Deletion of Trps1 regulatory elements recapitulates postnatal hip joint abnormalities and growth retardation of Trichorhinophalangeal syndrome in mice.

Trichorhinophalangeal syndrome (TRPS) is a genetic disorder caused by point mutations or deletions in the gene-encoding transcription factor TRPS1. TRPS patients display a range of skeletal dysplasias, including reduced jaw size, short stature, and a cone-shaped digit epiphysis. Certain TRPS patients experience early onset coxarthrosis that leads to a devastating drop in their daily activities. The etiologies of congenital skeletal abnormalities of TRPS were revealed through the analysis of Trps1 mutant mouse strains. However, early postnatal lethality in Trps1 knockout mice has hampered the study of postnatal TRPS pathology. Here, through epigenomic analysis we identified two previously uncharacterized candidate gene regulatory regions in the first intron of Trps1. We deleted these regions, either individually or simultaneously, and examined their effects on skeletal morphogenesis. Animals that were deleted individually for either region displayed only modest phenotypes. In contrast, the Trps1Δint/Δint mouse strain with simultaneous deletion of both genomic regions exhibit postnatal growth retardation. This strain displayed delayed secondary ossification center formation in the long bones and misshaped hip joint development that resulted in acetabular dysplasia. Reducing one allele of the Trps1 gene in Trps1Δint mice resulted in medial patellar dislocation that has been observed in some patients with TRPS. Our novel Trps1 hypomorphic strain recapitulates many postnatal pathologies observed in human TRPS patients, thus positioning this strain as a useful animal model to study postnatal TRPS pathogenesis. Our observations also suggest that Trps1 gene expression is regulated through several regulatory elements, thus guaranteeing robust expression maintenance in skeletal cells.

Histological and immunohistochemical studies of the fungiform and the circumvallate papillae through the life stages from 6- to 72-week-old Sprague-Dawley male rats.

Taste sensitivity decreases with age. Therefore, we investigated the histological and immunohistochemical changes in the receptive fields circumvallate papilla (CvP) and fungiform papilla (FfP) to explore the mechanism underlying age-related changes in taste sensitivity in 6- to 72-week-old rats. We analyzed papilla size, the thickness of the keratin layer of the papilla and stratified squamous epithelium, taste bud size, the keratin layer around the taste pores in the CvP and FfP, and the number and distribution of taste buds in the CvP coronal section. We further assessed the expression of marker proteins for Type II and III cells, phospholipase C subtype beta 2 (PLCß2), and synaptosomal-associated protein 25 (SNAP-25). The cellular activity of these taste cells was examined through co-localization with the senescence cell marker protein-30 (SMP30). There were no differences in the number of taste bud sections in the CvP among the age groups. However, the size of the CvP increased and the density of the taste bud area in the CvP area decreased with increasing age. In contrast, the number of cells with co-expression of SMP30, PLCß2, and SNAP-25 decreased with age. Furthermore, the morphological structures of the CvP, FfP, and taste buds in these regions changed with age, but not the overall taste bud number in the CvP coronal section. The decrease in cell count with co-expression of SMP30 and PLCß2, or SNAP-25 may indicate reduced cellular functions of taste cells with aging.

Modeling of intramembranous ossification using human pluripotent stem cell-derived paraxial mesoderm derivatives.

Vertebrates form their skeletal tissues from three distinct origins (the neural crest, paraxial mesoderm, and lateral plate mesoderm) through two distinct modes of ossification (intramembranous and endochondral ossification). Since the paraxial mesoderm generates both intramembranous and endochondral bones, it is thought to give rise to both osteoprogenitors and osteo-chondroprogenitors. However, it remains unclear what directs the paraxial mesoderm-derived cells toward these different fates in distinct skeletal elements during human skeletal development. To answer this question, we need experimental systems that recapitulate paraxial mesoderm-mediated intramembranous and endochondral ossification processes. In this study, we aimed to develop a human pluripotent stem cell (hPSC)-based system that models the human intramembranous ossification process. We found that spheroid culture of the hPSC-derived paraxial mesoderm derivatives generates osteoprogenitors or osteo-chondroprogenitors depending on stimuli. The former induced intramembranous ossification, and the latter endochondral ossification, in mouse renal capsules. Transcriptional profiling supported the notion that bone signatures were enriched in the intramembranous bone-like tissues. Thus, we developed a system that recapitulates intramembranous ossification, and that enables the induction of two distinct modes of ossification by controlling the cell fate of the hPSC-derived paraxial mesoderm derivatives.

