Anhedonic-like traits and lack of affective deficits in 18-month-old C57BL/6 mice: Implications for modeling elderly depression

The prevalence of depression increases with aging. We hypothesized that like humans, old animals exhibit anhedonic-like behavior, along with signs of behavioral despair. In rodents, anhedonia, a reduced sensitivity to reward, which is listed as a core feature of major depression in the DSM-IVR, can be measured by a decrease in intake of and preference for sweet solutions. Here, sucrose intake, forced swimming, immobility in the modified tail suspension test, novelty exploration, grooming, anxiety and locomotor activity were compared in naïve 3- and 18-month-old male C57BL/6 mice. The absolute amounts and the ratio of consumed 1% sucrose solution to water intake was significantly smaller in 18-month-old mice than in 3-month-old mice. The consumption of 5%-sucrose solution requiring high levels of drinking effort, novelty exploration in two setups and grooming behavior in the splash test were reduced in older animals. Analysis of other behaviors suggested that the above-mentioned signs of anhedonic-like traits were unlikely to be attributable to the potential effect of aging on metabolic needs for water, taste perception, motor capabilities or the induction of essential anxiety and neophobia. A 4-week treatment with the antidepressant imipramine (7 mg/kg/day) or dimebon, a compound with suggested neuroprotective proneurogenic properties (1 mg/kg/day) restored sucrose intake and preference in 18-month-old mice. Meanwhile, young and old mice showed no differences in the parameters of behavioral despair evaluated in the forced swim and modified tail suspension tests. Thus, the behavioral profile of aged mice parallels that of humans with elderly depression, in whom the symptoms of hedonic deficits typically outweigh affective disturbances. The assessment of anhedonic-like traits with the sucrose preference test in 18-month-old mice will be useful in preclinical studies of elderly depression.     

Leptin modulates behavioral responses to sweet substances by influencing peripheral taste structures

Leptin is a hormone that regulates body weight homeostasis mainly via the hypothalamic functional leptin receptor Ob-Rb. Recently, we proposed that the taste organ is a new peripheral target for leptin. Leptin selectively inhibits mouse taste cell responses to sweet substances and thereby may act as a sweet taste modulator. The present study further investigated leptin action on the taste system by examining expression of Ob-Rb in taste cells and behavioral responses to sweet substances in leptin-deficient ob/ob, and Ob-Rb-deficient db/db mice and their normal litter mates. RT-PCR analysis showed that Ob-Rb was expressed in taste cells in all strains tested. The db/db mice, however, had a RT-PCR product containing an abnormal db insertion that leads to an impaired shorter intracellular domain. In situ hybridization analysis showed that the hybridization signals for normal Ob-Rb mRNA were detected in taste cells in lean and ob/ob mice but not in db/db mice. Two different behavioral tests, one using sweet-bitter mixtures as taste stimuli and the other a conditioned taste aversion paradigm, demonstrated that responses to sucrose and saccharin were significantly decreased after ip injection of leptin in ob/ob and normal littermates, but not in db/db mice. These results suggest that leptin suppresses behavioral responses to sweet substances through its action on Ob-Rb in taste cells. Such taste modulation by leptin may be involved in regulation for food intake.     

Human taste bud density across adult age groups

Some of the subjective variability attributed to taste experience could be related to wide variations of taste bud density. Studies of taste perception show a direct relationship between sensation and the number of receptors. Taste bud densities are quantified in this study using light microscopy to reconstruct two regions of 18 human cadaver tongues. Specimens came from male and female cadavers representing three age groups: young adults, middle-aged adults, and older adults. The results show a range of more than 100-fold in taste bud density that is evenly distributed among age groups and sexes. The disparity is not attributable to the state of health of the adults prior to death, and it is corroborated in the literature. Differences in taste bud density that extend across age groups probably confound some inferences about the effects of aging on taste sensitivity that are derived from cross-sectional studies of human populations. It is not clear from the data whether or not human taste bud density in individuals and in populations is stable or changing with time.     

