Yoshihiro Murata

Discrimination of taste qualities among mouse fungiform taste bud cells

Multiple lines of evidence from molecular studies indicate that individual taste qualities are encoded by distinct taste receptor cells. In contrast, many physiological studies have found that a significant proportion of taste cells respond to multiple taste qualities. To reconcile this apparent discrepancy and to identify taste cells that underlie each taste quality, we investigated taste responses of individual mouse fungiform taste cells that express gustducin or GAD67, markers for specific types of taste cells. Type II taste cells respond to sweet, bitter or umami tastants, express taste receptors, gustducin and other transduction components. Type III cells possess putative sour taste receptors, and have well elaborated conventional synapses. Consistent with these findings we found that gustducin‐expressing Type II taste cells responded best to sweet (25/49), bitter (20/49) or umami (4/49) stimuli, while all GAD67 (Type III) taste cells examined (44/44) responded to sour stimuli and a portion of them showed multiple taste sensitivities, suggesting discrimination of each taste quality among taste bud cells. These results were largely consistent with those previously reported with circumvallate papillae taste cells. Bitter‐best taste cells responded to multiple bitter compounds such as quinine, denatonium and cyclohexamide. Three sour compounds, HCl, acetic acid and citric acid, elicited responses in sour‐best taste cells. These results suggest that taste cells may be capable of recognizing multiple taste compounds that elicit similar taste sensation. We did not find any NaCl‐best cells among the gustducin and GAD67 taste cells, raising the possibility that salt sensitive taste cells comprise a different population.

Action potential-enhanced ATP release from taste cells through hemichannels

Only some taste cells fire action potentials in response to sapid stimuli. Type II taste cells express many taste transduction molecules but lack well-elaborated synapses, bringing into question the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells on action potentials. To identify type II taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the alpha-gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a single GFP-positive taste cell. We monitored ATP release from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with saccharin, quinine, or glutamate on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in taste cells responsive to sweet, bitter, or umami tastants enhance ATP release through pannexin 1, not connexin-based hemichannels.

Multiple receptors underlie glutamate taste responses in mice

l-Glutamate is known to elicit a unique taste, umami, that is distinct from the tastes of sweet, salty, sour, and bitter. Recent molecular studies have identified several candidate receptors for umami in taste cells, such as the heterodimer T1R1/T1R3 and brain-expressed and taste-expressed type 1 and 4 metabotropic glutamate receptors (brain-mGluR1, brain-mGluR4, taste-mGluR1, and taste-mGluR4). However, the relative contributions of these receptors to umami taste reception remain to be elucidated. We critically discuss data from recent studies in which mouse taste cell, nerve fiber, and behavioral responses to umami stimuli were measured to evaluate whether receptors other than T1R1/T1R3 are involved in umami responses. We particularly emphasized studies of umami responses in T1R3 knockout (KO) mice and studies of potential effects of mGluR antagonists on taste responses. The results of these studies indicate the existence of substantial residual responses to umami compounds in the T1R3-KO model and a significant reduction of umami responsiveness after administration of mGluR antagonists. These findings thus provide evidence of the involvement of mGluRs in addition to T1R1/T1R3 in umami detection in mice and suggest that umami responses, at least in mice, may be mediated by multiple receptors.

Gurmarin sensitivity of sweet taste responses is associated with co-expression patterns of T1r2, T1r3, and gustducin.

Gurmarin (Gur) is a peptide that selectively suppresses sweet taste responses in rodents. The inhibitory effect of Gur differs among tongue regions and mouse strains. Recent studies demonstrated that co-expression levels of genes controlling sweet receptors (T1r2/T1r3 heterodimer) versus Galpha-protein, gustducin, are much lower in Gur-insensitive posterior circumvallate papillae than in Gur-sensitive anterior fungiform papillae. Here, we investigated the potential link of Gur-sensitivity with the co-expression for T1r2/T1r3 receptors and gustducin by comparing those of taste tissues of Gur-sensitive (B6, dpa congenic strains) and Gur-weakly-sensitive (BALB) strains. The results indicated that co-expression ratios among T1r2, T1r3, and gustducin in the fungiform papillae were significantly lower in Gur-weakly-sensitive BALB mice than in Gur-sensitive B6 and dpa congenic mice. This linkage between Gur-sensitivity and co-expression for T1r2/T1r3 receptors versus gustducin suggests that gustducin may be a key molecule involved in the pathway for Gur-sensitive sweet responses.

Common properties between synaptic plasticity in the main olfactory bulb and olfactory learning in young rats

Aversive olfactory learning was established in young rats after odor exposure paired with foot shock through a classical conditioning paradigm. Using behavioral pharmacology and Western blotting, we previously reported that plasticity in the main olfactory bulb (MOB) underlies aversive olfactory learning. Since long-term potentiation (LTP) observed in the hippocampus is believed to be a cellular substrate for aspects of memory, we attempted to induce LTP in the MOB. Using brain slices containing the MOB, we found that five tetani of the lateral olfactory tract evoked LTP that was blocked by the N-methyl-d-aspartate (NMDA) receptor antagonist AP5. Although three tetani induced no significant changes in control slices, with noradrenaline (NA) application they produced clear LTP (NA-mediated LTP), which was not dependent on NMDA receptors. NAs facilitating effect on LTP induction was blocked by the beta-adrenoceptor antagonist timolol but not by the alpha-adrenoceptor antagonist phentolamine, and was mimicked by the beta-adrenoceptor agonist isoproterenol. The l-type calcium channel blocker nifedipine completely blocked LTP as well as NA-mediated LTP. In addition, we found that aversive olfactory learning was impaired by beta-adrenoceptor antagonist, timolol but not by alpha-adrenoceptor antagonist, phentolamine, and only odor training established olfactory learning by isoproterenol infusion. Moreover, we found that nifedipine but not AP5 prevented olfactory learning formation. These common properties provided evidence for neural correlates between NA-mediated LTP aversive olfactory learning in young rats.