The flavor-enhancing action of glutamate and its mechanism involving the notion of kokumi.

The sodium salt of glutamic acid, or monosodium glutamate (MSG), has two effects in foods: one is to induce a unique taste called umami, which is one of the five basic tastes, and the other is to make food palatable (i.e., flavor-enhancing or seasoning effects). However, the mechanism behind how MSG makes food more palatable remains poorly understood, although many food scientists seem to believe that the umami taste itself plays an important role. Here, we propose an alternative notion regarding this topic based on previous and recent studies. When added to complex food compositions, MSG facilitates the binding of existing kokumi substances to kokumi receptors. In turn, these bound kokumi substances enhance the intensity of umami, sweet, salty, and fatty tastes, resulting in increased palatability accompanied by kokumi flavor, such as thickness, mouthfulness, and continuity. The requisite for sufficient palatability and kokumi flavor is a good balance of umami and kokumi substances. This framework gives a scientifically useful background for providing newly developed foods, including cultured meat and plant-based meat substitutes, with good taste characteristics.

Pregnane X receptor (PXR) represses osteoblast differentiation through repression of the Hedgehog signaling pathway.

The pregnane X receptor (PXR, NR1I2) belongs to the nuclear receptor family and functions as a xenobiotic and endobiotic sensor by binding to various molecules through its relatively flexible ligand-binding domain. In addition to these well-known canonical roles, we previously reported that PXR represses osteoblast differentiation. However, the mechanisms underlying the PXR-mediated repression of osteoblast differentiation remains unknown. In this study, we analyzed the changes in global gene expression profiles induced by PXR in calvarial osteoblasts cultured in standard fetal bovine serum (in which PXR induces repression of differentiation), and in those cultured in charcoal-stripped fetal bovine serum (in which PXR does not induce repression of differentiation). The comparison revealed that PXR attenuated the Hedgehog-mediated signaling in culture conditions that induced PXR-mediated repression of differentiation. Real-time PCR analysis showed that PXR repressed the Hedgehog signaling-induced genes such as Gli1 and Hhip, and conversely induced the Hedgehog signaling-repressed genes such as Cdon, Boc, and Gas1. Activation of Smo-mediated signaling in osteoblasts following treatment with a Smo agonist (SAG) significantly restored Gli-mediated transcriptional activity and osteoblast differentiation. Our results demonstrate the osteoblast-autonomous effects of PXR and identify a novel regulation of Hedgehog signaling by nuclear receptors.

The Effects of Roux-en-Y Gastric Bypass on Glucose- vs. Fructose-Associated Conditioned Flavor Preference.