On the presence of nonfunctional uterine estrogen receptors in middle-aged and old C57BL/6J mice

The nuclear binding kinetics of uterine 17 beta-estradiol (E2) receptors (UER) were studied throughout aging in intact and castrated (OVX) mice. When compared to young animals, 15- to 18-month-old mice showed a significant reduction in their total cytosolic (0.526 vs. 0.405 pmol/uterus; P less than 0.05) and nuclear (0.37 vs. 0.16 pmol/uterus; P less than 0.01) UER content, whereas the affinity (Ka) for estrogens remained constant (0.8-1.6 X 10(9) M-1). This age-related decrease in UER was preceded by a blunted and retarded nuclear binding of UER at 10-14 months of age, which was further accentuated after transition from perimenopause. Ovariectomy (OVX), whether performed neonatally or in adulthood, reduced the total concentration of cytosolic and nuclear UER in each age group studied, but did not prevent this reduced nuclear binding observed in middleaged mice. However, when standardized per tissue protein, the mean number of cytosolic UER from young and middle-aged, but not old, mice was reduced by 50% after neonatal OVX (176.5, 178.4, and 218.8 fmol/mg protein, respectively), whereas it remained unchanged when OVX was performed in adulthood and the animals subsequently studied at peri- and postmenopausal ages (326.3 and 283.3 fmol/mg protein, respectively). Daily administration of a physiological dose of E2 for 7 days to OVX mice of each age group induced maximal synthesis of UER in young animals, but not in peri- and postmenopausal ones; in peri- and postmenopausal animals, this was paralleled by reduced uterotropic responses despite similar increments in plasma E2. These results suggest an age-related, gonad-independent decline in the number of functional UER early in reproductive aging.     

Leptin as a modulator of sweet taste sensitivities in mice

Leptin acts as a potent inhibitory factor against obesity by regulating energy expenditure, food intake, and adiposity. The obese diabetic db/db mouse, which has defects in leptin receptor, displays enhanced neural responses and elevated behavioral preference to sweet stimuli. Here, we show the effects of leptin on the peripheral taste system. An administration of leptin into lean mice suppressed responses of peripheral taste nerves (chorda tympani and glossopharyngeal) to sweet substances (sucrose and saccharin) without affecting responses to sour, salty, and bitter substances. Whole-cell patch-clamp recordings of activities of taste receptor cells isolated from circumvallate papillae (innervated by the glossopharyngeal nerve) demonstrated that leptin activated outward K(+) currents, which resulted in hyperpolarization of taste cells. The db/db mouse with impaired leptin receptors showed no such leptin suppression. Taste tissue (circumvallate papilla) of lean mice expressed leptin-receptor mRNA and some of the taste cells exhibited immunoreactivities to antibodies of the leptin receptor. Taken together, these observations suggest that the taste organ is a peripheral target for leptin, and that leptin may be a sweet-sensing modulator (suppressor) that may take part in regulation of food intake. Defects in this leptin suppression system in db/db mice may lead to their enhanced peripheral neural responses and enhanced behavioral preferences for sweet substances.     

Sweet taste and obesity

For more than 50 years, there has been evidence for greater consumption of sweet- foods in overweight humans and animals, relative to those that have a normal weight. Furthermore, it has long been suggested that energy deficit resulting from dieting, while moving the individual from a higher weight set point, would result in heightened susceptibility to palatable tastants, namely to sweet tastants. This was the motivation behind the first studies comparing sweet taste perception between individuals with obesity and those of a normal weight. These studies, using direct measures of taste, have been characterized by significant methodological heterogeneity, contributing towards variability in results and conclusions. Nevertheless, some of these findings have been used to support the theory that patients with obesity have decreased taste perception, particularly for sweet tastants. A similar hypothesis has been proposed regarding evidence for reduced brain dopamine receptors in obesity and, in both cases, it is proposed that increased food consumption, and associated weight gain, result from the need to increase sensory and brain stimulation. However, the available literature is not conclusive on the association between obesity and reduced sweet taste perception, with both negative and contradictory findings in comparisons between individuals with obesity and normal weight control subjects, as well as within-subject comparisons before and after bariatric surgery. Nevertheless, following either Roux-en-Y gastric bypass or sleeve gastrectomy, there is evidence of changes in taste perception, particularly for reward-related measures of sweet tastants, that should be further tested and confirmed in large samples, using consensual methodology.     

