The gene Tas1r3 codes for the protein T1R3, which dimerizes with T1R2 to form a sweetener-binding receptor in taste cells. Tas1r3 influences sweetener preferences in mice, as shown by work with a 129.B6-Tas1r3 segregating congenic strain on a 129P3/J (129) genetic background; members of this strain vary in whether they do or do not have one copy of a donor fragment with the C57BL/6ByJ (B6) allele for Tas1r3 (B6/129 and 129/129 mice, respectively). Taste-evoked neural responses were measured in the nucleus of the solitary tract (NST), the first central gustatory relay, in B6/129 and 129/129 littermates, to examine how the activity dependent on the T1R2/T1R3 receptor is distributed across neurons and over time. Responses to sucrose were larger in B6/129 than in 129/129 mice, but only during a later, tonic response portion (>600 ms) sent to different cells than the earlier, phasic response. Similar results were found for artificial sweeteners, whose responses were best considered as complex spatiotemporal patterns. There were also group differences in burst firing of NST cells, with a significant positive correlation between bursting prevalence and sucrose response size in only the 129/129 group. The results indicate that sweetener transduction initially occurs through T1R3-independent mechanisms, after which the T1R2/T1R3 receptor initiates a separate, spatially distinct response, with the later period dominating sweet taste perceptions and driving sugar preferences. Furthermore, the current data suggest that burst firing is distributed across NST neurons nonrandomly and in a manner that may amplify weak incoming gustatory signals.

Different gustatory papilla types vary in their locations on the tongue. Distinctions have often made between types, but variation within fungiform papillae has seldom been explored. Here, regional differences in fungiform papillae were investigated by flowing solutions selectively over either an anterior fungiform (AF, tongue tip) or a posterior fungiform (PF, middle third) region as taste-evoked activity was measured in the chorda tympani nerve of C57BL/6J (B6) mice. Significantly larger responses were evoked by NaCl applied to the AF than PF region, and the ENaC blocker amiloride reduced the NaCl response size only for the former. Umami synergy, based on co-presenting MSG and IMP, was larger for the AF than PF region. The regions did not differ in response size to sour chemicals, but responses to l-lysine, l-arginine, sucrose, and tetrasodium pyrophosphate were larger for the AF than PF region. Thus, fungiform papillae on the tongue tip differed from those found further back in their transduction mechanisms for salty and umami compounds. Gustatory sensitivity also showed regional variation, albeit with a complex relationship to palatability and taste quality. Overall, the data support a regional organization for the mouse tongue, with different functional zones for the anterior, middle, and posterior thirds.

The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt’s disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.

Inflammatory responses play a key role in many neural pathologies, with localized signaling from the non-immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. The NLRP3 inflammasome requires two stages to contribute: priming and activation. The priming stage involves upregulation of inflammasome components while the activation stage results in the assembly and activation of the inflammasome complex. The priming step can be rate limiting and can connect insult to chronic inflammation, but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammation is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non-ischemic elevation of intraocular pressure increased mRNA for inflammasome components IL-1β, NLRP3, ASC, and CASP1 in rat and mouse retinas. The elevation was greater 1 day after the insult, with the rise in IL-1β most pronounced. The P2X7 receptor was implicated in the mechanosensitive priming of IL-1β mRNA in vivo, as the antagonist Brilliant Blue G (BBG) blocked the increased expression, the agonist BzATP mimicked the pressure-dependent rise in IL-1β, and the rise was absent in P2X7 knockout mice. In vitro measurements from optic nerve head astrocytes demonstrated an increased expression of IL-1β following stretch or swelling. This increase in IL-1β was eliminated by degradation of extracellular ATP with apyrase, or by the block of pannexin hemichannels with carbenoxolone, probenecid, or 10panx1 peptide. The rise in IL-1β expression was also blocked by P2X7 receptor antagonists BBG, A839977 or A740003. The rise in IL-1β was prevented by blocking transcription factor NFκB with Bay 11-7082, while the swelling-dependent fall in NFκB inhibitor IκB-α was reduced by A839977 and in P2X7 knockout mice. In summary, mechanical trauma to the retina primed NLRP3 inflammasome components, but only if there was ATP release through pannexin hemichannels, and autostimulation of the P2X7 receptor. As the P2X7 receptor can also trigger stage two of inflammasome assembly and activation, the P2X7 receptor may have a central role in linking mechanical strain to neuroinflammation.