In rodents, repeated single-bottle exposures to distinctly flavored isocaloric glucose and fructose solutions, two sugars with different metabolic pathways, eventually lead to a preference for the former. Because Roux-en-Y gastric bypass (RYGB) surgery decreases preference for and intake of sugar solutions in rats, we tested whether RYGB would curtail the conditioning of a preference for a glucose-paired vs. fructose-paired flavor. RYGB (♂ n=11; ♀ n=10) and sham-operated (SHAM; ♂ n=9; ♀ n=10) rats were trained with a single bottle (30 min/day) containing 8% glucose solution flavored with either 0.05% grape or cherry Kool-Aid (Glu/CSG) or 8% fructose solution with the alternative Kool-Aid flavor (Fru/CSF) in an alternating fashion for 8 days. To determine baseline preferences, a 4-day 30-min two-bottle test was used to assess preference for Glu/CSG vs. Fru/CSF before training. After training, 2-day 30-min two-bottle tests assessed preference for the a) Glu/CSG (CSG-flavored 8% glucose solution) vs Fru/CSF (CSF-flavored 8% fructose solution), b) CSG- vs. CSF-flavored mixture of 4% glucose & 4% fructose (isocaloric), c) CSG- vs. CSF-flavored 0.2% saccharin ("sweet", no calories), and d) CSG- vs. CSF-flavored water. During training, only male SHAM rats demonstrated progressively increased intake of Glu/CSG over Fru/CSF, and female SHAM rats displayed a trend. RYGB eliminated any difference in single-bottle intake of these solutions during training, regardless of sex. Like their male and female SHAM counterparts, male RYGB rats displayed a conditioned preference for the CSG-associated stimulus in Tests 1-3. Although female RYGB rats displayed acquisition of the conditioned flavor preference in Test 1, unlike the other groups, when the differential sugar cue between the two solutions was removed in Tests 2 and 3, female rats did not display a CSG preference. When the sugar and sweetener cues were both removed on Test 4, all groups displayed some generalization decrement. Thus, RYGB does not compromise the ability of rats to learn and express a glucose- vs. fructose-associated conditioned flavor preference when the exact CS used in training is presented in testing. The mechanistic basis for the sex difference in the effect of RYGB on the generalization decrement observed in this type of flavor preference learning warrants further study.

Senescence-accelerated mouse prone 8 (SAMP8) mice exhibit reduced entoconid in the lower second molar.

OBJECTIVE: The position and size of the major cusps in mammalian molars are arranged in a characteristic pattern that depends on taxonomy. In humans, the cusp which locates distally within each molar is smaller than the mesially located cusp, which is referred to as "distal reduction". Although this concept has been well-recognized, it is still unclear how this reduction occurs. Current study examined whether senescence-accelerating mouse prone 8 (SAMP8) mice could be a possible animal model for studying how the mammalian molar cusp size is determined. DESIGN: SAMP8 mice were compared with parental control (SAMR1) mice. Microcomputed tomography images of young and aged mice were captured to observe molar cusp morphologies. Cusp height from cement-enamel junction and mesio-distal length of molars were measured. The statistical comparison of the measurements was performed by Mann-Whitney U test. RESULTS: SAMP8 mice showed reduced development of the disto-lingual cusp (entoconid) of lower second molar when compared with SAMR1 mice. The enamel thickness and structure was disturbed at entoconid, and aged SAMP8 mice displayed severe wear of the entoconid in lower second molar. These phenotypes were observed on both sides of the lower second molar. CONCLUSIONS: In addition to the general senescence phenotype observed in SAMP8 mice, this strain may genetically possess molar cusp phenotypes which is determined prenatally. Further, SAMP8 mice would be a potential model strain to study the genetic causes of the distal reduction of molar cusp size.

Neural Isolation of the Olfactory Bulbs Severely Impairs Taste-Guided Behavior to Normally Preferred, But Not Avoided, Stimuli.

Here we systematically tested the hypothesis that motivated behavioral responsiveness to preferred and avoided taste compounds is relatively independent of the olfactory system in mice whose olfactory bulbs (main and accessory) were surgically disconnected from the rest of the brain [bulbotomy (BULBx)]. BULBx was confirmed histologically as well as functionally with the buried food test. In brief access taste tests, animals received 10-s trials of various concentrations of a taste compound delivered quasirandomly. BULBx C57BL/6 (B6) mice displayed severely blunted concentration-dependent licking for the disaccharide sucrose, the maltodextrin Maltrin, and the fat emulsion Intralipid relative to their sham-operated controls (SHAM B6). Licking for the noncaloric sweetener saccharin was also blunted by bulbotomy, but less so. As expected, mice lacking a functional "sweet" receptor [T1R2+T1R3 knockout (KO)] displayed concentration-dependent responsiveness to Maltrin and severely attenuated licking to sucrose. Like in B6 mice, responsiveness to both stimuli was exceptionally curtailed by bulbotomy. In contrast to these deficits in taste-guided behavior for unconditionally preferred stimuli, BULBx in B6 and KO mice did not alter concentration-dependent decreases for the representative avoided stimuli quinine and citric acid. Nor did it temper the intake of and preference for high concentrations of affectively positive stimuli when presented in long-term (23-h) two-bottle tests, demonstrating that the surgery does not lead to a generalized motivational deficit. Collectively, these behavioral results demonstrate that specific aspects of taste-guided ingestive motivation are profoundly disturbed by eliminating the anatomic connections between the main/accessory olfactory bulbs and the rest of the brain.