Endocannabinoids selectively enhance sweet taste

Endocannabinoids such as anandamide [N-arachidonoylethanolamine (AEA)] and 2-arachidonoyl glycerol (2-AG) are known orexigenic mediators that act via CB₁ receptors in hypothalamus and limbic forebrain to induce appetite and stimulate food intake. Circulating endocannabinoid levels inversely correlate with plasma levels of leptin, an anorexigenic mediator that reduces food intake by acting on hypothalamic receptors. Recently, taste has been found to be a peripheral target of leptin. Leptin selectively suppresses sweet taste responses in wild-type mice but not in leptin receptor-deficient db/db mice. Here, we show that endocannabinoids oppose the action of leptin to act as enhancers of sweet taste. We found that administration of AEA or 2-AG increases gustatory nerve responses to sweeteners in a concentration-dependent manner without affecting responses to salty, sour, bitter, and umami compounds. The cannabinoids increase behavioral responses to sweet-bitter mixtures and electrophysiological responses of taste receptor cells to sweet compounds. Mice genetically lacking CB₁ receptors show no enhancement by endocannnabinoids of sweet taste responses at cellular, nerve, or behavioral levels. In addition, the effects of endocannabinoids on sweet taste responses of taste cells are diminished by AM251, a CB₁ receptor antagonist, but not by AM630, a CB₂ receptor antagonist. Immunohistochemistry shows that CB₁ receptors are expressed in type II taste cells that also express the T1r3 sweet taste receptor component. Taken together, these observations suggest that the taste organ is a peripheral target of endocannabinoids. Reciprocal regulation of peripheral sweet taste reception by endocannabinoids and leptin may contribute to their opposing actions on food intake and play an important role in regulating energy homeostasis.     

Gut-expressed gustducin and taste receptors regulate secretion of glucagon-like peptide-1

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express alpha-gustducin. Ingestion of glucose by alpha-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from alpha-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses alpha-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin. We conclude that L cells of the gut "taste" glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut "taste cells" may provide an important treatment for obesity, diabetes and abnormal gut motility.     

Single Lgr5- or Lgr6-expressing taste stem/progenitor cells generate taste bud cells ex vivo

Leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) and its homologs (e.g., Lgr6) mark adult stem cells in multiple tissues. Recently, we and others have shown that Lgr5 marks adult taste stem/progenitor cells in posterior tongue. However, the regenerative potential of Lgr5-expressing (Lgr5⁺) cells and the identity of adult taste stem/progenitor cells that regenerate taste tissue in anterior tongue remain elusive. In the present work, we describe a culture system in which single isolated Lgr5⁺ or Lgr6⁺ cells from taste tissue can generate continuously expanding 3D structures (“organoids”). Many cells within these taste organoids were cycling and positive for proliferative cell markers, cytokeratin K5 and Sox2, and incorporated 5-bromo-2’-deoxyuridine. Importantly, mature taste receptor cells that express gustducin, carbonic anhydrase 4, taste receptor type 1 member 3, nucleoside triphosphate diphosphohydrolase-2, or cytokeratin K8 were present in the taste organoids. Using calcium imaging assays, we found that cells grown out from taste organoids derived from isolated Lgr5⁺ cells were functional and responded to tastants in a dose-dependent manner. Genetic lineage tracing showed that Lgr6⁺ cells gave rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR data demonstrated that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Together, our data demonstrate that functional taste cells can be generated ex vivo from single Lgr5⁺ or Lgr6⁺ cells, validating the use of this model for the study of taste cell generation.Taste bud cells are heterogeneous and undergo constant turnover (1); however, the origins and generation of taste buds in adult mammals remain largely unclear. Based on morphological and functional characteristics, there are at least three different types of mature taste bud cells [type 1 (glial-like cells), type 2 (receptor cells, including those responsible for sensing sweet, bitter, and umami stimuli), and type 3 (presynaptic cells, including sour sensors)], and well as one type of immature taste bud cell [type 4 (basal cells that are precursors of other types of mature taste cells)] (2, 3). Mature taste bud cells are postmitotic and short-lived, with average life spans estimated at 8–12 d (4, 5), although distinct subtypes of taste bud cells may have different life spans (1, 4, 5). At present, the stem cell population and the regenerative process from adult taste stem/progenitor cells to mature taste bud cells are not well characterized.Lgr5 (leucine-rich repeat-containing G protein-coupled receptor 5), encoded by a Wnt (wingless-type MMTV integration site family) target gene, marks adult stem/progenitor cells in taste tissue in posterior tongue that in vivo give rise to all major types of taste bud cells, as well as perigemmal cells (6, 7). Lgr5 is also known to mark actively cycling stem cells in small intestine, colon, stomach, and hair follicle, as well as quiescent stem cells in liver, pancreas, and cochlea (8). Isolated Lgr5⁺ adult stem cells from multiple tissues are able to generate so-called organoid structures ex vivo (9–11). For instance, Sato and colleagues (10) developed a 3D culture system to grow crypt-villus organoids from single intestinal stem cells; all differentiated cell types were found in these structures, indicating the multipotent nature of these cells. We hypothesized that Lgr5⁺ taste stem/progenitor cells in a 3D culture system would be capable of expanding and giving rise to taste receptor cells ex vivo. In the present study, we isolated Lgr5⁺ stem/progenitor cells from taste tissue and cultured them in a 3D culture system. Single Lgr5⁺ cells grew into organoid structures ex vivo in defined culture conditions, with the presence of both proliferating cells and differentiated mature taste cells in which taste signaling components are functionally expressed. When organoids were replated onto a 2D surface precoated with laminin and polylysine, cells grew out of the organoids and attached to the flat surface, and some cells retained the expressed taste signaling elements and responded to taste stimuli.Lgr5 marks adult taste stem/progenitor cells in posterior tongue, which was shown using an engineered mouse model in which enhanced green fluorescent protein (EGFP) and tamoxifen-inducible Cre recombinase (CreERT2) are knocked-in to replace the coding sequence of Lgr5 and act as surrogate markers for Lgr5 (6, 7). Although Lgr5 is present in fungiform papillae in anterior tongue during embryonic stages and early life, based on the intrinsic GFP signal from the Lgr5-EGFP transgene, Lgr5-EGFP signal could not be detected in fungiform papillae cells in adult mice (6, 7). Therefore, taste stem/progenitor cells remain to be identified in fungiform papillae in anterior tongue. We hypothesized that Lgr6, an Lgr5 homolog, may mark adult taste stem/progenitor cells in anterior tongue, prompted by the finding that Lgr6 is preferentially expressed in taste tissue, but not in the surrounding epithelium devoid of taste tissue (12). Using the Lgr6-EGFP-ires-CreERT2 mouse line (13), we here show that Lgr6 is expressed in cells at the basal area of taste buds in fungiform and circumvallate papillae. By genetic lineage tracing, we show that Lgr6⁺ cells give rise to taste bud cells in taste papillae in both anterior and posterior tongue. RT-PCR shows that Lgr5 and Lgr6 may mark the same subset of taste stem/progenitor cells both anteriorly and posteriorly. Similar to Lgr5⁺ cells, isolated Lgr6⁺ cells can build taste organoids that generate mature taste cells.     

Role of the ectonucleotidase NTPDase2 in taste bud function

Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.Taste buds, the sensory end organs of gustation, are unique among the special senses in using ATP as a key transmitter to activate their sensory nerve fibers (1). The gustatory nerves express the purinergic receptor subunits P2X2 and P2X3 (2), which rapidly depolarize the nerve terminal when exposed to ATP. An important feature of neurotransmission is the removal of transmitter from extracellular space to prevent desensitization of the receptors by prolonged exposure to the ligand. In the case of taste buds, removal of ATP is accomplished largely by one of the eight known ectonucleotidases (for a review, see ref. 3), nucleoside triphosphate diphosphohydrolase-2 (NTPDase2), a highly specific nucleoside triphosphate diphosphohydrolase (4, 5), which preferentially degrades ATP over ADP (6), (i.e., an ectoATPase, as defined histochemically by specificity for ATP).The NTPDase of taste buds is expressed by only one of the three principal types of cells within the bud. Each taste bud contains an onion-shaped cluster of 50–100 elongate taste cells, comprising morphologically and molecularly distinct cell types (for review, see ref. 7): type I, type II, and type III. The detection and transduction of different tastants is accomplished by type II and type III cells (for review, see refs. 7 and 8). Type II cells, also called “receptor” cells, express the G protein-coupled taste receptors and downstream effectors for bitter, sweet, and umami qualities. When activated, type II cells release ATP via a nonvesicular mechanism, likely involving ion channels (9–12). Type III cells, also called “presynaptic” cells, are implicated in transduction of sour and salty substances and are the only taste cells that possess conventional synaptic contacts with afferent nerve fibers (13–17). Type I cells, generally considered to have a support or “glial-like” function because they wrap around the other cell types, are the cells that express NTPDase2 (18-19).Once ATP is released into the extracellular space within a taste bud, it activates purinergic receptors on other taste cells as well as the ionotropic purinergic receptors on the taste nerves composed of P2X2 and P2X3 subunits. Various taste cells express ionotropic P2X2 and P2X7 receptors (20, 21) and metabotropic P2Y1 (22, 23), P2Y2, and P2Y4 (24) receptors. After release, ATP is degraded via NTPDase2 to ADP, which itself can activate the P2Y receptors. The ADP then is further degraded to AMP and finally to adenosine via other and less specific ectonucleotidases including ecto-5′-nucleotidase expressed in type III cells (25).In this study we have focused on the role of NTPDase2 in regulating synaptic function in taste buds. We report that genetic deletion of NTPDase2 results in the accumulation of ATP in the taste tissues and a concomitant significant decrease of neural taste responses, likely because of receptor desensitization.     