The consumption of amino acids by animals is controlled by both oral and postoral mechanisms. We used a genetic approach to investigate these mechanisms. Our studies have shown that inbred mouse strains differ in voluntary amino acid consumption, and these differences depend on sensory and nutritive properties of amino acids. Like humans, mice perceive some amino acids as having a sweet (sucrose-like) taste and others as having an umami (glutamate-like) taste. Mouse strain differences in the consumption of some sweet-tasting amino acids (d-phenylalanine, d-tryptophan, and l-proline) are associated with polymorphisms of a taste receptor, type 1, member 3 gene (Tas1r3), and involve differential peripheral taste responsiveness. Strain differences in the consumption of some other sweet-tasting amino acids (glycine, l-alanine, l-glutamine, and l-threonine) do not depend on Tas1r3 polymorphisms and so must be due to allelic variation in other, as yet unknown, genes involved in sweet taste. Strain differences in the consumption of l-glutamate may depend on postingestive rather than taste mechanisms. Thus, genes and physiologic mechanisms responsible for strain differences in the consumption of each amino acid depend on the nature of its taste and postingestive properties. Overall, mouse strain differences in amino acid taste and appetite have a complex genetic architecture. In addition to the Tas1r3 gene, these differences depend on other genes likely involved in determining the taste and postingestive effects of amino acids. The identification of these genes may lead to the discovery of novel mechanisms that regulate amino acid taste and appetite.

Neurons that fire in bursts have been well-characterized in vision and other neural systems, but not in taste systems. We therefore examined whether brain stem gustatory neurons fire in bursts during spontaneous activity and, if so, whether such cells differ from nonbursting cells in other characteristics. We looked at neurons in the nucleus of the solitary tract (NST) of C57BL/6ByJ (B6) and 129P3/J (129) mice, and in the NST and parabrachial nucleus (PBN) of Sprague-Dawley rats. Many NST cells fired frequently with short intervals characteristic of bursting, and such neurons differed from others in their responsiveness to taste compounds. In B6 mice and rats, there was a significant positive correlation between the prevalence of short-interval firing and the net spikes evoked by application of NaCl. In contrast, in 129 mice the prevalence of short intervals was positively correlated with the size of sucrose responses. We also compared breadth-of-tuning measures based on counting either all spikes or only those following short intervals, and we found narrower tuning for the latter in the NST of B6 mice and rats. There was little evidence of spontaneous bursting in the rat PBN, and firing patterns in this nucleus were not related to the size of taste-evoked responses. We suggest that bursting may be a strategy employed by the NST to amplify the postsynaptic impact of particular taste stimuli, depending on an animal's needs. Another function may be to sharpen breadth-of-tuning and thus enhance the contrast between stimuli of different taste qualities.

Different parts of the mouth vary in their taste responsiveness and gustatory transduction components.However, there have been few attempts to consider regional variation among areas innervated by a single nerve branch or containing only one type of gustatory papilla.Here, we examined whether taste-elicited responses of a single nerve, the chorda tympani (CT), depend on where taste solutions are delivered on the tongue in mice.In experiment 1, multiunit CT responses to NaCl and sucrose were larger if sapid taste solutions were applied to the tongue tip, which contains the anterior-most fungiform papillae, than if they were flowed over fungiform and foliate papillae on the posterior tongue.Further, the epithelial sodium channel (ENaC) blocker amiloride suppressed NaCl responses to a greater degree for the tongue tip.In experiment 2, CT nerve responses were compared between the tongue tip and a region further back that contained only fungiform papillae.NaCl and sucrose solutions applied to posterior fungiform papillae produced smaller responses than did those elicited by the same taste stimuli applied to anterior fungiform papillae on the tongue tip.Amiloride suppressed the response to NaCl delivered to the anterior fungiform but not posterior fungiform papillae.These results indicate that the CT response is tongue-region dependent in the mouse.Furthermore, the spatial location of a fungiform papilla provides important information about its properties, such as whether sodium taste transduction is mediated by amiloride-sensitive ENaCs.