The Basolateral Nucleus of the Amygdala Executes the Parallel Processes of Avoidance and Palatability in the Retrieval of Conditioned Taste Aversion in Male Rats.

Conditioned taste aversion (CTA) is an essential behavior for animal survival. Conditioned animals show avoidance and decreased palatability to a conditioned stimulus (CS) on CTA retrieval. In this study, we aimed to determine whether the basolateral nucleus of the amygdala (BLA) is involved in CTA retrieval and whether avoidance and palatability in CTA retrieval are processed in the BLA. We developed an experimental chamber for time-course analysis of the behavior to approach a spout and lick a CS. In this experimental chamber, we analyzed the behavior of male rats following microinjections of GABAA receptor agonist muscimol or saline into the BLA. The rats showed two types of approach behavior: they either (1) approached and licked the spout or (2) approached but did not lick the spout. Muscimol injection into the BLA decreased the frequency of the latter type of approach behavior, indicating that BLA inactivation reduced avoidance to the CS. The muscimol injection into the BLA also significantly increased the consumption of the CS. Lick microstructure analysis demonstrated that intra-BLA muscimol significantly increased licking burst number and size, indicating that BLA inactivation attenuated aversion to the CS as large burst licking is an indicator of high palatability. These results suggest that the increase in CS consumption with intra-BLA muscimol injection was due to alterations in approach and aversive responses to the CS. Therefore, we conclude that the BLA plays an essential role in CTA retrieval by parallel processing of avoidance and palatability.

T1R2+T1R3-independent chemosensory inputs contributing to behavioral discrimination of sugars in mice.

Simple sugars are thought to elicit a unitary sensation, principally via the "sweet" taste receptor type 1 taste receptor (T1R)2+T1R3, yet we previously found that rats with experience consuming two metabolically distinct sugars, glucose and fructose, subsequently licked more for glucose than fructose, even when postingestive influences were abated. The results pointed to the existence of an orosensory receptor that binds one sugar but not the other and whose signal is channeled into neural circuits that motivate ingestion. Here we sought to determine the chemosensory nature of this signal. First, we assessed whether T1R2 and/or T1R3 are necessary to acquire this behavioral discrimination, replicating our rat study in T1R2+T1R3 double-knockout (KO) mice and their wild-type counterparts as well as in two common mouse strains that vary in their sensitivity to sweeteners [C57BL/6 (B6) and 129X1/SvJ (129)]. These studies showed that extensive exposure to multiple concentrations of glucose and fructose in daily one-bottle 30-min sessions enhanced lick responses for glucose over fructose in brief-access tests. This was true even for KO mice that lacked the canonical "sweet" taste receptor. Surgical disconnection of olfactory inputs to the forebrain (bulbotomy) in B6 mice severely disrupted the ability to express this experience-dependent sugar discrimination. Importantly, these bulbotomized B6 mice exhibited severely blunted responsiveness to both sugars relative to water in brief-access lick tests, despite the fact that they have intact T1R2+T1R3 receptors. The results highlight the importance of other sources of chemosensory and postingestive inputs in shaping and maintaining "hardwired" responses to sugar.

Short-Term Exposure to a Calorically Dense Diet Alters Taste-Evoked Responses in the Chorda Tympani Nerve, But Not Unconditioned Lick Responses to Sucrose.

