duration on the previous day, and calcium imaging was performed in the taste bottle to the total access duration in two bot- during taste stimulation. Images were acquired using data tles. Mice in the Umami and Bitter groups that exhibited acquisition software (ver. 2.0.4; Inscopix) at 20 frames per an intake of less than 0.1  g during the 15-min test were sec, 25% of LED power, and a gain of 3.5, and behavioral excluded from the data because of the inability to prop- videos were recorded simultaneously by triggers from the erly assess their preference. After the 15-min test, the miniscope system. Six minutes after the start of imaging, mice were returned to the home cage and given ad  libi- mice were given 100  µL of bitter (1  mM quinine), sweet tum access to food and taste solution until the next test (50  mM sucrose), and umami (100  mM monosodium on the following day. glutamate and 10 mM disodium inosinate) taste solution alternately with neutral-taste water at 2-min intervals for Stereotaxic surgery for in vivo calcium imaging three trials (Fig. 2A). Mice surgeries were performed as described previ- Acquired imaging data were down-sampled (1/2 spa- ously with minor modifications [21, 22]. Each deeply tial binning), preprocessed, motion corrected, cropped, anesthetized mouse was fixed in a stereotactic frame and then additionally down-sampled (1/2 spatial and (Model 942; Kopf Instruments, CA, USA). For viral 1/2 temporal binning) using Mosaic Software (ver. 1.2.0; injection, skull surface was exposed, a glass capillary Inscopix). Processed images were loaded to Inscopix was inserted through a drilled small hole, and 500 nL of Data Processing Software (ver. 1.3.0; Inscopix), and then adeno-associated virus (AAV) solution (AAV1/2-CAG- calcium transients of individual neurons were extracted DIO-GCaMP6f-WPRE; 3.5 × 10 genomes/mL) [23] with a constrained non-negative matrix factorization was loaded into the right CeA (AP − 1.35, ML + 2.95, for microendoscopic data (CNMF-E) [25] with MAT- DV − 4.60) according to the atlas [24]. More than a week LAB (ver. R2018b; MathWorks, MA, USA). All extracted after viral injection, a second surgery was performed to traces were manually checked, and traces from multiple implant a customized 0.6-mm-diameter gradient index cells or non-cellular signals were excluded. Fluorescent (GRIN) lens probe (Inscopix, CA, USA) on the right traces from each neuron were z-scored, and taste-acti- CeA (AP − 1.45, ML + 3.00, DV − 4.40) using a custom- vated neurons were defined by the following formula: made implanter. The implanted lens probe was fixed to (averaged z-score for 30-s after each taste stimulation at the skull using UV-curable optical adhesive (NOA-81; Hamada et al. Molecular Brain (2023) 16:28 Page 4 of 16 three trials − averaged z-score for 30-s after water given (300 mm × 160 mm × 140 mm) with clean paper bedding at nine trials) > 0.5. without food and water under a dim light 1 h before taste stimulation as the adaptation experimental environment. Fos counting by fluorescence in situ hybridization Then, mice were presented with a bottle with stainless The preparation of complementary RNA (cRNA) probes steel sipper tubes (Drinko-measurer; DM-G1, O’Hara and fluorescence in  situ hybridization (FISH) were per - & Co., Ltd.) containing either water or umami solution formed as described previously [26], with some modifi - under free-moving condition. Thirty minutes after the cations. To construct Fos, nitric oxide synthase 1 (Nos1), first licking action, the animals were deeply anesthetized Prkcd, Sst, tyrosine hydroxylase (Th), calcitonin gene- with isoflurane (5%) and sacrificed for in  situ hybridiza - related peptide (Calca), and pituitary adenylate cyclase- tion. Brains were removed, frozen rapidly by dry ice, and activating polypeptide (Adcyap1) FISH probes, total stored at − 80 °C. The frozen brains were sectioned coro - RNA from the adult B6 mouse brain was reverse tran- nally at a thickness of 20 μm on a cryostat (HM525 NX, scribed by Prime Script II RTase (Takara Bio Inc., Shiga, Thermo Fisher Scientific, MA, USA) at the Bregma + 1.10 Japan), and the Fos (NM_010234.2, 1–1291 base), Nos1 to + 0.60 (IC), − 1.22 to − 1.58 (CeA), − 3.08 to − 3.40 (NM_008712.1, 2898–3648 base), Prkcd (NM_011103.3, (VTA), − 5.02 to − 5.40 (PB), and − 6.84 to − 7.92 (N TS). 238–2262 base), Sst (NM_009215.1, 7–550 base), Sections mounted onto glass slides were fixed with 4% Calca (NM_007587.2, 156–566 base), and Adcyap1 paraformaldehyde and treated with the following acetyla- (NM_009625, 1244–2103 base) sequences were ampli- tion and hybridization buffers. Acetylation buffer: 0.25% fied by polymerase chain reaction using PrimeSTAR MX acetic anhydride and 0.1  M triethanolamine-HCl (pH DNA Polymerase (Takara Bio Inc.) with specific primer 8.0); hybridization buffer: 50% formamide, 600 mM NaCl, sets (Table  1). The resulting polymerase chain reaction 33 mM Tris–HCl (pH 8.0), 1 × Denhardt’s stock solution, fragments were subcloned into pBlueScript II KS (+) 10% dextran sulfate, 1  mM EDTA, 0.1% N-Lauryl sarco- phagemids (Agilent, CA, USA). TH-inserted pBlueScript sine sodium salt, and 200  μg/mL tRNA. Hybridization plasmid was kindly gifted from Prof. Watanabe (Hok- was performed for 12–16 h at 63.5 °C with FITC-labeled kaido Univ.) [27]. Fluorescein isothiocyanate (FITC)- or Fos and DIG-labeled cell type-specific marker cRNA Digoxigenin (DIG)-labeled cRNA probes were prepared probes in hybridization buffer. Subsequently, sections using T3 or T7 RNA polymerase (Promega, WI, USA) were washed at 61.0  °C with 5 × standard saline citrate with a FITC or DIG RNA labeling mix (Roche Diagnos- (SSC) for 30 min, 50% formamide containing 4 × SSC for tics, Tokyo, Japan) at 37.0 °CC for 2 h. 15  min, 50% formamide containing 2 × SSC for 15  min, Six-week-old naïve and prolonged taste exposed mice and 0.1 × SSC for 30  min three times. Additional wash- were acclimated to test bottles for 3–4  days before ing steps were performed at room temperature using the stimulation. Following 19–21  h of water depriva- NTE buffer [0.5 M NaCl, 10 mM Tris–HCl (pH 8.0), and tion, each mouse was placed in the stimulation cage 5  mM EDTA] for 5  min, NTE buffer containing 20  mM iodoacetamide for 15  min, NTE buffer for 10  min, and TNT buffer [0.1 M Tris–HCl (pH 7.4) and 0.15 M NaCl] for 5 min (the latter was used as a washing buffer in sub - Table 1 Cloning primers for FISH probes sequent processes). Samples were incubated with DIG blocking buffer [1% blocking reagent (Roche Diagnos - Primer name Sequence tics) and 10% normal sheep serum (Merck Millipore, Fos_F gggctgcaggaattcCAG CGA GCA ACT GAG AAG AC MA, USA)] for 30 min, and incubated with 0.5% (wt/vol) Fos_R cccctcgaggtcgacTCT GAC TGC TCA CAG GGC CA TSA-blocking solution (Akoya Biosciences, MA, USA) Nos1_F cgggctgcaggaattcGGC TAA GAA AGT CTT CAA GG for 30 min. The detection of the FITC-labeled probe was Nos1_R ccccctcgaggtcgacACA TGT CTG GAG AGG AGC TG performed using a peroxidase-conjugated anti-FITC Prkcd_F cgggctgcaggaattcATG GCA CCC TTC CTG CGC ATC antibody (Roche Diagnostics), followed by processing Prkcd_R ccccctcgaggtcgacTTA AAT GTC CAG GAA TTG CTC with TSA plus the FITC System (Akoya Biosciences). Sst_F cgggctgcaggaattcTGA AGG AGA CGC TAC CGA AG After the inactivation of peroxidase by 1% hydroperox- Sst_R ccccctcgaggtcgacTGC AGG GTC AAG TTG AGC ATC ide, detection of the DIG-labeled probe was performed Calca_F tcccccgggctgcagATG GGC TTC CTG AAG TTC TC using a peroxidase-conjugated anti-DIG antibody (Roche Calca_R cccctcgaggtcgacTGC CAA AAT GGG ATT Diagnostics) with 4′,6-diamidino-2-phenylindole (DAPI) Adcyap1_F accgcggtggcggccgcTGG GTG CAC AAG GAT TGA A for 1  h, followed by processing with TSA plus the Cya- Adcyap1_R ccccctcgaggtcgacGGC AAG GGT AGG AAG GAG GG nine 3 System (Akoya Biosciences). Fluorescent images Lowercase letters indicate overlap sequence for cloning into pBluescriptII KS. were acquired using an FV1200 (Olympus, Tokyo, Japan) Underlines indicate restriction enzyme sites. Uppercase letters indicate the recognition sequence for each gene Hamada  et al. Molecular Brain (2023) 16:28 Page 5 of 16 microscope equipped with a dry objective (UPlanSAPO water. The preference ratio of umami in the Umami 10X/0.40, Olympus) for analysis of FISH signal intensity. group was significantly higher than 50% (p < 0.0001; For cell counting, imaging analysis was performed Fig.  1C). In addition, comparison of the preference using expanded ImageJ version Fiji (NIH). ROI areas were ratios among the three groups revealed that the Umami determined by the marker expression pattern. Images group showed a significantly high preference ratio of were converted into the binary pattern using auto-umami (F = 30.09, p < 0.0001; Umami vs. Water group, 2,21 threshold algorithms (“Triangle” for Fos and “Moments” p < 0.0001; Umami vs. Bitter group, p < 0.0001; Water vs. after background subtraction for Prkcd, Sst, Calca, and Bitter group, p = 0.9934; Fig.  1C), which indicated that Adcyap1) and particles more than 10 µm were analyzed. prolonged exposure to umami increased its preference. The irrelevance signals, such as non-match to the DAPI We also analyzed access duration to the water and umami signal and two or three divided signals in one nucleus, bottles to assess exploring behavior to each tastant. All were corrected manually. Fos and marker double-positive groups showed significant increase in access duration to neurons were counted as cells with more than 4 pixels the umami bottle at several time points when analyzed overlapped. Data were normalized by the ROI area or every 5  min (Additional file  3: Fig. S1A–C). Therefore, the number of marker-positive cells. In data tabulation, potential neophobia to the unexperienced tastants, which Fos FISH counts were analyzed for each slice, and the 4 may have been observed in the first 5  min, would have slices with middle value for each individual were used for been canceled or at least negligible in our experimental tabulation in order to reduce variation among individu- condition. In total access duration during the whole test als. The four slices with middle value were also adopted session, both the Water and Umami groups contacted for analyses of the double-staining with Fos and cell-type the umami bottle longer duration than the water bot- marker genes in the CeA and PB. tle (Water group, p = 0.0315; Umami group, p = 0.0008; Fig. 1D). The Bitter group showed a tendency of increased Quantification and statistical analysis access duration to the umami bottle (p = 0.0676; Fig. 1D). The intake and access duration were analyzed using The ratio of access duration to the umami bottle in the paired t-test. The preference ratio and access ratio were Water and Umami groups was significantly high com - analyzed by one-way ANOVA followed by Tukey’s post pared with the chance rate (50%), and that of the Bitter hoc test and one sample t-test. The Fos FISH cell count- group was slightly but not significantly higher than 50% ing data were analyzed by unpaired t-test. (Water group, p = 0.0483; Umami group, p < 0.0001; Bit- ter group, p = 0.0676; Fig.  1E). These observations indi - Results cate that not only the Umami group but also the Water Chronic umami or bitter exposure induced increased and Bitter groups showed interest in umami. The Umami preference for umami or decreased aversion to bitter group, however, had a significantly higher access ratio To investigate the influence of prolonged experience of to the umami bottle compared with that of the other umami and bitter tastants on taste preference, mice were groups (F = 15.12, p < 0.0001; Umami vs. Water group, 2,21 reared with ad libitum water (Water group), umami solu- p = 0.0002; Umami vs. Bitter group, p = 0.0008; Water vs. tion (Umami group), or bitter solution (Bitter group) for Bitter group, p = 0.9063; Fig. 1E). 