Age-associated increases in intensity discrimination for taste

Impaired taste sensation in the aging person may affect the appreciation of food and beverages and compromise nutritional status. Changes in flavor perception may be due to altered ability to discriminate between intensities of suprathreshold taste stimuli. An interesting question is whether all taste qualities (sweet, salty, sour, bitter) show similar age-associated decline in intensity discrimination. Taste intensity discrimination has been shown to be significantly poorer in elderly than in young women for the bitter stimulus caffeine, but not for the sweet stimulus sucrose. The present experiment investigated effects of taste substance and age on taste intensity discrimination by assessing Weber ratios (WRs) for citric acid and sodium chloride (NaCl) in 60 people, half elderly, and half women. Results indicate a significant effect of age on WRs for citric acid and NaCl, suggesting the importance of suprathreshold intensity discrimination for taste perception in the elderly.     

R-spondin substitutes for neuronal input for taste cell regeneration in adult mice

Taste bud cells regenerate throughout life. Taste bud maintenance depends on continuous replacement of senescent taste cells with new ones generated by adult taste stem cells. More than a century ago it was shown that taste buds degenerate after their innervating nerves are transected and that they are not restored until after reinnervation by distant gustatory ganglion neurons. Thus, neuronal input, likely via neuron-supplied factors, is required for generation of differentiated taste cells and taste bud maintenance. However, the identity of such a neuron-supplied niche factor(s) remains unclear. Here, by mining a published RNA-sequencing dataset of geniculate ganglion neurons and by in situ hybridization, we demonstrate that R-spondin-2, the ligand of Lgr5 and its homologs Lgr4/6 and stem-cell-expressed E3 ligases Rnf43/Znrf3, is expressed in nodose-petrosal and geniculate ganglion neurons. Using the glossopharyngeal nerve transection model, we show that systemic delivery of R-spondin via adenovirus can promote generation of differentiated taste cells despite denervation. Thus, exogenous R-spondin can substitute for neuronal input for taste bud cell replenishment and taste bud maintenance. Using taste organoid cultures, we show that R-spondin is required for generation of differentiated taste cells and that, in the absence of R-spondin in culture medium, taste bud cells are not generated ex vivo. Thus, we propose that R-spondin-2 may be the long-sought neuronal factor that acts on taste stem cells for maintaining taste tissue homeostasis.