Genetic ablation of calcium homeostasis modulator 1 (CALHM1), which releases adenosine triphosphate from Type 2 taste cells, severely compromises the behavioral and electrophysiological responses to tastes detected by G protein-coupled receptors, such as sweet and bitter. However, the contribution of CALHM1 to salty taste perception is less clear. Here, we evaluated several salty taste-related phenotypes of CALHM1 knockout (KO) mice and their wild-type (WT) controls: 1) In a conditioned aversion test, CALHM1 WT and KO mice had similar NaCl avoidance thresholds. 2) In two-bottle choice tests, CALHM1 WT mice showed the classic inverted U-shaped NaCl concentration-preference function but CALHM1 KO mice had a blunted peak response. 3) In brief-access tests, CALHM1 KO mice showed less avoidance than did WT mice of high concentrations of NaCl, KCl, NH(4)Cl, and sodium lactate (NaLac). Amiloride further ameliorated the NaCl avoidance of CALHM1 KO mice, so that lick rates to a mixture of 1000 mM NaCl + 10 µM amiloride were statistically indistinguishable from those to water. 4) Relative to WT mice, CALHM1 KO mice had reduced chorda tympani nerve activity elicited by oral application of NaCl, NaLac, and sucrose but normal responses to HCl and NH(4)Cl. Chorda tympani responses to NaCl and NaLac were amiloride sensitive in WT but not KO mice. These results reinforce others demonstrating that multiple transduction pathways make complex, concentration-dependent contributions to salty taste perception. One of these pathways depends on CALHM1 to detect hypertonic NaCl in the mouth and signal the aversive taste of concentrated salt.

Laboratory rats and mice prefer some concentrations of tri- and tetrasodium pyrophosphate (Na3HP2O7 and Na4P2O7) to water, but how they detect pyrophosphates is unknown. Here, we assessed whether T1R3 is involved. We found that relative to wild-type littermate controls, Tas1r3 knockout mice had stronger preferences for 5.6-56mM Na3HP2O7 in 2-bottle choice tests, and they licked more 17.8-56mM Na3HP2O7 in brief-access tests. We hypothesize that pyrophosphate taste in the intact mouse involves 2 receptors: T1R3 to produce a hedonically negative signal and an unknown G protein-coupled receptor to produce a hedonically positive signal; in Tas1r3 knockout mice, the hedonically negative signal produced by T1R3 is absent, leading to a heightened avidity for pyrophosphate.

Dietary exposure to sugars increases the preference for and intake of sugar solutions in mice. We used brief-access lick tests and multiunit electrophysiological recordings from the nucleus of the solitary tract (NST) to investigate the role of taste in diet-induced changes in sucrose responsiveness. We exposed C57BL/6J (B6) and 129X1/SvJ (129) mice to either a sucrose diet (chow, water, and a 500mM sucrose solution) or a control diet (chow and water) for 3 days. In B6 mice, exposure to the sucrose diet decreased the appetitive response (i.e., number of trials initiated) but had no effect on the consummatory response (i.e., rate of licking) to 500mM sucrose and 20mM saccharin. In 129 mice, exposure to the sucrose diet increased the appetitive response but had no effect on the consummatory response to the sweetener solutions. In the NST recordings, the B6 mice exhibited larger multiunit responses to sweeteners than 129 mice, but there was no effect of the sucrose diet in either strain. Our results indicate that sucrose exposure alters the appetitive response of B6 and 129 mice to sweeteners in diametrically opposed ways and that these changes are mediated by structures in the gustatory neuraxis above the NST (e.g., ventral forebrain).