3 weeks in the immediate post-weaning period (Fig. 1A), To further confirm that the experiments with umami which did not affect body weight gain (Water group, (100  mM monosodium glutamate, MSG) reflects umami 4.45 ± 0.32  g; Umami group, 3.96 ± 0.22  g; Bitter group, effects rather than sodium effects, we also investigated the 4.10 ± 0.42  g). After prolonged taste exposure, we per- influence of the prolonged experience of umami (100 mM formed the two-bottle test between water and umami monopotassium glutamate, MPG). Mice were reared with to assess umami preference. The Umami group exhib - ad libitum water (Water group), MPG-based umami solu- ited a significant increase in intake of umami solution tion (MPG group) for 3 weeks in the immediate post-wean- compared with water, whereas the Water and Bitter ing period (Additional file  3: Fig. S2A), which did not affect groups showed no difference in water and umami intake body weight gain (Water group, 5.32 ± 0.36 g; MPG group, (Water group, p = 0.6588; Umami group, p = 0.0003; Bit- 5.67 ± 0.35 g). We found that MPG experience for 3 weeks ter group, p = 1.0000; Fig. 1B). Total intake of both water enhanced umami intake similar to MSG experience (Water and umami was comparable between the three groups group, p = 0.0261; Umami group, p = 0.0047; Additional (Water group, 0.76 ± 0.08 g; Umami group, 1.04 ± 0.13  g; file  3: Fig. S2B, Fig.  1B). Although both the Water and Bitter group, 0.94 ± 0.15  g). We calculated the ratio of MPG groups showed attraction to umami, the MPG group umami intake to total intake of water and umami as a exhibited higher preference ratio (MPG vs. Water group, preference ratio, so that a preference ratio higher than p = 0.1336; Additional file  3: Fig. S2C). In addition, the the 50% value indicated that umami was preferred over MPG group exhibited increased access duration and access Hamada et al. Molecular Brain (2023) 16:28 Page 6 of 16 Two-bottle test taste exposure habituation Watergroup water water water vs umami vs bitter 4 days 3 weeks Umami group umami Bitter group bitter 2 days 2 days water vs umami water water B CD E umami umami #### #### ### ### 2.0 *** †††† †††† *** 1.5 60 60 1.0 0.5 20 20 0 0 0.0 0 water vs bitter water water FG HI bitter ### bitter ## 2.0 200 # # 1.5 150 *** * *** * 1.0 100 40 † 40 †††† †††† 0.5 50 20 20 0.0 0 0 0 Fig. 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) ratio compared to the Water group (Additional file  3: Fig. MSG and MPG suggest that prolonged umami exposure S2D, E). These results regarding access duration are simi - increased the preference for umami. The following experi - lar to those obtained using MSG. Taken together, these ments were conducted with MSG-based umami solution experiments support the idea that our experiments with that induced remarkable changes in umami preference in MSG also reflect the effect of umami. These results using behavioral experiments. I( ntake g) I( ntake g) P) reference ratio (% P) reference ratio (% A( ccess duration s) A( ccess duration s) A) ccess ratio (% A) ccess ratio (% Hamada  et al. Molecular Brain (2023) 16:28 Page 7 of 16 Next, we performed the two-bottle test with water and such as the NTS, PB, and IC [5, 29, 30]. Especially, it has bitter solution to assess whether prolonged umami or bit- been reported that Prkcd-positive neurons in the CeA are a ter exposure affected bitter aversiveness. The Water and population that responds to aversive tastant [2]. Therefore, Umami groups consumed significantly less bitter solu - one intriguing possibility is that there are cell-type specific tion than water (Water group, p = 0.0004; Umami group, responses to the negative and positive taste qualities within p = 0.0165; Fig.  1F) and showed small preference ratios of the CeA, and neuronal activity changes occur in this circuit bitter compared to 50% (Water group, p < 0.0001; Umami may lead to the modification of outcome behavior toward group, p = 0.0113; Fig.  1G). In contrast, the Bitter group the tastant. However, how each tastant, such as umami, consumed as much bitter solution as water (p = 0.7539; regulates CeA activity, and the correspondence between Fig.  1F) and exhibited bitter preference ratio around cells encoding each taste qualities has not been fully eluci- 50% (p = 0.8361; Fig.  1G), which was significantly high dated, even under untreated naïve conditions. To investi- compared with that of the other groups (F = 12.37, gate innate responses to various tastants in the CeA, we first 2,21 p = 0.0003; Umami vs. Water groups, p = 0.1372; Umami performed in  vivo calcium imaging for two major geneti- vs. Bitter groups, p = 0.0128; Water vs. Bitter groups, cally identified CeA cell populations, Prkcd-positive and p = 0.0002; Fig.  1G), indicating that the Bitter group Sst-positive neurons. Mice were sequentially given water showed no aversion to bitter. The total intake of water and and bitter, sweet, and umami tastant solutions as shown in bitter was comparable between the three groups (Water Fig.  2A. Some neurons showed responses prior to presen- group, 0.56 ± 0.07  g; Umami group, 0.49 ± 0.05  g; Bitter tation of tastant solutions (Additional file  1: Movie S1 and group, 0.66 ± 0.08  g). Although access duration to the bit- Additional file  2: Movie S2). So we evaluated the difference ter bottle was significantly less than that to the water bottle in responses to water and each tastant solution to mini- in the Water and Umami groups, the Bitter group accessed mize the influences of physical stimuli such as oral inser - the bitter and water bottles for almost the same duration tion of a ball tip needle and non-taste-specific responses (Water group, p = 0.0005; Umami group, p = 0.0198; Bitter to drinking itself, and to extract taste-specific response group, p = 0.8296; Fig.  1H; Additional file  3: Fig. S1D–F). neurons. In Prkcd-positive neurons, the largest popula- The access duration ratio to the bitter bottle in the Bit - tions (19.7%) responded to bitter tastant (Fig.  2B), as was ter group was significantly higher than that of the other reported in previous Fos-labeling studies [2, 31]. Notably, a groups (F = 9.17, p = 0.0014; Umami vs. Water group, comparative number of neurons (18.8%) also responded to 2,21 p = 0.2014; Umami vs. Bitter group, p = 0.0370; Water vs. umami, and a smaller number of neurons (7.2%) responded Bitter group, p = 0.0010; Fig. 1I). These results suggest that to sweet tastant (Fig.  2B, D, Additional file  3: Fig. S4A, B). prolonged bitter exposure decreased aversion to bitter, Furthermore, we found that 17.8%, 11.0%, and 11.0% of Sst- which was innately aversive. positive neurons responded to umami, bitter, and sweet tastants, respectively (Fig.  2C, Additional file  3: Fig. S5A, The CeA is composed of neurons with heterogeneous B). Interestingly, one-third of sweet-response and one-fifth response properties for various tastants of umami-response Sst-positive neurons also responded These changes in taste preference/avoidance due to pro - to umami and sweet tastants, respectively, both of which longed taste exposure were considered as an adaptation are thought to be attractive tastants (Fig. 2C, E, Additional accompanied by neuroplasticity. We next sought to deter- file  3: Fig. S5B). Taken together, both Prkcd-positive and mine the areas of the brain that display neuronal activity Sst-positive neurons are not unique populations to respond associated with these behavioral changes. Recent studies to a particular tastant, but are composed of mixed cells that have reported that the CeA plays a pivotal role in emotional respond to negative and positive tastants, although there is behavioral selection [28]. In addition, the CeA receives a bias in the tendency of the responding tastant. direct input from multiple nuclei of the gustatory circuit (See figure on next page.) Fig. 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 Hamada et al. Molecular Brain (2023) 16:28 Page 8 of 16 water REC umami bitter sweet 6 min 2 min Prkcd-cre activated umami umami bitter sweet other 7.2 31 18.8 19.7 2 6 10 34 80.3 1 81.2 92.8 sweetbitter % % Sst-cre umami umami bitter sweet 11.0 11.0 17.8 7 2 13 18 82.2 89.0 89.0 sweetbitter % % Prkcd-cre umami activateds bitter activated weet activated -30-20 -100 10 20 30 -30-20 -100 10 20 30 -30-20 -100 10 20 30 Time (s) Time (s) Time (s) Sst-cre umami activateds bitter activated weet activated -30-20 -100 10 20 30 -30-20 -100 10 20 30 -30-20 -100 10 20 30 Time (s) Time (s) Time (s) Fig. 2 (See legend on previous page.) Z-score Z-score Z-score Z-score Z-score Z-score Hamada  et al. Molecular Brain (2023) 16:28 Page 9 of 16 Prkcd‑positive neurons in the CeA were activated ratio of Fos-positive neurons in the Calca- or Adcyap1- by umami after prolonged umami exposure positive neurons, because these neurons are known to Prkcd-positive neurons of the CeA were thought to innervate the CeA [32, 33]. The number of Fos-positive respond to bitter and suppress appetitive behavior [2, neurons in the PB was not significant between the Water 31], but our calcium imaging results indicate that a part and Umami groups (PB, p = 0.2181; Fig.  4D). On the of Prkcd-positive neurons also respond to attractive taste other hand, while Fos-positive neurons in the Calca- umami. To elucidate the umami taste information pro- positive neurons in the PB was comparable between cessing in more detail, we investigated the responses of two groups, Fos-positive neurons in the Adcyap-positive neurons in the CeA and upstream nuclei of the gusta- neurons was increased in the Umami-tastant provided tory circuit: the NTS, PB, VTA, and IC. To evaluate the group (Calca, p = 0.9617; Adcyap, p = 0.0495; Fig.  4E, F). neuronal activities of these nuclei with regard to umami The NTS showed no difference in Fos-positive neurons, tastant, we performed Fos counting studies by fluores - but Fos-positive neurons in the VTA and IC increased cence in  situ hybridization (FISH). For the identification in the Umami group (NTS, p = 0.5137; V TA , p = 0.0174; of these nuclei, we also used molecular marker genes, IC, p = 0.0476; Fig.  4G–I). These results suggest that the including protein kinase Prkcd and peptide hormone Sst nuclei in higher gustatory circuit, such as CeA, VTA, and in the CeA, nitric oxide synthase Nos1 in the IC, tyros- IC are more activated by the umami administration than ine hydroxylase (Th) in the VTA and NTS, and peptide NTS and PB, which are the primary nuclei. hormones Calca and Adcyap1 in the PB (Additional Next, to determine changes in neuronal activity by pro- file  3: Fig. S6A). The Fos antisense probe detected Fos- longed taste exposure, mice received water or umami positive neurons in the pentylenetetrazole-treated mouse solution ad  libitum for 3  weeks, and Fos FISH assay was hippocampus, but not in the vehicle-treated mouse hip- performed after taste stimulation (Fig.  5). As observed pocampus. Sense probes did not detect the signal in mice in the single taste stimulation (Fig.  4), Fos expression in hippocampi from both treatment groups (Additional the CeA was markedly increased by umami stimulation file 3: Fig. S6B). in prolonged taste exposure mice (p = 0.0008; Fig.  5A). Initially, to investigate the immediate neuronal activ- Interestingly, there was no difference in the ratio of Fos - ity of the tastant, mice were individually housed with positive neurons in the Sst-positive neurons, while the restricted feeding for over an hour and restricted drink- ratio of Fos-positive neurons in the Prkcd-positive neu- ing for 19–21  h before the taste experiment. To assess rons was significantly increased in the Umami group the innate taste response, naïve mice were exposed the (Prkcd, p = 0.0001; Sst, p = 0.2778; Fig.  5B, C). Among water, umami, or bitter solutions (Fig.  3). However, the the higher gustatory nuclei, no difference was observed mice provided with bitter solution did not drink it (water, except for the VTA, unlike the single taste administration 0.37 ± 1.10  g; umami, 0.58 ± 0.11  g; bitter, 0.03 ± 0.01  g; (PB, p = 0.8423; Calca, p = 0.8279, Adcyap1, p = 0.1059; F = 11.29, p = 0.0004, one-way ANOVA; Umami NTS, p = 0.2047; IC, p = 0.2740; Fig.  5D–G, H). Intrigu- 2,22 vs. Water group, p = 0.2188; Umami vs. Bitter group, ingly, the VTA showed a decrease in the Fos-positive p = 0.0004; Bitter vs. Water group, p = 0.0137, Tukey ’s neurons in the prolonged umami administration (VTA, post hoc test). Therefore, we did not perform Fos FISH p = 0.0276; Fig. 5H). These results suggest that prolonged experiments in mice provided with bitter solution exposure to umami taste induces some plastic changes (Fig.  4). The Fos -positive neurons were increased in the in the gustatory circuit, particularly in the CeA, in a cell CeA in umami-stimulated mice compared with water- type-specific manner. stimulated mice (p < 0.0001; Fig.  4A). In addition, we investigated cell type-specific neuronal activity in the Discussion CeA by analyzing Fos and Prkcd- or Sst-double-positive The modification of taste preference by previous taste neurons. The ratios of Fos and Sst double-positive neu- experiences has been studied in animals. In rodents, both rons per Sst-positive neurons in the Umami group was attractive and aversive taste exposure increases intake significantly higher than those in the Water group, while of the exposed taste; exposure to umami in adulthood Fos-positive neurons in the Prkcd-positive neurons was or sweet in the lactation period enhances its palatability comparable between these groups (Sst, p = 0.016398; [14, 34], and exposure to bitter in post-weaning or adult- Prkcd, p = 0.4373; Fig.  4B, C). Next, we performed the hood, or sour in the lactation period reduces its aversive- Fos FISH assay in the CeA upstream gustatory nuclei (PB, ness [10, 12]. Our results are consistent with this body of NTS, VTA, and IC). In the IC, we focused on the area evidence and showed that prolonged exposure to umami between Bregma + 1.1  mm and + 0.6 mm as the umami and bitter in the post-weaning juvenile period also field, because the umami field is between the bitter and increases the preference for the exposed taste. Further- sweet hot fields [5, 17]. In the PB, we also calculated the more, we found that prolonged exposure to umami did Hamada et al. Molecular Brain (2023) 16:28 Page 10 of 16 Taste stimulation Fos FISH Water Sst Prkcd or Fos Adcyap1 + Calca Umami 30 min Th Nos1 DAPI Fos Prkcd CeA B C PB VTA IC NTS CeA BLA DAPI Fos Th DAPI Fos Adcyap1 NTS PB scp DAPI Fos Th DAPI Fos Nos1 VTA IC Fig. 