The sense of taste allows humans and other animals to test a food before ingesting it. It helps distinguish nutritional foods with an attractive taste (e.g., sweet, umami) from potentially harmful or toxic food items with an aversive taste (e.g., sour, bitter) (1–4). In the oral cavity, tastants (chemicals in food) are detected by specific receptors expressed in taste cells that are clustered in taste buds, onion-shaped structures found in the tongue and soft palate in the oral cavity (1–4). Taste buds are the basic units for processing taste information peripherally; each comprises a heterogeneous population of about 50 to 100 taste cells (1–4). Like the surrounding epithelial cells, taste bud cells undergo constant turnover throughout life; they are replenished by new cells generated from adult taste stem/progenitor cells, such as Lgr5⁺ and/or Lgr6⁺ cells (5–9).Taste bud cells are innervated by gustatory neurons that relay taste information to the brain to initiate taste sensation/perception. Intact gustatory nerves are also essential for taste stem cells to generate new cells to maintain the integrity of taste buds. More than a century ago it was demonstrated that transection of the gustatory nerves leads to degeneration of the taste buds they innervate (10, 11). The restoration of taste buds occurs only after reinnervation by gustatory ganglion neurons (12, 13). Thus, neuronal input is required for taste bud cell replenishment and taste bud maintenance. This most likely involves a neuron-supplied niche factor(s) that regulates adult taste stem/progenitor cell activity, but the identity of such a factor(s) is unknown.Recent work has suggested that sonic hedgehog (Shh) is a key neuron-supplied factor for direct patterning of taste organ regeneration (14). However, Shh is produced by both taste cells and gustatory neurons. Castillo-Azofeifa et al. (15) showed that disturbance of taste bud homeostasis occurs only when both sources of Shh, from gustatory neurons and from taste cells, are eliminated. Furthermore, in an ex vivo organoid culture system we developed, no exogenous Shh is required for taste stem/progenitor cells to produce mature taste cells. Thus, despite the requirement of Shh signaling for taste bud maintenance (16–19), Shh itself may not be the principal gustatory-neuron-produced factor for maintenance of taste buds and regeneration of taste bud cells.To search for the principal gustatory-neuron-produced factor, we focused on the Lgr5 pathway because of the expression of Lgr4/5/6 in taste stem/progenitor cells (20). We asked if the ligand of Lgr4/5/6, R-spondin, may act as the principal gustatory-neuron-produced factor for regulating taste tissue homeostasis. Mechanistically, Lgr5 and its analogs (Lgr4/Lgr6) interact with all four R-spondin proteins promiscuously to regulate activity of stem cells (e.g., intestinal Lgr5⁺ stem cells) (21). Aside from Lgrs, all four R-spondin proteins also interact with the stem-cell-expressed E3 ligases Rnf43/Znrf3 to neutralize their ligase activity (21).Here, by mining of the published single-cell RNA-sequencing (RNAseq) data of gustatory neurons (22), we show that Rspo2 is predominantly expressed in gustatory neurons, and we validate the expression of Rspo2 in gustatory neurons by in situ hybridization and qPCR. We demonstrate that systemic supply of exogenous Rspo1 and Rspo2 via adenovirus can maintain taste bud integrity after gustatory nerve (glossopharyngeal) transection and that cultured taste organoids require R-spondin (either Rspo1 or Rspo2) to produce taste cells. Our work suggests that R-spondin (primarily Rspo2) may be the neuron-produced factor that regulates Lgr5⁺- and/or Lgr6⁺-expressing adult taste stem/progenitor cell activity to maintain taste tissue homeostasis.     

Age-related changes of activity of acid phosphatase in PHA-stimulated lymphocytes

Peripheral blood lymphocytes of 70-year-old individuals as well as spleen cells of 18-month-old Balb/c mice were characterized by diminished activity of acid phosphatase in relation to the activity of that enzyme in cells from young subjects. Simultaneously performed histochemical tests revealed that aging process in both species examined was accompanied by a reduction of the number of cells, disclosing the activity of acid phosphatase. Age-related differences with regard to the level of acid phosphatase became more pronounced after stimulation of cells with PHA. The decrease of acid phosphatase activity during aging is discussed in relation to the function of lymphocytes.     

Late taste disorders in bone marrow transplantation: clinical evaluation with taste solutions in autologous and allogeneic bone marrow recipients.

The aim of this work was to determine the type and the significance of taste disorders in allogeneic bone marrow transplanted patients. In a retrospective study the taste threshold of a cohort of 15 allogeneic bone marrow transplanted patients, 4-51 months after transplantation (mean: 30.6 +/- 15.8), was compared to the taste threshold of 8 autologous bone marrow recipients, 4-48 months after transplantation (mean: 24.12 +/- 12.18), and to the taste threshold of a group of 20 consecutive normal subjects. Allogeneic bone marrow transplanted patients showed a significant hypogeusia for salt (Pearson's chi square p = 0.0002; Yates' correction p = 0.0007) and sour (Pearson's chi square p = 0.001; Yates' correction p = 0.008). No significant variations were observed for sweet and bitter. Autologous bone marrow recipients did not show any significant variation of taste acuity for sweet, salt or sour; a constant reduction of the taste threshold for bitter was observed, but the values were not significantly different from normal (Pearson's chi square p = 0.47; Yates' correction p = 0.83). So, late and selective taste disorders are observed in allogeneic bone marrow transplanted patients. Since the severity of the disorders is not strictly related to the severity of chronic oral G.V.H.D., taste analysis could discover the slightest, clinically undetectable cases of chronic oral G.V.H.D. The mechanism of immune aggression on the sensorial taste cells is poorly understood. Further trials are needed to define variations of taste acuity not only after allogeneic bone marrow transplantation, but also in systemic immune diseases.     