Upon presentation of a calorically dense diet, rats display hyperphagia driven by increased meal size. The increased meal size and hyperphagia are most robust across the first several days of diet exposure before changes in body weight are evident, thus it is plausible that one of the factors that drives the hyperphagia may be enhanced orosensory responsivity. Here, electrophysiological responses to an array of taste stimuli were recorded from the chorda tympani nerve, a branch of the facial nerve that innervates taste receptors in the anterior tongue, of rats presented a high-energy (45% fat and 17% sucrose) diet for 3 days. Responses in the high-energy diet group were significantly higher for 0.01, 0.03, 0.06 and 0.3 M sucrose; 0.05 M Na-saccharin; and 0.01 M quinine compared with those of chow-fed controls. Another cohort of animals was tested in 30-min brief-access taste sessions (10-s trials) to a sucrose concentration series across the first 6 days of high-energy diet presentation. Both groups responded in a concentration-dependent manner. No significant group differences in unconditioned licking or trials initiated were revealed. Results from a third cohort of rats showed that responses to sucrose in a brief-access taste test also remained largely unchanged as a function of 3-day access to a sucrose solution. Taken together, these findings suggest that 3 days of high-energy diet exposure results in alterations to peripheral gustatory signaling yet these changes do not necessarily generalize to changes in responsiveness to sucrose, as least as measured in this procedure.

Taste preference changes throughout different life stages in male rats.

Taste preference, a key component of food choice, changes with aging. However, it remains unclear how this occurs. To determine differences in taste preference between rats in different life stages, we examined the consumption of taste solutions and water using a two-bottle test. Male Sprague-Dawley rats of different ages were used: juvenile (3-6 weeks), young adult (8-11 weeks), adult (17-20 weeks), middle-aged (34-37 weeks), and old-aged (69-72 weeks). The intakes of the high and low concentration solutions presented simultaneously were measured. We observed that the old-aged group had lower preference ratios for 0.3 M sucrose and 0.1 M MSG in comparison with other groups. The preference ratio for 0.03 mM QHCl was higher in the middle-aged group than in the three younger groups and higher in the old-aged group than the juvenile group. The taste preferences for HCl and NaCl did not significantly differ among the age groups. The old-aged group tended to prefer high concentrations of sucrose, QHCl, NaCl, and MSG to low concentrations, indicating age-related decline in taste sensitivity. We also aimed to investigate differences between life stages in the electrophysiological responses of the chorda tympani nerve, one of the peripheral gustatory nerves, to taste stimuli. The electrophysiological recordings showed that aging did not alter the function of the chorda tympani nerve. This study showed that aging induced alterations in taste preference. It is likely that these alterations are a result of functional changes in other peripheral taste nerves, the gastrointestinal system, or the central nervous system.

Polymer-brush-afforded SPIO Nanoparticles Show a Unique Biodistribution and MR Imaging Contrast in Mouse Organs.

INTRODUCTION: To investigate the biodistribution and retention properties of the new super paramagnetic iron oxide (new SPIO: mean hydrodynamic diameter, 100 nm) nanoparticles, which have concentrated polymer brushes in the outer shell and are difficult for phagocytes to absorb, and to compare the new SPIO with clinically approved SPIO (Resovist: mean hydrodynamic diameter, 57 nm). MATERIALS AND METHODS: 16 male C57BL/6N mice were divided in two groups according to the administered SPIO (n = 8 for each group; intravenous injection does, 0.1 ml). In vivo magnetic resonance imaging (MRI) was performed before and one hour, one day, one week and four weeks after SPIO administration by two dimensional-the fast low angle shot (2D-FLASH) sequence at 11.7T. Ex vivo high-resolution images of fixed organs were also obtained by (2D-FLASH). After the ex vivo MRI, organs were sectioned and evaluated histologically to confirm the biodistribution of each particle precisely. RESULTS: The new SPIO was taken up in small amounts by liver Kupffer cells and showed a unique in vivo MRI contrast pattern in the kidneys, where the signal intensity decreased substantially in the boundaries between cortex and outer medulla and between outer and inner medulla. We found many round dark spots in the cortex by ex vivo MRI in both groups. Resovist could be detected almost in the cortex. The shapes of the dark spots were similar to those observed in the new SPIO group. Transmission electron microscopy revealed that Resovist and the new SPIO accumulated in different cells of glomeruli, that is, endothelial and mesangial cells, respectively. CONCLUSION: The new SPIO was taken up in small amounts by liver tissue and showed a unique MRI contrast pattern in the kidney. The SPIO were found in the mesangial cells of renal corpuscles. Our results indicate that the new SPIO may be potentially be used as a new contrast agent for evaluation of kidney function as well as immunune function.