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) * Hamada  et al. Molecular Brain (2023) 16:28 Page 11 of 16 AB C CeA Water WaterUmami WaterUmami Umami Water Water Umami Umami 0 10 20 30 40 0 5 10 15 20 25 (Fos + Sst) / Sst (%) (Fos + Prkcd) / Prkcd (%) PB DE F WaterUmami WaterUmami Water Water Umami Umami 0 5 10 15 0 5 10 15 (Fos + Calca) / Calca (%) (Fos + Adcyap1) / Adcyap1 (%) VTA IC NTS H I 250 140 * 200 20 150 15 100 10 0 0 0 Fig. 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 mm 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, G–I Fos FISH assay in the PB, NTS, VTA, and IC, which are upstream regions of the CeA. Fos‑positive cell counts/1 mm 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) Fos cells / mm F/ os cells mm F/ os cells mm Fos cells / mm Fos cells / mm *** Hamada et al. Molecular Brain (2023) 16:28 Page 12 of 16 CeA A B C Water WaterUmami WaterUmami 200 *** Umami Water Water Umami Umami 0 5 10 15 0 5 10 15 20 25 (Fos + Sst) / Sst (%) (Fos + Prkcd) / Prkcd (%) DE F PB WaterUmami WaterUmami Water Water Umami Umami 0 4 8 12 0 4 8 12 (Fos + Adcyap1) / Adcyap1 (%) (Fos + Calca) / Calca (%) G NTS VTA IC 80 100 0 0 0 Fig. 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 mm 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, G–I Fos FISH assay in the PB, NTS, VTA, and IC, which are upstream regions of the CeA. Fos‑positive cell counts/1 mm 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) F/ os cells mm Fos cells / mm F/ os cells mm F/ os cells mm F/ os cells mm Hamada  et al. Molecular Brain (2023) 16:28 Page 13 of 16 not affect bitter preference, and vice versa for bitter expo - not sweet tastant solution [2]. Furthermore, Kim et  al. sure, which suggests that there is little crossover effect of employed free access to bitter tastant solution after 24 h different taste qualities. In contrast to our results, it has of water deprivation and found that Fos-positive neurons also been reported that bitter exposure during lactation were increased specifically in Prkcd-positive neurons in has no significant effect on bitter ingestion [10]. Fur - the capsular part of the CeA compared with mice pro- thermore, sweet exposure in the post-weaning period vided with neutral-taste water [31]. Collectively, the taste reduces its hedonic valence in adulthood [13]. These lines specificity of the CeA cell-types remains to be ambigu - of evidence suggest that appropriate time window of taste ous. Therefore, in order to clarify not only the respon - experiences may be critical for the increment of taste siveness to various tastants but also the correspondency preference. It would be an interesting future study to of responding cells, here we attempted to consecutive examine whether there is a critical period to induce such recording of neuronal responses to various tastants using experience-dependent changes in taste preference. The calcium imaging, and identified that the Prkcd- and Sst- studies described above mainly focused on preference for positive neuronal populations consisted of both cells the exposed taste, but not other tastes, especially oppo- responding to negative and positive tastants, respectively. site valence tastes such as umami and bitter. We therefore The CeA receives direct inputs from bitter-responsive examined the influence on bitter and umami prefer - neurons in the PB and bitter-responsive neuron hot- ence in the Umami and Bitter groups and showed that spots located in the caudal part of the IC [2, 5], suggest- preference for the unexposed taste is unchanged, which ing that the bitter-responsive neurons of the CeA can be suggests that taste preference is modified in a manner activated by these inputs. Since the IC also possesses a selective to the exposed taste. sweet-responsive neuron hotspot on the rostral side and We used MSG- and MPG-based umami solutions mainly projects to the basolateral amygdala (BLA), it is for the two-bottle tests. The Water group did not show possible that sweet stimulation, at least in part, is indi- a strong umami preference using MSG-based umami rectly transmitted to the CeA through the BLA [5, 17]. In (Fig. 1C, E), while it did using MPG-based umami (Addi- addition, it has been reported that an umami-responsive tional file  3: Fig. S2C, E). The reason for this is unclear. neuron hotspot also exists in the IC between the bitter- One can speculate that there may be some interaction and sweet-responsive neuron hotspots [17], and there between sodium and umami signals. For example, a pre- are direct inputs to the CeA from these areas of the IC vious study demonstrated that some Satb2-positive neu- [36], suggesting that umami information can be trans- rons in PB respond to both sodium chloride and umami mitted to the CeA directly from the umami hotspot of stimuli [16]. These Satb2 neurons enhance taste percep - the IC. Furthermore, the VTA dopamine neurons pro- tion and affect licking behavior. One possibility is that jects directly to the CeA, especially to the medial part of the umami solution containing 100 mM MSG influences the CeA (CeM), where Prkcd-positive neurons are less taste perception and umami intake. Therefore, the slight abundant and Sst-positive neurons are more abundant difference in preference between MSG- and MPG-based [15, 37]. Therefore, it is also possible that umami infor - umami in the Water group may be due to the difference mation is relayed to Sst-positive neurons in the CeA via in the neuronal activities in the PB to umami and sodium VTA dopamine neurons. Interestingly, some dopaminer- signals. The interaction and plasticity mechanisms are gic neurons in the VTA project to the IC, consolidating essential topics for future investigation. aversive taste memory [38]. In order to elucidate through In the present study, we first targeted and investigated which nuclei the information for each taste is relayed to the CeA neurons in response to various tastants because the CeA, further studies in combination with circuit trac- these neurons receive direct and indirect inputs from ing are required. Together, our findings suggest that taste multiple nuclei of the gustatory circuit, and play a criti- stimuli are represented in a more complex manner in the cal role in encoding negative or positive valence. The CeA than previously thought. taste response of the CeA neurons has been investigated It is noteworthy that the ratio of the Fos-positive neu- in rodents by several previous Fos-labeling studies; how- rons in the CeA were high in the Sst-positive neurons ever, it should be noted that each experiment employed in single umami administration, while they were pre- a partially different method of taste stimulation. Otsubo dominant in the Prkcd-positive neurons in prolonged et al. reported that the Fos-like immunoreactivity of neu- umami administration. At least, our calcium imag- rons in the CeA was increased by both forcibly sweet and ing showed that there is a population that responds to umami stimulations after 24-h fasting compared with umami in Prkcd-positive neurons, suggesting that pro- salty stimulation [35]. In contrast, Cai et  al. reported longed umami administration enhanced the activity of that Fos-positive neurons in the CeA were increased by these neurons as well. Because the Prkcd-positive and forcible intraoral infusion of bitter tastant solution but Prkcd-negative (mainly Sst-positive) neurons are both Hamada et al. Molecular Brain (2023) 16:28 Page 14 of 16 inhibitory neurons and form reciprocally connected quinine, the bitter substance used in the present study, microcircuits in the CeA [39, 40], one possible underly- has been identified [48], and it is possible that the sen - ing mechanism is that Sst- and Prkcd-positive neurons sitivity of some of these receptors was changed. Periph- are plastically regulated in a different manner via mutual eral nerves have been reported to sense bitterness and inhibition, resulting in opposite plastic changes. In fact, nutrition and contribute to preference [49]. In addi- Sst- and Prkcd-positive neurons exhibit contradictory tion, it has also been suggested that changes in periph- responses in fear learning and pain-like behavior [41, 42]. eral taste bud structure are accompanied by changes Furthermore, among Sst-positive neurons, different sub - in preference due to taste experience [34]. In the pre- regions within CeA have different plasticity phenotypes sent study, we were unable to examine the peripheral [43]. Another possibility is that the inputs to the Sst- and involvement in changes in taste preference, but this will Prkcd-positive neurons are different, thereby acute and need to be examined in the future. chronic taste experiences have different effects on these Experimental systems such as conditioned taste aver- cell-types. Indeed, excitatory synaptic inputs from the IC sion and conditioned place aversion exist for the mecha- to the lateral and capsular part of the CeA are greater in nism that makes animals dislike what they like, and these Sst-positive neurons [44], whereas PB inputs are larger systems have been widely studied worldwide [2, 3, 50, in Sst-negative neurons in the capsular part of the CeA, 51]. Conversely, there are few experimental systems that but those are larger in Sst-positive neurons in the CeM examine the mechanism of liking something one dislikes, [43]. Also, the VTA dopaminergic neurons project pre- and further research on the mechanism of increased pref- dominantly to the CeM, where Sst-positive neurons are erence by bitter taste experience may lead to the elucida- rich. Therefore, prolonged umami administration may tion of a complementary mechanism. Unfortunately, in cause experiment-related plasticity in the CeA to act on the present study, Fos FISH analysis was not possible for Sst- and Prkcd-positive neurons differentially, resulting bitter taste in the free-moving condition. In future stud- in changes in the balance between these neurons which ies, we would like to examine changes in Fos expression may influence the palatability of umami. To support this patterns associated with single and prolonged changes notion, it is known that the satiety-related peptide hor- in bitter taste exposure by either lowering the concentra- mone cholecystokinin (CCK) is released by umami [45]. tion of bitter taste or by forced drinking. Furthermore, CCK from the peripheral tissue can activate Prkcd-pos- we would like to research changes in umami preference itive neurons in the CeA [46]. These lines of evidence behavior through the inhibition of Prkcd-positive neurons suggest that umami experience is involved in activation by the artificial circuit manipulation of neuronal