Autophagic activity in thymus and liver during aging

Impaired or deficient autophagy is believed to cause or contribute to aging, as well as several age-related pathologies. Thymic epithelial cells had a high constitutive level of autophagy. The autophagic process may play a supporting role or even a crucial role in the presentation of self-Ags in the thymus to shape the T-cell repertoires. Autophagic activity in the liver is important for the balance of energy and nutrients for basic cell functions. The abundance of autophagic structure in both cortical and medullary thymic epithelial cells and liver with mouse age has not been examined in detail. Here, we demonstrated that the architecture of mouse thymus and liver markedly changed with age. We found that the expression of LC3 detected by immunofluorescence and Western blot analysis was greatly decreased in thymus and liver of 12-month-old mice. The same level of reduction was observed in thymus and liver of 24-month-old mice. Ultrastructure analysis by an electron microscope revealed that the number of autophagic structure/vacuole in total thymic epithelial cells and hepatocytes decrease with age. The age-related decrease of autophagic structure in thymic epithelial cells may cause the reduction of immunocompetent T-cell pool in aged mice. The age-related decrease of autophagy in liver may induce accumulation of cellular materials in liver of aged mice.     

Glucose transporters and ATP-gated K+ (KATP) metabolic sensors are present in type 1 taste receptor 3 (T1r3)-expressing taste cells

Although the heteromeric combination of type 1 taste receptors 2 and 3 (T1r2 + T1r3) is well established as the major receptor for sugars and noncaloric sweeteners, there is also evidence of T1r-independent sweet taste in mice, particularly so for sugars. Before the molecular cloning of the T1rs, it had been proposed that sweet taste detection depended on (a) activation of sugar-gated cation channels and/or (b) sugar binding to G protein-coupled receptors to initiate second-messenger cascades. By either mechanism, sugars would elicit depolarization of sweet-responsive taste cells, which would transmit their signal to gustatory afferents. We examined the nature of T1r-independent sweet taste; our starting point was to determine if taste cells express glucose transporters (GLUTs) and metabolic sensors that serve as sugar sensors in other tissues. Using RT-PCR, quantitative PCR, in situ hybridization, and immunohistochemistry, we determined that several GLUTs (GLUT2, GLUT4, GLUT8, and GLUT9), a sodium-glucose cotransporter (SGLT1), and two components of the ATP-gated K(+) (K(ATP)) metabolic sensor [sulfonylurea receptor (SUR) 1 and potassium inwardly rectifying channel (Kir) 6.1] were expressed selectively in taste cells. Consistent with a role in sweet taste, GLUT4, SGLT1, and SUR1 were expressed preferentially in T1r3-positive taste cells. Electrophysiological recording determined that nearly 20% of the total outward current of mouse fungiform taste cells was composed of K(ATP) channels. Because the overwhelming majority of T1r3-expressing taste cells also express SUR1, and vice versa, it is likely that K(ATP) channels constitute a major portion of K(+) channels in the T1r3 subset of taste cells. Taste cell-expressed glucose sensors and K(ATP) may serve as mediators of the T1r-independent sweet taste of sugars.     

Alterations of cell cycle distribution in the bone marrow of aging mice measured by flow cytometry

The effect of aging on the cell cycle distribution in bone marrow cells was measured by flow cytometry with special reference to the lability in bone marrow physiology. Female C3H mice were examined every 3 h during a 24-h period at the age of 16, 21 and 26 months, vs 3-month-old control mice. Considerable circadian fluctuations were found in the different cell cycle phases in young mice. The rhythmicity patterns were different when comparing different months. In aging mice the variations were dampened, while consistent age-related phase shifts were not seen. The maximal 24-h mean numbers of cells in all three cell phases, but especially the S and G2 phases were reached at 21 months. The relative number of S and G2 phases were significantly reduced in 26-month-old mice, indicating an age-related shift of the proliferative capacity. The present findings are discussed in relation to age-related changes in granulopoiesis and the increase of myelotoxic effects during cancer chemotherapy in aging individuals.     

