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.

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 MgCl2 and saccharin solutions but not representative salty, sour, bitter, or umami taste compounds. A genome scan of B6 x PWK F2 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.

The C57BL/6ByJ (B6) strain of mice exhibits higher preferences than does the 129P3/J (129) strain for a variety of sweet tasting compounds. We measured gustatory afferent responses of the whole chorda tympani nerve in these two strains using a broad array of sweeteners and other taste stimuli. Neural responses were greater in B6 than in 129 mice to the sugars sucrose and maltose, the polyol D-sorbitol and the non-caloric sweeteners Na saccharin, acesulfame-K, SC-45647 and sucralose. Lower neural response thresholds were also observed in the B6 strain for most of these stimuli. The strains did not differ in their neural responses to amino acids that are thought to taste sweet to mice, with the exception of L-proline, which evoked larger responses in the B6 strain. Aspartame and thaumatin, which taste sweet to humans but are not strongly preferred by B6 or 129 mice, did not evoke neural responses that exceeded threshold in either strain. The strains generally did not differ in their neural responses to NaCl, quinine and HCl. Thus, variation between the B6 and 129 strains in the peripheral gustatory system may contribute to differences in their consumption of many sweeteners.

C57BL/6ByJ (B6) and 129P3/J (129) mice have different alleles of Tas1r3, which is thought to influence gustatory transduction of sweeteners, but studies have provided conflicting results regarding differences in sweetness perception between these strains. Single-unit taste-evoked activity was measured in the nucleus of the solitary tract (NST) in anesthetized B6 and 129 mice to address this controversy and to provide the first electrophysiological characterization of this nucleus in mice. Neurons had properties similar to those of NST cells in other species, including mean breadth-of-tuning of 0.8 Ϯ 0.0. There were no strain differences in neural responses at 600 or 900 ms after onset, but, with a 5 s evoked period, responses to the sweeteners sucrose, maltose, acesulfame-K, SC-45647, and D-phenylalanine were significantly larger in B6 relative to 129 mice. The strains did not differ in their mean response to NaSaccharin, but it evoked an across-neuron pattern of activity that was more similar to that of sucrose and less similar to that of NaCl in B6 mice compared with 129 mice. Neurons were classified as sucrose, NaCl, or HCl responsive, with the former more common in B6 than 129 mice. Relative to other neurons, sucrose-responsive cells had delayed but more sustained sweetener responses in both strains. The results suggest that B6 mice perceive some sweeteners as more intense, but NaSaccharin as sweeter and less salty, relative to 129 mice. Furthermore, activity evoked by sweeteners includes a phasic response sent to different NST cells than a later tonic response, and only the latter differs between B6 and 129 mice.

Sodium-deprived rats develop a salt appetite and show changes in gustatory responses to NaCl in the periphery and brain stem; salt-sensitive neurons respond less to hypertonic NaCl than do corresponding cells in replete controls. By administering DOCA and renin, we generated a need-free sodium appetite quickly enough to permit us to monitor the activity of individual neurons in the nucleus of the solitary tract before and after its creation, permitting a more powerful within-subjects design. Subjects received DOCA pretreatment followed by an intracerebroventricular infusion of renin. In animals that were tested behaviorally, this resulted in elevated intake of 0.5 M NaCl. In neural recordings, renin caused decreased responding to hypertonic NaCl across all neurons and in the salt-sensitive neurons that were most responsive to NaCl before infusion. Most sugar-sensitive cells, in contrast, gave increased phasic responses to NaCl. These results confirm that sodium appetite is accompanied by decreased responding to NaCl in salt-sensitive neurons, complemented by increased activity in sugar-sensitive cells, even when created rapidly and independently of need.

No abstract

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.

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.