activity of the CeA Prkcd-positive neurons via the CCK path- during prolonged umami exposure. way. Although the causal relationship between drinking behavior and activity of Prkcd-positive neurons is not Abbreviations clear due to the limitations of our experimental methods, CeA Central amygdala these neurons may intricately regulate umami prefer- NTS Nucleus of the solitary tract PB Parabrachial nucleus ence and drinking control. Intriguingly, a previous study VTA Ventral tegmental area reported that the CeA Prkcd-positive neurons are criti- IC Insular cortex cally involved in chronic alcohol-drinking behavior in AAV Adeno‑associated virus GRIN Gradient index rats [47]. Therefore, one possibility is that the Prkcd-pos- cRNA Complementary RNA itive neurons are involved in experience-dependent plas- FISH Fluorescence in situ hybridization tic changes such as prolonged umami intake and chronic NOS1 N itric oxide synthase 1 Prkcd Protein kinase C delt alcohol drinking. It would be an interesting future study Sst Asomatostatin to examine the molecular mechanisms of the synaptic Th Tyrosine hydroxylase plasticity in the Prkcd-positive neurons and their physi- Calca Calcitonin gene‑related peptide Adcyap1 P ituitary adenylate cyclase‑activating polypeptide ological consequences. FITC Fluorescein isothiocyanate Although the present study focused on the plasticity DIG Digoxigenin to the central nervous system caused by taste experi- SSC Standard saline citrate DAPI 4′,6‑Diamidino ‑2‑phenylindole ences, it is also possible that changes in the periphery BLA Basolateral amygdala influence taste preference. In general, animals avoid bit - CeM Medial part of the CeA ter tastes, but frequent ingestion decreases the avoid-MSG Monosodium glutamate MPG Monopotassium glutamate ance behavior. The decrease in aversiveness may be due CCK Cholecystokinin to fewer bitter taste receptors. The bitter receptor of Hamada  et al. Molecular Brain (2023) 16:28 Page 15 of 16 Funding Supplementary Information This work was supported in part by JSPS Grants‑in‑Aid for Scientific Research The online version contains supplementary material available at https:// doi. [JP19H04062, JP21K18564, and JP22H03542 to AMW; JP19H03324 to TO; org/ 10. 1186/ s13041‑ 023‑ 01017‑x. JP21H05091 and JP20H03339 to ST‑K; JP20K15929 and JP22K06483 to SU; JP16H06276 (AdAMS) to HB and ST‑K; JP17H06312 to HB; JP20K15936 to MN; Additional file 1: Movie S1. Representative calcium imaging movie of JP21K16374 to TN], Core Research for Evolutional Science and Technology Prkcd-cre mice (right) and simultaneously recorded behavioral movie Japan Science and Technology Agency (CREST‑ JST: JPMJCR1751 to AMW and (left). The tastant solution was poured at the timing when the text color is TO), Japan Agency for Medical Research and Development (AMED) Brain reversed. The movie plays at 2× speed. Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) (JP19dm0207081 to SH and AMW ), JST (Moonshot R and D) (JPMJMS2024 to Additional file 2: Movie S2. Representative calcium imaging movie of AMW ), JST‑Mirai Program (JPMJMI21G6 to ST ‑K), and a Grant ‑in‑Aid for Scien‑ Sst-cre mice (right) and simultaneously recorded behavioral movie (left). tific Research on Innovation Areas grant (JP19H05014 to TO). The tastant solution was poured at the timing when the text color is reversed. The movie plays at 2× speed. Availability of data and materials Additional file 3: Figure S1. Time course of access duration in two ‑bottle All data are available upon request to the corresponding author. test. A–C Access duration to water or umami bottles every 5 min in 2 days two‑bottle tests in Water (A), Umami (B) and Bitter (C) groups. D–F Access Declarations duration to water or bitter bottles every 5 min in 2 days two‑bottle tests in Water (D), Umami (E) and Bitter (F) groups. Data are represented as Ethics approval and consent to participate mean ± SEM. Water group, n = 8; Umami group, n = 9; Bitter group, n = 7. All experimental protocols in this study that included the use of animals were *p < 0.05, **p < 0.01, ***p < 0.001 (Paired t‑test). Figure S2. Preference for approved by the Institutional Animal Care and Use Committee of The Jikei MPG‑based umami in the two ‑bottle test in prolonged taste exposure University (Kashiwa City, Japan) (Approval number 2018‑072, 2019‑010) and mice. A Experimental paradigm of prolonged taste exposure and two‑ Nagoya University (approval number R210154). 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 Consent for publication ratio of the umami intake to the total intake. D Access duration to water Not applicable. or umami bottle. E Access ratio of umami bottle. Each circle represents results from one mouse. Data are represented as mean ± SEM. Water Competing interests group, n = 10; MPG group, n = 10. *p < 0.05, **p < 0.01 (paired t‑test); † †† $ The authors declare no competing interests. p < 0.05, p < 0.01 (one sample t‑test); p < 0.05 ( Welch’s t‑test followed by correction with Bonferroni method). Figure S3. A Schematic of viral injections and lens implantation into the CeA for calcium imaging. B Rep‑ Received: 18 November 2022 Accepted: 3 March 2023 resentative image of GCaMP6f expression and lens probe tract of Prkcd-cre mouse brain. Scale bar, 200 µm. C Implanted lens probe locations of four Prkcd-cre (magenta) and four Sst-cre (light blue) mice. The values indicate anterior–posterior distances from bregma. Figure S4. Heatmaps indicate responses to umami (upper), bitter (middle), and sweet (lower) tastant solution of 3 trials each (A), and average responses of 3 trials for each References tastant (B) in total extracted cell population from Prkcd‑ cre mice (223 1. Yarmolinsky DA, Zuker CS, Ryba NJP. Common sense about taste: from cells) aligned in descending order by response value for umami (upper), mammals to insects. Cell. 2009;139(2):234–44. bitter (middle), and sweet (lower) described in the methods section. Red 2. Cai H, Haubensak W, Anthony TE, Anderson DJ. Central amygdala PKC‑ lines on the left of each row correspond to neurons activated in each δ+ neurons mediate the influence of multiple anorexigenic signals. taste. Figure S5. Heatmaps indicate responses to umami (upper), bitter Nat Neurosci. 2014;17(9):1240–8. (middle), and sweet (lower) tastant solution of 3 trials each (A), and 3‑trial 3. Fu O, Iwai Y, Kondoh K, Misaka T, Minokoshi Y, Nakajima K‑I. SatB2‑ average responses for each tastant (B) in total extracted cell population expressing neurons in the parabrachial nucleus encode sweet taste. from Sst‑ cre mice (191 cells) aligned in descending order by response Cell Rep. 2019;27(6):1650‑1656.e4. value for umami (upper), bitter (middle), and sweet (lower) described 4. Tan H‑E, Sisti AC, Jin H, Vignovich M, Villavicencio M, Tsang KS, in the methods section. Red lines on the left of each row correspond to et al. The gut–brain axis mediates sugar preference. Nature. neurons activated in each taste. Figure S6. Validation of FISH probes. 2020;580(7804):511–6. (A) Validation of probes for the brain region or cell type‑specific markers 5. Wang L, Gillis‑Smith S, Peng Y, Zhang J, Chen X, Salzman CD, et al. The Prkcd, Sst, Nos1, Th, Calca, and Adcyap1. Brain region or cell type‑specific coding of valence and identity in the mammalian taste system. 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Experience-dependent changes in affective valence of taste in male mice

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Abstract

Taste plays an essential role in the evaluation of food quality by detecting potential harm and benefit in what ani‑ mals are about to eat and drink. While the affective valence of taste signals is supposed to be innately determined, taste preference can also be drastically modified by previous taste experiences of the animals. However, how the experience‑ dependent taste preference is developed and the neuronal mechanisms involved in this process are poorly understood. Here, we investigate the effects of prolonged exposure to umami and bitter tastants on taste preference using two‑bottle tests in male mice. Prolonged umami exposure significantly enhanced umami preference with no changes in bitter preference, while prolonged bitter exposure significantly decreased bitter avoidance with no changes in umami preference. Because the central amygdala (CeA) is postulated as a critical node for the valence processing of sensory information including taste, we examined the responses of cells in the CeA to sweet, umami, and bitter tastants using in vivo calcium imaging. Interestingly, both protein kinase C delta (Prkcd)-positive and Somatostatin (Sst)‑positive neurons in the CeA showed an umami response comparable to the bitter response, and no difference in cell type ‑specific activity patterns to different tastants was observed. Meanwhile, fluorescence in situ hybridization with c-Fos antisense probe revealed that a single umami experience significantly activates the CeA and several other gustatory‑related nuclei, and especially CeA Sst‑positive neurons were strongly activated. Intriguingly, after prolonged umami experience, umami tastant also significantly activates the CeA neurons, but the Prkcd ‑positive neurons instead of Sst‑positive neurons were highly activated. These results suggest a relationship between amygdala activity and experience‑ dependent plasticity developed in taste preference and the involvement of the genetically defined neural populations in this process. Keywords Gustatory circuit, Umami, Bitter, Amygdala, Taste preference, Plasticity, Calcium imaging † 3 Shun Hamada, Kaori Mikami, Shuhei Ueda and Masashi Nagase contributed Department of Neuroscience I, Research Institute of Environmental equally to this work Medicine, Nagoya University, Nagoya 464‑8601, Japan Department of Molecular/Cellular Neuroscience, Nagoya University *Correspondence: Graduate School of Medicine, Nagoya 466‑8550, Japan Ayako M. Watabe Department of Neurochemistry, Graduate School of Medicine, The awatabe@jikei.ac.jp University of Tokyo, Tokyo 113‑0033, Japan Department of Biochemistry, Faculty of Medicine, University of Yamanashi, Yamanashi 409‑3898, Japan Institute of Clinical Medicine and Research, Research Center for Medical Sciences, The Jikei University School of Medicine, 163‑1 Kashiwashita, Kashiwa, Chiba 277‑8567, Japan © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hamada et al. Molecular Brain (2023) 16:28 Page 2 of 16 (CeA) directly or indirectly [3, 15, 16]. Recent studies Introduction have demonstrated that taste information is processed by The taste system is crucial for animals to detect poten - a labeled-line system, such that information about each tial benefits, e.g., nutrients, and potential harm, e.g., taste quality has a discrete pathway from its taste recep- toxins, in what they are about to eat and drink [1, 2]. tors to the corresponding neuronal taste circuits [17, 18]. There are five basic taste qualities: sweet, sour, salty, bit - While some neurons in the central taste pathway are ter, and umami. The taste system has an emotional and tuned to particular taste qualities, other neurons respond motivational aspect, and affective taste, such as sweet more broadly to multiple tastes [19]. Furthermore, some and umami, drives approaching and appetitive behav- neurons change in their responsive profiles according to iors, while aversive taste, such as bitter and sour, drives experience and time [20], which suggests some plastic- avoidance behaviors [3–5]. The attractive and aversive ity in the taste coding rule. Therefore, neural plasticity valence of taste signals is innately determined. For exam- within the described taste circuitry can regulate expe- ple, human newborn infants exhibit affective behav - rience-dependent changes in the affective and aversive iors such as lip sucking, elevation of the corners of the valence of particular taste signals. mouth, and rhythmic tongue protrusions when exposed In the present study, we have addressed this issue to sweet or umami solutions [6, 7]. They also show aver - by establishing a behavioral paradigm for experience- sive responses such as nose wrinkling and grimacing dependent plasticity in the taste preference for attractive when exposed to bitter or sour solutions. Rats and mice (umami) and aversive (bitter) taste quality, and examined also exhibit affective behaviors such as