c-Fos expression in the parabrachial nucleus following intraoral bitter stimulation in the rat with dietary-induced zinc deficiency.

Zinc deficiency causes various symptoms including taste disorders. In the present study, changes in expression of c-Fos immunoreactivity in neurons of the parabrachial nucleus (PBN), one of the relay nuclei for transmission of gustatory information, after bitter stimulation to the dorsal surface of the tongue were examined in zinc-deficient rats. Experimental zinc-deficient animals were created by feeding a low-zinc diet for 4weeks, and showed the following symptoms of zinc deficiency: low body weight, low serum zinc content and behavioral changes to avoid bitter stimulation. In normal control animals, intraoral application of 1mM quinine caused increased numbers of c-Fos-immunoreactive (c-Fos-IR) neurons in the external lateral subnucleus and external medial subnucleus of the PBN (elPBN and emPBN, respectively) compared with application of distilled water. However, in the zinc-deficient animals, the numbers of c-Fos-IR neurons in the elPBN and emPBN did not differ significantly between application of quinine and distilled water. After feeding the zinc-deficient animals a normal diet for 4weeks, the symptoms of zinc deficiency recovered, and the expression of c-Fos-IR neurons following intraoral bitter stimulation became identical to that in the normal control animals. The present results indicate that dietary zinc deficiency causes alterations to neuronal activities in the gustatory neural circuit, and that these neuronal alterations can be reversed by changing to a normal diet.

11.7 T MR Imaging Revealed Dilatation of Virchow-Robin Spaces within Hippocampus in Maternally Lipopolysaccharide-exposed Rats.

PURPOSE: 11.7 Tesla MRI was examined to detect Virchow-Robin spaces (VRSs) smaller than 100 µm in the rat brain. The effects of maternal exposure to lipopolysaccharide (LPS) were evaluated on basis of the number of dilated VRSs in the offspring rat brain. METHODS: T2-weighted MRI with an in-plane resolution up to 78 µm (repetition time = 5000 ms, echo time = 35 ms, slice thickness = 250 µm, imaging plane, coronal) was applied to identify VRSs. The dilated VRSs were counted in the rat brain at 5 and 10 weeks of age. The dams of half the number in each group were treated with LPS during pregnancy, and the remaining half was employed as control. LPS injection in gestation period was used to simulate maternal infections, the method of which was widely accepted as a rat model inducing neuropsychiatric disorders in the offspring. Effects of LPS exposure on the offspring rat brain were statistically investigated. RESULTS: VRSs as small as 78 µm were successfully detected by the ultra high-field MRI. All dilated VRSs were observed within lacunosum molecular layer of hippocampus, and molecular and granular layers of dentate gyrus around hippocampal fissure. In juvenile rats (5 weeks of age), the number of dilated VRSs was significantly increased in the prenatal LPS exposed rat brain (12.9 ± 2.4, n = 7) than in the control (5.3 ± 1.5, n = 7, P < 0.05), while in young adult rats (10 weeks of age), there was no significant difference in the number between the prenatal LPS exposed rat brain (3.6 ± 0.9, n = 5) and the control (2.6 ± 0.4, n = 5). CONCLUSION: The results of the present study suggested that maternal infection might cause dilatation of VRSs through neural damages especially in the dentate gyrus of the offspring rats. Thus, ultra high-field MRI can offer a promising diagnostic tool capable of determining the location of neonatal brain damage caused by maternal infections.