Quantitative anatomical study of taste buds in fungiform papillae of young and old Fischer rats

To determine if differences in neural taste responses relate to taste bud loss in old age, taste buds were counted in fungiform papillae of Fischer 344 rats aged 4 to 6 months, 20 to 24 months, and 30 to 37 months. Papillae anterior to the intermolar eminence on one half of the tongue were examined in serial sections. Presence or absence of a taste bud was noted and taste bud diameter was measured. Average percentages of papillae that contained a taste bud in the three groups were 99.6, 99.3, and 94.7%. This is a significant age-related difference but actual number of taste buds lost in the oldest rats was small. Taste bud diameter did not differ with age and general anatomical characteristics of buds were similar in all groups. Thus, anatomical observations on taste bud maintenance in rats over a wide age range, coupled with neurophysiological data, demonstrate that the integrity of the peripheral gustatory system is not altered greatly in old age.     

Estradiol and soy extract increase the production of new cells in the dentate gyrus of old rats

In young rodents, estradiol increases cell proliferation in the dentate gyrus of the hippocampus. However, it is unknown if the old brain retains this response to estradiol. Here we assessed the generation of new cells in the dentate gyrus of old rats after administration of estradiol or a soy extract, since soy is used as an alternative to hormonal replacement therapy in postmenopausal women. In a first experiment, 12-month-old animals were ovariectomized and studied at 14, 18 or 22 months of age. The production of new cells, assessed by the incorporation of bromodeoxyuridine (BrdU), was similar in 14- and 18-month-old rats. However, there was a significant reduction in the number of BrdU-immunoreactive cells at 22 months of age. In a second experiment, 22-month-old ovariectomized animals were treated for 10 weeks with a weekly s.c. injection of 150 microg estradiol valerianate or with 60 mg/kg per day soy extract added to the drinking water. Both treatments increased significantly the production of new cells in the dentate gyrus. These findings indicate that the brains of old rats retain the ability to increase the production of new cells in response to estradiol and soy extracts.     

Luteinizing hormone-releasing hormone (LHRH) agonist restoration of age-associated decline of thymus weight, thymic LHRH receptors, and thymocyte proliferative capacity

The presence of specific LHRH-binding sites within the rat thymus gland and the ability of LHRH and its agonistic and antagonistic analogs to directly modulate thymus function prompted us to study the possible changes in the number of thymic LHRH-binding sites during aging-induced physiological immunosenescence. Moreover, the effects of chronic treatment of aging rats with a potent LHRH agonist (LHRH-A) on thymic LHRH receptors, thymus weight and histology, as well as thymocyte proliferative capacity were assessed. For comparison, the effects of castration on the same parameters was also investigated. The process of aging is accompanied by a sharp reduction in LHRH-A-binding sites within the thymus gland of both female and male rats. Starting at 7 months of age, a 50% decrease in thymic LHRH-A binding was followed, at 11-13 months of age, by a nearly 65% inhibition of receptor numbers. In 16- to 19-month-old rats, LHRH-A binding was almost completely lost. Thymus weight was 30% reduced in 7-month-old animals, while a 50% reduction in thymic size was reached at 11 months of age in males and 13 months in female rats. A further decrease in thymic mass was observed at 16 and 19 months. Chronic (45-day) treatment of aging (15-16 months old) female and male rates with the potent LHRH-A, [D-Trp6,Des-Gly10]LHRH-N-ethylamide, reversed the age-related decreases in both thymus weight and thymic LHRH-binding sites. Similarly, surgical removal of testicular hormones by castration restored thymus weight and increased LHRH-A binding in the thymus of aged rats. While thymus histology in 3-month-old rats was characterized by a clear demarcation of cortical and medullary regions, only thymic remnants were present in 16- to 17-month-old animals. Castration of old rats resulted in a partial restoration of thymic structure, while chronic treatment of aging rats with the LHRH-A produced a homogeneous organization of both cortical and medullary compartments accompanied by a marked increase in the width of the cortical layer, densely packed with lymphocytes. While the process of aging was accompanied by an almost complete loss of the proliferative response of thymocytes to optimal concentrations of the mitogen Concanavalin-A, thymocyte cultures from old rats treated with LHRH-A or from castrated animals, displayed significantly greater proliferative responses. Furthermore, the combination of both manipulations resulted in a further significant increase in thymocyte proliferative capacity.(ABSTRACT TRUNCATED AT 400 WORDS)