rhythmic and lat - the neuronal correlates in mice using in  vivo calcium eral tongue protrusions when administered with sweet or imaging and fluorescence in  situ hybridization in multi - umami solutions [7–9]. They also show aversive behav - ple brain regions. iors, such as gasping, chin rubbing, and handshaking, when exposed to quinine solution. Materials and methods The attractive and aversive valence of taste can also be Animals acquired so that experiences can modify the preference Adult male C57BL/6J mice (Japan SLC, Inc., Shizuoka, for certain tastants. For instance, many people may expe- Japan) were group housed (3–4 mice per cage) on a rience the development of an acquired taste for coffee, 12  h light/12  h dark cycle and provided with food (CE- beer, or even quinine. Likewise, in rats and mice, expo- 2, CLEA Japan, Inc., Tokyo, Japan) and water ad libitum, sure to sour and bitter substances before and after wean- unless otherwise noted. Protein kinase C delta (Prkcd)- ing leads to a significant preference for those substances cre mice [Tg(Prkcd-glc-1/CFP,-cre)EH124Gsat; stock in adulthood [10–12]. Thus, the unconditioned avoidance #011559-UCD] and somatostatin (Sst)-cre mice [Sst of sour and bitter can be modulated by early-life experi- tm2.1(cre)Zjh/J; stock #013044] were obtained from the ences. Compared with that of unfavored tastes, litera- Mutant Mouse Resource & Research Center and the ture on the modification of hedonic valence of favored Jackson Laboratory, respectively, and were maintained tastes is limited. It has been shown that rats exposed to heterozygous on a C57BL/6J background. Adult male overconsumption of sucrose during adolescence display mice over 3  months old were used for in  vivo calcium reduced sweet consumption and hedonic perception in imaging studies. All experimental protocols in this study adulthood [13]. Furthermore, Ackroff et al. demonstrated that included the use of animals were approved by the that prior umami experience significantly enhances pref - Institutional Animal Care and Use Committee of The erence for umami solutions in mice [14]. These findings Jikei University (Kashiwa City, Japan) (Approval number suggest that both attractive and aversive valence of taste 2018-072, 2019-010) and Nagoya University (approval signals can be subject to influence from previous expe - number R210154). All experiments complied with the riences; however, the neuronal mechanisms underlying Guidelines for Proper Conduct of Animal Experiments these observations are not well understood. by the Science Council of Japan (2006) and those recom- Taste signals first arise via taste receptor cells in the mended by the International Association for the Study of taste buds, which detect tastant chemicals and activate Pain. All efforts were made to reduce the number of ani - matching ganglion neurons. These signals are transmit - mals used and the suffering of the animals. ted through gustatory nerves, including the chorda tym- pani and glossopharyngeal nerves, to the nucleus of the Prolonged taste exposure and two‑bottle tests solitary tract (NTS), which relays information to the pon- Mice in the prolonged taste exposure groups had ad libi- tine parabrachial nucleus (PB) in rodents. These neurons tum access to food and one of the following taste solu- then activate the ventral tegmental area (VTA), the ven- tions instead of water beginning at 4  weeks of age, tral posteromedial nucleus of the thalamus, and the insu- immediately after weaning, for 3  weeks. The umami lar cortex (IC), which all project to the central amygdala Hamada  et al. Molecular Brain (2023) 16:28 Page 3 of 16 solution contained 100  mM monosodium l-glutamate Norland Products, NJ, USA). Exposed skull was coated (Sigma-Aldrich, Darmstadt, Germany) or 100 mM mono- with super-bond (C&B Kit; Sun Medical, Shiga, Japan), potassium l-glutamate (Sigma-Aldrich, Darmstadt, Ger - additionally covered with dental cement (REPAIRSIN; many) and 10  mM disodium inosine-5′-monophosphate GC, Tokyo, Japan), and a stainless steel bar was attached (Sigma-Aldrich) mixture. The bitter solution contained for head fixation. GCaMP6f fluorescence was periodi - 0.3 mM quinine hydrochloride (FUJIFILM Wako, Osaka, cally evaluated using a miniature integrated microscope Japan). Mice that experienced prolonged taste exposure system (nVista HD 2.0; Inscopix), and when sufficient were subjected to a two-bottle test following 19–21  h of GCaMP6f expression was confirmed, the microscope water deprivation. Mice were acclimated to the stainless baseplate was mounted using blue light curing resin steel sipper tubes in the two-bottle test chamber (Drinko- (Flow-It ALC; Pentron, CT, USA). The sites of viral injec - measurer; DM-G1, trapezoid-shaped test chamber tion and lens probe implantation were confirmed histo - with 55  mm upper side × 205  mm lower side × 135  mm logically after imaging experiments (Additional file  3: Fig. depth × 200  mm height; TOP-3002WW, O’Hara & Co., S3). Ltd, Tokyo, Japan) within the sound-attenuating box (660 mm width × 460 mm depth × 690 mm height; TOP- In vivo calcium imaging during taste stimulation 4011, O’Hara & Co., Ltd) for 15  min per day for 4  days. More than 5  days after baseplate mounting, the mice The two-bottle test was performed with the bottle posi - were ready for imaging. After several days of imaging tions switched for 2 days to avoid side preference. On test studies for natural experiences, imaging experiments day 1 and day 2, an umami solution bottle and a water for taste stimuli-evoked responses were performed. A bottle were presented. On day 3 and day 4, a bitter solu- detailed procedure of the precedent imaging studies will tion bottle and a water bottle were presented. Access be described elsewhere. A day before the experiment, the duration to each bottle was measured as nose poking miniscope was attached to each mouse and each mouse time to the bottle and analyzed by Operant task Studio was head-fixed on a running disc wheel for 30  min to V2 (O’Hara & Co., Ltd). A preference ratio was calculated acclimatize, and then 100 µL of water was given six times as the ratio of the taste solution (umami or bitter) intake at 2-min intervals using an oral gavage ball tip needle. On to the total (taste solution and water) intake. An access the experimental day, mice were habituated for 30 min as ratio was determined as the ratio of the access duration on the previous day, and calcium imaging was performed in the taste bottle to the total access duration in two bot- during taste stimulation. Images were acquired using data tles. Mice in the Umami and Bitter groups that exhibited acquisition software (ver. 2.0.4; Inscopix) at 20 frames per an intake of less than 0.1  g during the 15-min test were sec, 25% of LED power, and a gain of 3.5, and behavioral excluded from the data because of the inability to prop- videos were recorded simultaneously by triggers from the erly assess their preference. After the 15-min test, the miniscope system. Six minutes after the start of imaging, mice were returned to the home cage and given ad  libi- mice were given 100  µL of bitter (1  mM quinine), sweet tum access to food and taste solution until the next test (50  mM sucrose), and umami (100  mM monosodium on the following day. glutamate and 10 mM disodium inosinate) taste solution alternately with neutral-taste water at 2-min intervals for Stereotaxic surgery for in vivo calcium imaging three trials (Fig. 2A). Mice surgeries were performed as described previ- Acquired imaging data were down-sampled (1/2 spa- ously with minor modifications [21, 22]. Each deeply tial binning), preprocessed, motion corrected, cropped, anesthetized mouse was fixed in a stereotactic frame and then additionally down-sampled (1/2 spatial and (Model 942; Kopf Instruments, CA, USA). For viral 1/2 temporal binning) using Mosaic Software (ver. 1.2.0; injection, skull surface was exposed, a glass capillary Inscopix). Processed images were loaded to Inscopix was inserted through a drilled small hole, and 500 nL of Data Processing Software (ver. 1.3.0; Inscopix), and then adeno-associated virus (AAV) solution (AAV1/2-CAG- calcium transients of individual neurons were extracted DIO-GCaMP6f-WPRE; 3.5 × 10 genomes/mL) [23] with a constrained non-negative matrix factorization was loaded into the right CeA (AP − 1.35, ML + 2.95, for microendoscopic data (CNMF-E) [25] with MAT- DV − 4.60) according to the atlas [24]. More than a week LAB (ver. R2018b; MathWorks, MA, USA). All extracted after viral injection, a second surgery was performed to traces were manually checked, and traces from multiple implant a customized 0.6-mm-diameter gradient index cells or non-cellular signals were excluded. Fluorescent (GRIN) lens probe (Inscopix, CA, USA) on the right traces from each neuron were z-scored, and taste-acti- CeA (AP − 1.45, ML + 3.00, DV − 4.40) using a custom- vated neurons were defined by the following formula: made implanter. The implanted lens probe was fixed to (averaged z-score for 30-s after each taste stimulation at the skull using UV-curable optical adhesive (NOA-81; Hamada et al. Molecular Brain (2023) 16:28 Page 4 of 16 three trials − averaged z-score for 30-s after water given (300 mm × 160 mm × 140 mm) with clean paper bedding at nine trials) > 0.5. without food and water under a dim light 1 h before taste stimulation as the adaptation experimental environment. Fos counting by fluorescence in situ hybridization Then, mice were presented with a bottle with stainless The preparation of complementary RNA (cRNA) probes steel sipper tubes (Drinko-measurer; DM-G1, O’Hara and fluorescence in  situ hybridization (FISH) were per - & Co., Ltd.) containing either water or umami solution formed as described previously [26], with some modifi - under free-moving condition. Thirty minutes after the cations. To construct Fos, nitric oxide synthase 1 (Nos1), first licking action, the animals were deeply anesthetized Prkcd, Sst, tyrosine hydroxylase (Th), calcitonin gene- with isoflurane (5%) and sacrificed for in  situ hybridiza - related peptide (Calca), and pituitary adenylate cyclase- tion. Brains were removed, frozen rapidly by dry ice, and activating polypeptide (Adcyap1) FISH probes, total stored at − 80 °C. The frozen brains were sectioned coro - RNA from the adult B6 mouse brain was reverse tran- nally at a thickness of 20 μm on a cryostat (HM525 NX, scribed by Prime Script II RTase (Takara Bio Inc., Shiga, Thermo Fisher Scientific, MA, USA) at the Bregma + 1.10 Japan), and the Fos (NM_010234.2, 1–1291 base), Nos1 to + 0.60 (IC), − 1.22 to − 1.58 (CeA), − 3.08 to − 3.40 (NM_008712.1, 2898–3648 base), Prkcd (NM_011103.3, (VTA), − 5.02 to − 5.40 (PB), and − 6.84 to − 7.92 (N TS). 