In vivo magnetic resonance imaging at 11.7 Tesla visualized the effects of neonatal transection of infraorbital nerve upon primary and secondary trigeminal pathways in rats.

Using 11.7T ultra high-field T2-weighted MRI, the present study aimed to investigate pathological changes of primary and secondary trigeminal pathways following neonatal transection of infraorbital nerve in rats. The trigeminal pathways consist of spinal trigeminal tract, trigeminal sensory nuclear complex, medial lemniscus, ventromedial portion of external medullary lamina and ventral posterior nucleus of thalamus. By selecting optimum parameters of MRI such as repetition time, echo time, and slice orientation, this study visualized the trigeminal pathways in rats without any contrast agents. Pathological changes due to the nerve transection were found at 8 weeks of age as a marked reduction of the areas of the trigeminal pathways connecting from the injured nerve. In addition, T2-weighted MR images of the trigeminal nerve trunk and the spinal trigeminal tract suggest a communication of CSF through the trigeminal nerve between the inside and outside of the brain stem. These results support the utility of ultra high-field MRI system for noninvasive assessment of effects of trigeminal nerve injury upon the trigeminal pathways.

Activation of efferents from the basolateral amygdala during the retrieval of conditioned taste aversion.

The basolateral amygdala (BLA) is critical in the retrieval of conditioned taste aversion (CTA). Although BLA neurons have axonal connections with several brain regions, it is unclear which efferent pathways are functional in CTA. The present study investigated the involvement of efferents from BLA in CTA retrieval with manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI). Rats receiving intraoral saccharin infusion paired with intraperitoneal administration of lithium chloride (LiCl) were presented with saccharin (C-S and BC-S groups) or water (C-W group) on the test day. The BC-S group was administered with LiCl 15 min before saccharin presentation on the conditioning day (backward conditioning, BC). Another two groups were injected with saline (S-S and S-W groups) instead of LiCl. On the test day, 50 nL of 40-mM manganese chloride (MnCl2) was injected into BLA before the intraoral fluid infusion. Using MRI, we analyzed Mn(2+) movements, which indicated the activation of efferent neurons. The C-S group showed the highest activities in several efferents from BLA. Of them, the activities of the efferents to the nucleus accumbens core (NAcC), the anterior part of the bed nucleus of the stria terminalis (aBNST), and the central amygdala (CeA) were larger in the C-S group than in the Q group, which was presented with a normally aversive quinine solution. Although rats equivalently rejected conditioned aversive saccharin and quinine, the aversive responses in the C-S group, and not the Q group, were due to CTA retrieval. Therefore, our results indicated that BLA efferents to NAcC, aBNST, and CeA were specifically activated during CTA retrieval, suggesting that these efferents are key components in the neural mechanisms of CTA.

Hedonics of taste influence the gastric emptying in rats.

It is known that taste sensation plays important roles in various functions including appetite, nutriental choices, food intake and digestion. To study the modulation of digestive functions by taste stimulation, we measured the gastric emptying of experimental foods with appetitive or aversive tastes in Wistar male rats. Rats were randomly divided into 3 groups: each group was trained to eat a mash mixture (8 g) made up with powdered food (4 g) and a solution (4 g) for 30 min per day. The solution was either distilled water, saccharin solution as a palatable taste, or quinine solution as an aversive taste. On the test day, the contents of the stomach of each rat were measured before and 30, 150, or 300 min after the start of eating the mash. The results showed that the food output from the stomach was increased by the palatable mash and was decreased by the aversive mash in comparison with non-adulterated mash 150 min after eating. There were no significant differences in gastric emptying among the 3 groups after deafferentation of the two peripheral taste nerve branches, the chorda tympani and glossopharyngeal nerves, or after intragastric infusions of each mash. After the establishment of conditioned taste aversions to saccharin, saccharin mash became ineffective in increasing gastric emptying. These results show that hedonically positive tastes increase, but hedonically negative tastes decrease gastric function in terms of gastric emptying.