Multiple receptor systems for umami taste in mice.

Recent molecular studies proposed that the T1r1/T1r3 heterodimer, mGluR1 and mGluR4 might function as umami taste receptors in mice. However, the roles of each of these receptors in umami taste are not yet clear. In this paper, we summarize recent data for T1r3, mGluR1, and mGluR4 as umami taste receptors and discuss receptor systems responsible for umami detection in mice.

Modulation and Transmission of Sweet Taste Information for Energy Homeostasis

Perception of sweet taste is important for animals to detect external energy source of calories. In mice, sweet‐sensitive cells possess a leptin receptor. Increase of plasma leptin with increasing internal energy storage in the adipose tissue suppresses sweet taste responses via this receptor. Data from our recent studies indicate that leptin may also modulate sweet taste sensation in humans with a diurnal variation in sweet sensitivity. This leptin modulation of sweet taste information to the brain may influence individuals’ preference and ingestive behavior, thereby playing important roles in regulation of energy homeostasis.

Contribution of the inositol 1,4,5-trisphosphate transduction cascade to the detection of “bitter” compounds in blowflies

Bitter taste detection is very important for many species including flies, because it prevents the ingestion of toxic food. Although it has been known that flies have specific bitter-sensitive taste cells in their contact chemosensilla, the mechanism by which those cells transduce the chemical signal into electrical activity has remained elusive. In this study, we first confirmed that type D4 and D5 tarsal chemosensilla of the blowfly Phormia regina responded well to bitter substances. Then, recording impulses from type D4 chemosensilla, we examined the possibility that a G-protein-coupled inositol 1,4,5-trisphosphate (IP(3))-dependent transduction cascade is of importance in the bitter-sensitive taste cells. We found that the response to bitter substances was depressed by specific inhibitors of G-protein, phospholipase C, or IP(3) receptor in the tarsal taste receptor cells. These results suggest that G-proteins mediate the IP(3) pathway in the transduction cascade in bitter-sensitive receptor cells.

Histone acetylation in the olfactory bulb of young rats facilitates aversive olfactory learning and synaptic plasticity.

Epigenetic mechanisms play an important role in memory formation and synaptic plasticity. Specifically, histone-associated heterochromatin undergoes changes in structure during the early stages of long-term memory formation. In keeping with the classical conditioning paradigm, young rats have been shown to exhibit aversion to an odor stimulus initially presented during foot shock. We previously showed that synaptic plasticity at the dendrodendritic synapses between mitral and granule cells in the olfactory bulb (OB) underlies this aversive olfactory learning. However, the epigenetic mechanisms involved are not well characterized. Therefore, we examined whether intrabulbar infusion of trichostatin A (TSA), a histone deacetylase inhibitor, facilitates olfactory learning in young rats. TSA infusion during odor-shock training enhanced a conditioned odor aversion in a dose-dependent manner and prolonged the learned aversion. Western blot and immunohistochemical analyses showed that the level of histone H4 acetylation significantly increased until 4 h after odor-shock training in both mitral and granule cells in the OB, whereas histone H3 acetylation returned to the control level at 2 h after the training. We also obtained evidence that TSA infusion elevated acetylation of histone H4 or H3. Furthermore, in vitro electrophysiological analysis using slices of the OB revealed that application of TSA significantly enhanced the long-term potentiation induced in synaptic transmission from mitral to granule cells at dendrodendritic synapses. Taken together, these results provide evidence that histone H4 and H3 acetylation in the OB is an epigenetic mechanism associated with aversive olfactory learning in young rats.

Regulation of synaptic currents by mGluR2 at reciprocal synapses in the mouse accessory olfactory bulb

The throughput of information from the accessory olfactory bulb (AOB) to downstream structures is controlled by reciprocal dendrodendritic inhibition of mitral cells by granule cells. Given the high expression levels of mGluR2, a metabotropic glutamate receptor, in the AOB and the fact that the activation of mGluR2 permits the formation of a specific olfactory memory, we reasoned that mGluR2 might play an important role in regulating dendrodendritic inhibition. To test this hypothesis, we examined the effects of pharmacological and genetic manipulations of mGluR2 on synaptic responses measured from mitral or granule cells in slice preparations from 23‐ to 36‐day‐old Balb/c mice. To evoke dendrodendritic inhibition, a depolarizing voltage step from –70 to 0 mV or a threshold current stimulus adjusted to elicit action potential(s) was applied to a mitral cell using either a nystatin‐perforated or conventional whole‐cell configuration. We found that an agonist for group II metabotropic glutamate receptors (mGluR2/mGluR3), DCG‐IV [(2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine], suppressed, whereas the mGluR2/mGluR3 antagonist LY341495 [(αS)‐α‐amino‐α‐[(1S,2S)‐2‐carboxycyclopropyl]‐9H‐xanthine‐9‐propanoic acid] enhanced dendrodendritic inhibition. Genetic ablation of mGluR2 markedly impaired the effects of DCG‐IV and LY341495 on dendrodendritic inhibition. DCG‐IV reduced both the frequency and the amplitude of spontaneous miniature excitatory postsynaptic currents recorded from granule cells. Additionally, DCG‐IV inhibited high‐voltage‐activated calcium currents in both mitral and granule cells. These results suggest that mGluR2 reduces dendrodendritic inhibition by inhibiting synaptic transmission between mitral cells and granule cells in the AOB.