238–2262 base), Sst (NM_009215.1, 7–550 base), Sections mounted onto glass slides were fixed with 4% Calca (NM_007587.2, 156–566 base), and Adcyap1 paraformaldehyde and treated with the following acetyla- (NM_009625, 1244–2103 base) sequences were ampli- tion and hybridization buffers. Acetylation buffer: 0.25% fied by polymerase chain reaction using PrimeSTAR MX acetic anhydride and 0.1  M triethanolamine-HCl (pH DNA Polymerase (Takara Bio Inc.) with specific primer 8.0); hybridization buffer: 50% formamide, 600 mM NaCl, sets (Table  1). The resulting polymerase chain reaction 33 mM Tris–HCl (pH 8.0), 1 × Denhardt’s stock solution, fragments were subcloned into pBlueScript II KS (+) 10% dextran sulfate, 1  mM EDTA, 0.1% N-Lauryl sarco- phagemids (Agilent, CA, USA). TH-inserted pBlueScript sine sodium salt, and 200  μg/mL tRNA. Hybridization plasmid was kindly gifted from Prof. Watanabe (Hok- was performed for 12–16 h at 63.5 °C with FITC-labeled kaido Univ.) [27]. Fluorescein isothiocyanate (FITC)- or Fos and DIG-labeled cell type-specific marker cRNA Digoxigenin (DIG)-labeled cRNA probes were prepared probes in hybridization buffer. Subsequently, sections using T3 or T7 RNA polymerase (Promega, WI, USA) were washed at 61.0  °C with 5 × standard saline citrate with a FITC or DIG RNA labeling mix (Roche Diagnos- (SSC) for 30 min, 50% formamide containing 4 × SSC for tics, Tokyo, Japan) at 37.0 °CC for 2 h. 15  min, 50% formamide containing 2 × SSC for 15  min, Six-week-old naïve and prolonged taste exposed mice and 0.1 × SSC for 30  min three times. Additional wash- were acclimated to test bottles for 3–4  days before ing steps were performed at room temperature using the stimulation. Following 19–21  h of water depriva- NTE buffer [0.5 M NaCl, 10 mM Tris–HCl (pH 8.0), and tion, each mouse was placed in the stimulation cage 5  mM EDTA] for 5  min, NTE buffer containing 20  mM iodoacetamide for 15  min, NTE buffer for 10  min, and TNT buffer [0.1 M Tris–HCl (pH 7.4) and 0.15 M NaCl] for 5 min (the latter was used as a washing buffer in sub - Table 1 Cloning primers for FISH probes sequent processes). Samples were incubated with DIG blocking buffer [1% blocking reagent (Roche Diagnos - Primer name Sequence tics) and 10% normal sheep serum (Merck Millipore, Fos_F gggctgcaggaattcCAG CGA GCA ACT GAG AAG AC MA, USA)] for 30 min, and incubated with 0.5% (wt/vol) Fos_R cccctcgaggtcgacTCT GAC TGC TCA CAG GGC CA TSA-blocking solution (Akoya Biosciences, MA, USA) Nos1_F cgggctgcaggaattcGGC TAA GAA AGT CTT CAA GG for 30 min. The detection of the FITC-labeled probe was Nos1_R ccccctcgaggtcgacACA TGT CTG GAG AGG AGC TG performed using a peroxidase-conjugated anti-FITC Prkcd_F cgggctgcaggaattcATG GCA CCC TTC CTG CGC ATC antibody (Roche Diagnostics), followed by processing Prkcd_R ccccctcgaggtcgacTTA AAT GTC CAG GAA TTG CTC with TSA plus the FITC System (Akoya Biosciences). Sst_F cgggctgcaggaattcTGA AGG AGA CGC TAC CGA AG After the inactivation of peroxidase by 1% hydroperox- Sst_R ccccctcgaggtcgacTGC AGG GTC AAG TTG AGC ATC ide, detection of the DIG-labeled probe was performed Calca_F tcccccgggctgcagATG GGC TTC CTG AAG TTC TC using a peroxidase-conjugated anti-DIG antibody (Roche Calca_R cccctcgaggtcgacTGC CAA AAT GGG ATT Diagnostics) with 4′,6-diamidino-2-phenylindole (DAPI) Adcyap1_F accgcggtggcggccgcTGG GTG CAC AAG GAT TGA A for 1  h, followed by processing with TSA plus the Cya- Adcyap1_R ccccctcgaggtcgacGGC AAG GGT AGG AAG GAG GG nine 3 System (Akoya Biosciences). Fluorescent images Lowercase letters indicate overlap sequence for cloning into pBluescriptII KS. were acquired using an FV1200 (Olympus, Tokyo, Japan) Underlines indicate restriction enzyme sites. Uppercase letters indicate the recognition sequence for each gene Hamada  et al. Molecular Brain (2023) 16:28 Page 5 of 16 microscope equipped with a dry objective (UPlanSAPO water. The preference ratio of umami in the Umami 10X/0.40, Olympus) for analysis of FISH signal intensity. group was significantly higher than 50% (p < 0.0001; For cell counting, imaging analysis was performed Fig.  1C). In addition, comparison of the preference using expanded ImageJ version Fiji (NIH). ROI areas were ratios among the three groups revealed that the Umami determined by the marker expression pattern. Images group showed a significantly high preference ratio of were converted into the binary pattern using auto-umami (F = 30.09, p < 0.0001; Umami vs. Water group, 2,21 threshold algorithms (“Triangle” for Fos and “Moments” p < 0.0001; Umami vs. Bitter group, p < 0.0001; Water vs. after background subtraction for Prkcd, Sst, Calca, and Bitter group, p = 0.9934; Fig.  1C), which indicated that Adcyap1) and particles more than 10 µm were analyzed. prolonged exposure to umami increased its preference. The irrelevance signals, such as non-match to the DAPI We also analyzed access duration to the water and umami signal and two or three divided signals in one nucleus, bottles to assess exploring behavior to each tastant. All were corrected manually. Fos and marker double-positive groups showed significant increase in access duration to neurons were counted as cells with more than 4 pixels the umami bottle at several time points when analyzed overlapped. Data were normalized by the ROI area or every 5  min (Additional file  3: Fig. S1A–C). Therefore, the number of marker-positive cells. In data tabulation, potential neophobia to the unexperienced tastants, which Fos FISH counts were analyzed for each slice, and the 4 may have been observed in the first 5  min, would have slices with middle value for each individual were used for been canceled or at least negligible in our experimental tabulation in order to reduce variation among individu- condition. In total access duration during the whole test als. The four slices with middle value were also adopted session, both the Water and Umami groups contacted for analyses of the double-staining with Fos and cell-type the umami bottle longer duration than the water bot- marker genes in the CeA and PB. tle (Water group, p = 0.0315; Umami group, p = 0.0008; Fig. 1D). The Bitter group showed a tendency of increased Quantification and statistical analysis access duration to the umami bottle (p = 0.0676; Fig. 1D). The intake and access duration were analyzed using The ratio of access duration to the umami bottle in the paired t-test. The preference ratio and access ratio were Water and Umami groups was significantly high com - analyzed by one-way ANOVA followed by Tukey’s post pared with the chance rate (50%), and that of the Bitter hoc test and one sample t-test. The Fos FISH cell count- group was slightly but not significantly higher than 50% ing data were analyzed by unpaired t-test. (Water group, p = 0.0483; Umami group, p < 0.0001; Bit- ter group, p = 0.0676; Fig.  1E). These observations indi - Results cate that not only the Umami group but also the Water Chronic umami or bitter exposure induced increased and Bitter groups showed interest in umami. The Umami preference for umami or decreased aversion to bitter group, however, had a significantly higher access ratio To investigate the influence of prolonged experience of to the umami bottle compared with that of the other umami and bitter tastants on taste preference, mice were groups (F = 15.12, p < 0.0001; Umami vs. Water group, 2,21 reared with ad libitum water (Water group), umami solu- p = 0.0002; Umami vs. Bitter group, p = 0.0008; Water vs. tion (Umami group), or bitter solution (Bitter group) for Bitter group, p = 0.9063; Fig. 1E). 3 weeks in the immediate post-weaning period (Fig. 1A), To further confirm that the experiments with umami which did not affect body weight gain (Water group, (100  mM monosodium glutamate, MSG) reflects umami 4.45 ± 0.32  g; Umami group, 3.96 ± 0.22  g; Bitter group, effects rather than sodium effects, we also investigated the 4.10 ± 0.42  g). After prolonged taste exposure, we per- influence of the prolonged experience of umami (100 mM formed the two-bottle test between water and umami monopotassium glutamate, MPG). Mice were reared with to assess umami preference. The Umami group exhib - ad libitum water (Water group), MPG-based umami solu- ited a significant increase in intake of umami solution tion (MPG group) for 3 weeks in the immediate post-wean- compared with water, whereas the Water and Bitter ing period (Additional file  3: Fig. S2A), which did not affect groups showed no difference in water and umami intake body weight gain (Water group, 5.32 ± 0.36 g; MPG group, (Water group, p = 0.6588; Umami group, p = 0.0003; Bit- 5.67 ± 0.35 g). We found that MPG experience for 3 weeks ter group, p = 1.0000; Fig. 1B). Total intake of both water enhanced umami intake similar to MSG experience (Water and umami was comparable between the three groups group, p = 0.0261; Umami group, p = 0.0047; Additional (Water group, 0.76 ± 0.08 g; Umami group, 1.04 ± 0.13  g; file  3: Fig. S2B, Fig.  1B). Although both the Water and Bitter group, 0.94 ± 0.15  g). We calculated the ratio of MPG groups showed attraction to umami, the MPG group umami intake to total intake of water and umami as a exhibited higher preference ratio (MPG vs. Water group, preference ratio, so that a preference ratio higher than p = 0.1336; Additional file  3: Fig. S2C). In addition, the the 50% value indicated that umami was preferred over MPG group exhibited increased access duration and access Hamada et al. Molecular Brain (2023) 16:28 Page 6 of 16 Two-bottle test taste exposure habituation Watergroup water water water vs umami vs bitter 4 days 3 weeks Umami group umami Bitter group bitter 2 days 2 days water vs umami water water B CD E umami umami #### #### ### ### 2.0 *** †††† †††† *** 1.5 60 60 1.0 0.5 20 20 0 0 0.0 0 water vs bitter water water FG HI bitter ### bitter ## 2.0 200 # # 1.5 150 *** * *** * 1.0 100 40 † 40 †††† †††† 0.5 50 20 20 0.0 0 0 0 Fig. 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) ratio compared to the Water group (Additional file  3: Fig. MSG and MPG suggest that prolonged umami exposure S2D, E). These results regarding access duration are simi - increased the preference for umami. The following experi - lar to those obtained using MSG. Taken together, these ments were conducted with MSG-based umami solution experiments support the idea that our experiments with that induced remarkable changes in umami preference in MSG also reflect the effect of umami. These results using behavioral experiments. I( ntake g) I( ntake g) P) reference ratio (% P) reference ratio (% A( ccess duration s) A( ccess duration s) A) ccess ratio (% A) ccess ratio (% Hamada  et al. Molecular Brain (2023) 16:28 Page 7 of 16 Next, we performed the two-bottle test with water and such as the NTS, PB, and IC [5, 29, 30]. Especially, it has bitter solution to assess whether prolonged umami or bit- been reported that Prkcd-positive neurons in the CeA are a ter exposure affected bitter aversiveness. The Water and population that responds to aversive tastant [2]. Therefore, Umami groups consumed significantly less bitter solu - one intriguing possibility is that there are cell-type specific tion than water (Water group, p = 0.0004; Umami group, responses to the negative and positive taste qualities within p = 0.0165; Fig.  1F) and showed small preference ratios of the CeA, and neuronal activity changes occur in this circuit bitter compared to 50% (Water group, p < 0.0001; Umami may lead to the modification of outcome behavior toward group, p = 0.0113; Fig.  1G). In contrast, the Bitter group the tastant. However, how each tastant, such as umami, consumed as much bitter solution as water (p = 0.7539; regulates CeA activity, and the correspondence between Fig.  1F) and exhibited bitter preference ratio around cells encoding each taste qualities has not been fully eluci- 50% (p = 0.8361; Fig.  1G), which was significantly high dated, even under untreated naïve conditions. To investi- compared with that of the other groups (F = 12.37, gate innate responses to various tastants in the CeA, we first 2,21 p = 0.0003; Umami vs. Water groups, p = 0.1372; Umami performed in  vivo calcium imaging for two major geneti- vs. Bitter groups, p = 0.0128; Water vs. Bitter groups, cally identified CeA cell populations, Prkcd-positive and p = 0.0002; Fig.  1G), indicating that the Bitter group Sst-positive neurons. Mice were sequentially given water showed no aversion to bitter. The total intake of water and and bitter, sweet, and umami tastant solutions as shown in bitter was comparable between the three groups (Water Fig.  2A. Some neurons showed responses prior to presen- group, 0.56 ± 0.07  g; Umami group, 0.49 ± 0.05  g; Bitter tation of tastant solutions (Additional file  1: Movie S1 and group, 0.66 ± 0.08  g). Although access duration to the bit- Additional file  2: Movie S2). So we evaluated the difference ter bottle was significantly less than that to the water bottle in responses to water and each tastant solution to mini- in the Water and Umami groups, the Bitter group accessed mize the influences of physical stimuli such as oral inser - the bitter and water bottles for almost the same duration tion of a ball tip needle and non-taste-specific responses (Water group, p = 0.0005; Umami group, p = 0.0198; Bitter to drinking itself, and to extract taste-specific response group, p = 0.8296; Fig.  1H; Additional file  3: Fig. S1D–F). neurons. In Prkcd-positive neurons, the largest popula- The access duration ratio to the bitter bottle in the Bit - tions (19.7%) responded to bitter tastant (Fig.  2B), as was ter group was significantly higher than that of the other reported in previous Fos-labeling studies [2, 31]. Notably, a groups (F = 9.17, p = 0.0014; Umami vs. Water group, comparative number of neurons (18.8%) also responded to 2,21 p = 0.2014; Umami vs. Bitter group, p = 0.0370; Water vs. umami, and a smaller number of neurons (7.2%) responded Bitter group, p = 0.0010; Fig. 1I). These results suggest that to sweet tastant (Fig.  2B, D, Additional file  3: Fig. S4A, B). prolonged bitter exposure decreased aversion to bitter, Furthermore, we found that 17.8%, 11.0%, and 11.0% of Sst- which was innately aversive. positive neurons responded to umami, bitter, and sweet tastants, respectively (Fig.  2C, Additional file  3: Fig. S5A, The CeA is composed of neurons with heterogeneous B). Interestingly, one-third of sweet-response and one-fifth response properties for various tastants of umami-response Sst-positive neurons also responded These changes in taste preference/avoidance due to pro - to umami and sweet tastants, respectively, both of which longed taste exposure were considered as an adaptation are thought to be attractive tastants (Fig. 2C, E, Additional accompanied by neuroplasticity. We next sought to deter- file  3: Fig. S5B). Taken together, both Prkcd-positive and mine the areas of the brain that display neuronal activity Sst-positive neurons are not unique populations to respond associated with these behavioral changes. Recent studies to a particular tastant, but are composed of mixed cells that have reported that the CeA plays a pivotal role in emotional respond to negative and positive tastants, although there is behavioral selection [28]. In addition, the CeA receives a bias in the tendency of the responding tastant. direct input from multiple nuclei of the gustatory circuit (See figure on next page.) Fig. 