The molecular mechanisms of sodium taste transduction are not completely understood, especially those responsible for the portion of NaCl's taste in rodents that is not blocked by amiloride. As a prelude to conducting genetic analyses of peripheral NaCl taste responsiveness, we performed multiunit electrophysiological recordings from the chorda tympani (CT) nerve in C57BL/6J (B6) and A/J mice. Mice were anesthetized, the CT was accessed, and taste solutions were flowed over the tongue in order to measure the integrated whole-nerve response. NaCl was delivered before and during application of 100μM amiloride. Pre-amiloride responses were significantly larger in A/J than B6 mice for 1-8mM NaCl. Responses to NaCl were suppressed significantly by amiloride in both strains and to similar degrees. However, the size of the amiloride-insensitive NaCl response component was significantly larger in A/J mice than in B6 mice for NaCl at 2-16mM. These data help to explain the prior observation that the strains differ in behavioral taste thresholds for NaCl. Specifically, the results suggest that perception of sodium-specific taste by mice depends on the ratio of amiloride-sensitive and -insensitive responses in the CT, rather than on the absolute level of the whole-nerve response to NaCl or on the size of the amiloride-sensitive component alone. Because the B6 and A/J mice differed in the size of their amiloride-insensitive components, they may prove useful in future genetic work designed to characterize the underlying transduction mechanisms.

We used the C57BL/6J (B6) and PWD/PhJ (PWD) mouse strains to investigate the controls of calcium intake. Relative to the B6 strain, the PWD strain had higher preferences in two-bottle choice tests for CaCl(2), calcium lactate (CaLa), MgCl(2), citric acid and quinine hydrochloride, but not for sucrose, KCl or NaCl. We also measured taste-evoked chorda tympani (CT) nerve activity in response to oral application of these compounds. Electrophysiological results paralleled the preference test results, with larger responses in PWD than in B6 mice for those compounds that were more highly preferred for the former strain. The strain differences were especially large for tonic, rather than phasic, chorda tympani activity. These data establish the PWD strain as a "calcium-preferring" strain and suggest that differences between B6 and PWD mice in taste transduction or a related peripheral event contributes to the differences between the strains in preferences for calcium solutions.

Calcium and magnesium are essential for survival but it is unknown how animals detect and consume enough of these minerals to meet their needs. To investigate this, we exploited the PWK/PhJ (PWK) strain of mice, which, in contrast to the C57BL/6J (B6) and other inbred strains, displays strong preferences for calcium solutions. We found that the PWK strain also has strong preferences for MgCl(2) and saccharin solutions but not representative salty, sour, bitter, or umami taste compounds. A genome scan of B6 x PWK F(2) mice linked a component of the strain difference in calcium and magnesium preference to distal chromosome 4. The taste receptor gene, Tas1r3, was implicated by studies with 129.B6ByJ-Tas1r3 congenic and Tas1r3 knockout mice. Most notably, calcium and magnesium solutions that were avoided by wild-type B6 mice were preferred (relative to water) by B6 mice null for the Tas1r3 gene. Oral calcium elicited less electrophysiological activity in the chorda tympani nerve of Tas1r3 knockout than wild-type mice. Comparison of the sequence of Tas1r3 with calcium and saccharin preferences in inbred mouse strains found 1) an inverse correlation between calcium and saccharin preference scores across primarily domesticus strains, which was associated with an I60T substitution in T1R3, and 2) a V689A substitution in T1R3 that was unique to the PWK strain and thus may be responsible for its strong calcium and magnesium preference. Our results imply that, in addition to its established roles in the detection of sweet and umami compounds, T1R3 functions as a gustatory calcium-magnesium receptor.

Sugars evoke a distinctive perceptual quality (“sweetness” in humans) and are generally highly preferred. The neural basis for these phenomena is reviewed for rodents, in which detailed electrophysiological measurements have been made. A receptor has been identified that binds sweeteners and activates G-protein-mediated signaling in taste receptor cells, which leads to changes in neural firing rates in the brain, where perceptions of taste quality, intensity, and palatability are generated. Most cells in gustatory nuclei are broadly tuned, so quality perception presumably arises from patterns of activity across neural populations. However, some manipulations affect only the most sugar-oriented cells, making it useful to consider them as a distinct neural subtype. Quality perception may also arise partly due to temporal patterns of activity to sugars, especially within sugar-oriented cells that give large but delayed responses. Non-specific gustatory neurons that are excited by both sugars and unpalatable stimuli project to ventral forebrain areas, where neural responses provide a closer match with behavioral preferences. This transition likely involves opposing excitatory and inhibitory influences by different subgroups of gustatory cells. Sweeteners are generally preferred over water, but the strength of this preference can vary across time or between individuals, and higher preferences for sugars are often associated with larger taste-evoked responses.