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 Hamada et al. Molecular Brain (2023) 16:28 Page 8 of 16 water REC umami bitter sweet 6 min 2 min Prkcd-cre activated umami umami bitter sweet other 7.2 31 18.8 19.7 2 6 10 34 80.3 1 81.2 92.8 sweetbitter % % Sst-cre umami umami bitter sweet 11.0 11.0 17.8 7 2 13 18 82.2 89.0 89.0 sweetbitter % % Prkcd-cre umami activateds bitter activated weet activated -30-20 -100 10 20 30 -30-20 -100 10 20 30 -30-20 -100 10 20 30 Time (s) Time (s) Time (s) Sst-cre umami activateds bitter activated weet activated -30-20 -100 10 20 30 -30-20 -100 10 20 30 -30-20 -100 10 20 30 Time (s) Time (s) Time (s) Fig. 2 (See legend on previous page.) Z-score Z-score Z-score Z-score Z-score Z-score Hamada  et al. Molecular Brain (2023) 16:28 Page 9 of 16 Prkcd‑positive neurons in the CeA were activated ratio of Fos-positive neurons in the Calca- or Adcyap1- by umami after prolonged umami exposure positive neurons, because these neurons are known to Prkcd-positive neurons of the CeA were thought to innervate the CeA [32, 33]. The number of Fos-positive respond to bitter and suppress appetitive behavior [2, neurons in the PB was not significant between the Water 31], but our calcium imaging results indicate that a part and Umami groups (PB, p = 0.2181; Fig.  4D). On the of Prkcd-positive neurons also respond to attractive taste other hand, while Fos-positive neurons in the Calca- umami. To elucidate the umami taste information pro- positive neurons in the PB was comparable between cessing in more detail, we investigated the responses of two groups, Fos-positive neurons in the Adcyap-positive neurons in the CeA and upstream nuclei of the gusta- neurons was increased in the Umami-tastant provided tory circuit: the NTS, PB, VTA, and IC. To evaluate the group (Calca, p = 0.9617; Adcyap, p = 0.0495; Fig.  4E, F). neuronal activities of these nuclei with regard to umami The NTS showed no difference in Fos-positive neurons, tastant, we performed Fos counting studies by fluores - but Fos-positive neurons in the VTA and IC increased cence in  situ hybridization (FISH). For the identification in the Umami group (NTS, p = 0.5137; V TA , p = 0.0174; of these nuclei, we also used molecular marker genes, IC, p = 0.0476; Fig.  4G–I). These results suggest that the including protein kinase Prkcd and peptide hormone Sst nuclei in higher gustatory circuit, such as CeA, VTA, and in the CeA, nitric oxide synthase Nos1 in the IC, tyros- IC are more activated by the umami administration than ine hydroxylase (Th) in the VTA and NTS, and peptide NTS and PB, which are the primary nuclei. hormones Calca and Adcyap1 in the PB (Additional Next, to determine changes in neuronal activity by pro- file  3: Fig. S6A). The Fos antisense probe detected Fos- longed taste exposure, mice received water or umami positive neurons in the pentylenetetrazole-treated mouse solution ad  libitum for 3  weeks, and Fos FISH assay was hippocampus, but not in the vehicle-treated mouse hip- performed after taste stimulation (Fig.  5). As observed pocampus. Sense probes did not detect the signal in mice in the single taste stimulation (Fig.  4), Fos expression in hippocampi from both treatment groups (Additional the CeA was markedly increased by umami stimulation file 3: Fig. S6B). in prolonged taste exposure mice (p = 0.0008; Fig.  5A). Initially, to investigate the immediate neuronal activ- Interestingly, there was no difference in the ratio of Fos - ity of the tastant, mice were individually housed with positive neurons in the Sst-positive neurons, while the restricted feeding for over an hour and restricted drink- ratio of Fos-positive neurons in the Prkcd-positive neu- ing for 19–21  h before the taste experiment. To assess rons was significantly increased in the Umami group the innate taste response, naïve mice were exposed the (Prkcd, p = 0.0001; Sst, p = 0.2778; Fig.  5B, C). Among water, umami, or bitter solutions (Fig.  3). However, the the higher gustatory nuclei, no difference was observed mice provided with bitter solution did not drink it (water, except for the VTA, unlike the single taste administration 0.37 ± 1.10  g; umami, 0.58 ± 0.11  g; bitter, 0.03 ± 0.01  g; (PB, p = 0.8423; Calca, p = 0.8279, Adcyap1, p = 0.1059; F = 11.29, p = 0.0004, one-way ANOVA; Umami NTS, p = 0.2047; IC, p = 0.2740; Fig.  5D–G, H). Intrigu- 2,22 vs. Water group, p = 0.2188; Umami vs. Bitter group, ingly, the VTA showed a decrease in the Fos-positive p = 0.0004; Bitter vs. Water group, p = 0.0137, Tukey ’s neurons in the prolonged umami administration (VTA, post hoc test). Therefore, we did not perform Fos FISH p = 0.0276; Fig. 5H). These results suggest that prolonged experiments in mice provided with bitter solution exposure to umami taste induces some plastic changes (Fig.  4). The Fos -positive neurons were increased in the in the gustatory circuit, particularly in the CeA, in a cell CeA in umami-stimulated mice compared with water- type-specific manner. stimulated mice (p < 0.0001; Fig.  4A). In addition, we investigated cell type-specific neuronal activity in the Discussion CeA by analyzing Fos and Prkcd- or Sst-double-positive The modification of taste preference by previous taste neurons. The ratios of Fos and Sst double-positive neu- experiences has been studied in animals. In rodents, both rons per Sst-positive neurons in the Umami group was attractive and aversive taste exposure increases intake significantly higher than those in the Water group, while of the exposed taste; exposure to umami in adulthood Fos-positive neurons in the Prkcd-positive neurons was or sweet in the lactation period enhances its palatability comparable between these groups (Sst, p = 0.016398; [14, 34], and exposure to bitter in post-weaning or adult- Prkcd, p = 0.4373; Fig.  4B, C). Next, we performed the hood, or sour in the lactation period reduces its aversive- Fos FISH assay in the CeA upstream gustatory nuclei (PB, ness [10, 12]. Our results are consistent with this body of NTS, VTA, and IC). In the IC, we focused on the area evidence and showed that prolonged exposure to umami between Bregma + 1.1  mm and + 0.6 mm as the umami and bitter in the post-weaning juvenile period also field, because the umami field is between the bitter and increases the preference for the exposed taste. Further- sweet hot fields [5, 17]. In the PB, we also calculated the more, we found that prolonged exposure to umami did Hamada et al. Molecular Brain (2023) 16:28 Page 10 of 16 Taste stimulation Fos FISH Water Sst Prkcd or Fos Adcyap1 + Calca Umami 30 min Th Nos1 DAPI Fos Prkcd CeA B C PB VTA IC NTS CeA BLA DAPI Fos Th DAPI Fos Adcyap1 NTS PB scp DAPI Fos Th DAPI Fos Nos1 VTA IC Fig. 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) * Hamada  et al. Molecular Brain (2023) 16:28 Page 11 of 16 AB C CeA Water WaterUmami WaterUmami Umami Water Water Umami Umami 0 10 20 30 40 0 5 10 15 20 25 (Fos + Sst) / Sst (%) (Fos + Prkcd) / Prkcd (%) PB DE F WaterUmami WaterUmami Water Water Umami Umami 0 5 10 15 0 5 10 15 (Fos + Calca) / Calca (%) (Fos + Adcyap1) / Adcyap1 (%) VTA IC NTS H I 250 140 * 200 20 150 15 100 10 0 0 0 Fig. 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 mm 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, G–I Fos FISH assay in the PB, NTS, VTA, and IC, which are upstream regions of the CeA. Fos‑positive cell counts/1 mm 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) Fos cells / mm F/ os cells mm F/ os cells mm Fos cells / mm Fos cells / mm *** Hamada et al. Molecular Brain (2023) 16:28 Page 12 of 16 CeA A B C Water WaterUmami WaterUmami 200 *** Umami Water Water Umami Umami 0 5 10 15 0 5 10 15 20 25 (Fos + Sst) / Sst (%) (Fos + Prkcd) / Prkcd (%) DE F PB WaterUmami WaterUmami Water Water Umami Umami 0 4 8 12 0 4 8 12 (Fos + Adcyap1) / Adcyap1 (%) (Fos + Calca) / Calca (%) G NTS VTA IC 80 100 0 0 0 Fig. 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 mm 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, G–I Fos FISH assay in the PB, NTS, VTA, and IC, which are upstream regions of the CeA. Fos‑positive cell counts/1 mm 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) F/ os cells mm Fos cells / mm F/ os cells mm F/ os cells mm F/ os cells mm Hamada  et al. Molecular Brain (2023) 16:28 Page 13 of 16 not affect bitter preference, and vice versa for bitter expo - not sweet tastant solution [2]. Furthermore, Kim et  al. sure, which suggests that there is little crossover effect of employed free access to bitter tastant solution after 24 h different taste qualities. In contrast to our results, it has of water deprivation and found that Fos-positive neurons also been reported that bitter exposure during lactation were increased specifically in Prkcd-positive neurons in has no significant effect on bitter ingestion [10]. Fur - the capsular part of the CeA compared with mice pro- thermore, sweet exposure in the post-weaning period vided with neutral-taste water [31]. Collectively, the taste reduces its hedonic valence in adulthood [13]. These lines specificity of the CeA cell-types remains to be ambigu - of evidence suggest that appropriate time window of taste ous. Therefore, in order to clarify not only the respon - experiences may be critical for the increment of taste siveness to various tastants but also the correspondency preference. It would be an interesting future study to of responding cells, here we attempted to consecutive examine whether there is a critical period to induce such recording of neuronal responses to various tastants using experience-dependent changes in taste preference. The calcium imaging, and identified that the Prkcd- and Sst- studies described above mainly focused on preference for positive neuronal populations consisted of both cells the exposed taste, but not other tastes, especially oppo- responding to negative and positive tastants, respectively. site valence tastes such as umami and bitter. We therefore The CeA receives direct inputs from bitter-responsive examined the influence on bitter and umami prefer - neurons in the PB and bitter-responsive neuron hot- ence in the Umami and Bitter groups and showed that spots located in the caudal part of the IC [2, 5], suggest- preference for the unexposed taste is unchanged, which ing that the bitter-responsive neurons of the CeA can be suggests that taste preference is modified in a manner activated by these inputs. Since the IC also possesses a selective to the exposed taste. sweet-responsive neuron hotspot on the rostral side and We used MSG- and MPG-based umami solutions mainly projects to the basolateral amygdala (BLA), it is for the two-bottle tests. The Water group did not show possible that sweet stimulation, at least in part, is indi- a strong umami preference using MSG-based umami rectly transmitted to the CeA through the BLA [5, 17]. In (Fig. 1C, E), while it did using MPG-based umami (Addi- addition, it has been reported that an umami-responsive tional file  3: Fig. S2C, E). The reason for this is unclear. neuron hotspot also exists in the IC between the bitter- One can speculate that there may be some interaction and sweet-responsive neuron hotspots [17], and there between sodium and umami signals. For example, a pre- are direct inputs to the CeA from these areas of the IC vious study demonstrated that some Satb2-positive neu- [36], suggesting that umami information can be trans- rons in PB respond to both sodium chloride and umami mitted to the CeA directly from the umami hotspot of stimuli [16]. These Satb2 neurons enhance taste percep - the IC. Furthermore, the VTA dopamine neurons pro- tion and affect licking behavior. One possibility is that jects directly to the CeA, especially to the medial part of the umami solution containing 100 mM MSG influences the CeA (CeM), where Prkcd-positive neurons are less taste perception and umami intake. Therefore, the slight abundant and Sst-positive neurons are more abundant difference in preference between MSG- and MPG-based [15, 37]. Therefore, it is also possible that umami infor - umami in the Water group may be due to the difference mation is relayed to Sst-positive neurons in the CeA via in the neuronal activities in the PB to umami and sodium VTA dopamine neurons. Interestingly, some dopaminer- signals. The interaction and plasticity mechanisms are gic neurons in the VTA project to the IC, consolidating essential topics for future investigation. aversive taste memory [38]. In order to elucidate through In the present study, we first targeted and investigated which nuclei the information for each taste is relayed to the CeA neurons in response to various tastants because the CeA, further studies in combination with circuit trac- these neurons receive direct and indirect inputs from ing are required. Together, our findings suggest that taste multiple nuclei of the gustatory circuit, and play a criti- stimuli are represented in a more complex manner in the cal role in encoding negative or positive valence. The CeA than previously thought. taste response of the CeA neurons has been investigated It is noteworthy that the ratio of the Fos-positive neu- in rodents by several previous Fos-labeling studies; how- rons in the CeA were high in the Sst-positive neurons ever, it should be noted that each experiment employed in single umami administration, while they were pre- a partially different method of taste stimulation. Otsubo dominant in the Prkcd-positive neurons in prolonged et al. reported that the Fos-like immunoreactivity of neu- umami administration. At least, our calcium imag- rons in the CeA was increased by both forcibly sweet and ing showed that there is a population that responds to umami stimulations after 24-h fasting compared with umami in Prkcd-positive neurons, suggesting that pro- salty stimulation [35]. In contrast, Cai et  al. reported longed umami administration enhanced the activity of that Fos-positive neurons in the CeA were increased by these neurons as well. Because the Prkcd-positive and forcible intraoral infusion of bitter tastant solution but Prkcd-negative (mainly Sst-positive) neurons are both Hamada et al. Molecular Brain (2023) 16:28 Page 14 of 16 inhibitory neurons and form reciprocally connected quinine, the bitter substance used in the present study, microcircuits in the CeA [39, 40], one possible underly- has been identified [48], and it is possible that the sen - ing mechanism is that Sst- and Prkcd-positive neurons sitivity of some of these receptors was changed. Periph- are plastically regulated in a different manner via mutual eral nerves have been reported to sense bitterness and inhibition, resulting in opposite plastic changes. In fact, nutrition and contribute to preference [49]. In addi- Sst- and Prkcd-positive neurons exhibit contradictory tion, it has also been suggested that changes in periph- responses in fear learning and pain-like behavior [41, 42]. eral taste bud structure are accompanied by changes Furthermore, among Sst-positive neurons, different sub - in preference due to taste experience [34]. In the pre- regions within CeA have different plasticity phenotypes sent study, we were unable to examine the peripheral [43]. Another possibility is that the inputs to the Sst- and involvement in changes in taste preference, but this will Prkcd-positive neurons are different, thereby acute and need to be examined in the future. chronic taste experiences have different effects on these Experimental systems such as conditioned taste aver- cell-types. Indeed, excitatory synaptic inputs from the IC sion and conditioned place aversion exist for the mecha- to the lateral and capsular part of the CeA are greater in nism that makes animals dislike what they like, and these Sst-positive neurons [44], whereas PB inputs are larger systems have been widely studied worldwide [2, 3, 50, in Sst-negative neurons in the capsular part of the CeA, 51]. Conversely, there are few experimental systems that but those are larger in Sst-positive neurons in the CeM examine the mechanism of liking something one dislikes, [43]. Also, the VTA dopaminergic neurons project pre- and further research on the mechanism of increased pref- dominantly to the CeM, where Sst-positive neurons are erence by bitter taste experience may lead to the elucida- rich. Therefore, prolonged umami administration may tion of a complementary mechanism. Unfortunately, in cause experiment-related plasticity in the CeA to act on the present study, Fos FISH analysis was not possible for Sst- and Prkcd-positive neurons differentially, resulting bitter taste in the free-moving condition. In future stud- in changes in the balance between these neurons which ies, we would like to examine changes in Fos expression may influence the palatability of umami. To support this patterns associated with single and prolonged changes notion, it is known that the satiety-related peptide hor- in bitter taste exposure by either lowering the concentra- mone cholecystokinin (CCK) is released by umami [45]. tion of bitter taste or by forced drinking. Furthermore, CCK from the peripheral tissue can activate Prkcd-pos- we would like to research changes in umami preference itive neurons in the CeA [46]. These lines of evidence behavior through the inhibition of Prkcd-positive neurons suggest that umami experience is involved in activation by the artificial circuit manipulation of neuronal activity of the CeA Prkcd-positive neurons via the CCK path- during prolonged umami exposure. way. Although the causal relationship between drinking behavior and activity of Prkcd-positive neurons is not Abbreviations clear due to the limitations of our experimental methods, CeA Central amygdala these neurons may intricately regulate umami prefer- NTS Nucleus of the solitary tract PB Parabrachial nucleus ence and drinking control. Intriguingly, a previous study VTA Ventral tegmental area reported that the CeA Prkcd-positive neurons are criti- IC Insular cortex cally involved in chronic alcohol-drinking behavior in AAV Adeno‑associated virus GRIN Gradient index rats [47]. Therefore, one possibility is that the Prkcd-pos- cRNA Complementary RNA itive neurons are involved in experience-dependent plas- FISH Fluorescence in situ hybridization tic changes such as prolonged umami intake and chronic NOS1 N itric oxide synthase 1 Prkcd Protein kinase C delt alcohol drinking. It would be an interesting future study Sst Asomatostatin to examine the molecular mechanisms of the synaptic Th Tyrosine hydroxylase plasticity in the Prkcd-positive neurons and their physi- Calca Calcitonin gene‑related peptide Adcyap1 P ituitary adenylate cyclase‑activating polypeptide ological consequences. FITC Fluorescein isothiocyanate Although the present study focused on the plasticity DIG Digoxigenin to the central nervous system caused by taste experi- SSC Standard saline citrate DAPI 4′,6‑Diamidino ‑2‑phenylindole ences, it is also possible that changes in the periphery BLA Basolateral amygdala influence taste preference. In general, animals avoid bit - CeM Medial part of the CeA ter tastes, but frequent ingestion decreases the avoid-MSG Monosodium glutamate MPG Monopotassium glutamate ance behavior. The decrease in aversiveness may be due CCK Cholecystokinin to fewer bitter taste receptors. The bitter receptor of Hamada  et al. Molecular Brain (2023) 16:28 Page 15 of 16 Funding Supplementary Information This work was supported in part by JSPS Grants‑in‑Aid for Scientific Research The online version contains supplementary material available at https:// doi. [JP19H04062, JP21K18564, and JP22H03542 to AMW; JP19H03324 to TO; org/ 10. 1186/ s13041‑ 023‑ 01017‑x. JP21H05091 and JP20H03339 to ST‑K; JP20K15929 and JP22K06483 to SU; JP16H06276 (AdAMS) to HB and ST‑K; JP17H06312 to HB; JP20K15936 to MN; Additional file 1: Movie S1. Representative calcium imaging movie of JP21K16374 to TN], Core Research for Evolutional Science and Technology Prkcd-cre mice (right) and simultaneously recorded behavioral movie Japan Science and Technology Agency (CREST‑ JST: JPMJCR1751 to AMW and (left). The tastant solution was poured at the timing when the text color is TO), Japan Agency for Medical Research and Development (AMED) Brain reversed. The movie plays at 2× speed. Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS) (JP19dm0207081 to SH and AMW ), JST (Moonshot R and D) (JPMJMS2024 to Additional file 2: Movie S2. Representative calcium imaging movie of AMW ), JST‑Mirai Program (JPMJMI21G6 to ST ‑K), and a Grant ‑in‑Aid for Scien‑ Sst-cre mice (right) and simultaneously recorded behavioral movie (left). tific Research on Innovation Areas grant (JP19H05014 to TO). The tastant solution was poured at the timing when the text color is reversed. The movie plays at 2× speed. Availability of data and materials Additional file 3: Figure S1. Time course of access duration in two ‑bottle All data are available upon request to the corresponding author. test. A–C Access duration to water or umami bottles every 5 min in 2 days two‑bottle tests in Water (A), Umami (B) and Bitter (C) groups. D–F Access Declarations duration to water or bitter bottles every 5 min in 2 days two‑bottle tests in Water (D), Umami (E) and Bitter (F) groups. Data are represented as Ethics approval and consent to participate mean ± SEM. Water group, n = 8; Umami group, n = 9; Bitter group, n = 7. All experimental protocols in this study that included the use of animals were *p < 0.05, **p < 0.01, ***p < 0.001 (Paired t‑test). Figure S2. Preference for approved by the Institutional Animal Care and Use Committee of The Jikei MPG‑based umami in the two ‑bottle test in prolonged taste exposure University (Kashiwa City, Japan) (Approval number 2018‑072, 2019‑010) and mice. A Experimental paradigm of prolonged taste exposure and two‑ Nagoya University (approval number R210154). 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 Consent for publication ratio of the umami intake to the total intake. D Access duration to water Not applicable. or umami bottle. E Access ratio of umami bottle. Each circle represents results from one mouse. Data are represented as mean ± SEM. Water Competing interests group, n = 10; MPG group, n = 10. *p < 0.05, **p < 0.01 (paired t‑test); † †† $ The authors declare no competing interests. p < 0.05, p < 0.01 (one sample t‑test); p < 0.05 ( Welch’s t‑test followed by correction with Bonferroni method). Figure S3. A Schematic of viral injections and lens implantation into the CeA for calcium imaging. B Rep‑ Received: 18 November 2022 Accepted: 3 March 2023 resentative image of GCaMP6f expression and lens probe tract of Prkcd-cre mouse brain. Scale bar, 200 µm. C Implanted lens probe locations of four Prkcd-cre (magenta) and four Sst-cre (light blue) mice. The values indicate anterior–posterior distances from bregma. Figure S4. Heatmaps indicate responses to umami (upper), bitter (middle), and sweet (lower) tastant solution of 3 trials each (A), and average responses of 3 trials for each References tastant (B) in total extracted cell population from Prkcd‑ cre mice (223 1. 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Journal

Molecular Brain – Springer Journals

Published: Mar 11, 2023

Keywords: Gustatory circuit; Umami; Bitter; Amygdala; Taste preference; Plasticity; Calcium imaging

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