Stuart A. McCaughey

Involvement of T1R3 in calcium-magnesium taste

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 TASTE OF SUGARS

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.

Salty Taste Deficits in CALHM1 Knockout Mice

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.

Calcium deprivation increases the palatability of calcium solutions in rats

Calcium-deprived rats have elevated intakes of CaCl2, other calcium salts, and some non-calcium compounds. We used taste reactivity to examine the effects of calcium deprivation on the palatability of CaCl2 and other solutions. Nine male Sprague-Dawley rats were calcium-deprived by maintenance on a low-calcium diet, and eight replete rats were used as controls. All rats were videotaped during intraoral infusion of the following solutions: 30 and 300 mM CaCl2, 30 mM calcium lactate, 100 and 600 mM NaCl, 30 mM MgCl2, 1 mM quinine.HCl, 2.5 mM sodium saccharin, and deionized water. We counted individual orofacial and somatic movements elicited by the infusions and used them to calculate total ingestive and aversive scores. Relative to controls, calcium-deprived rats gave a significantly larger number of tongue protrusions and had higher total ingestive scores for CaCl2, calcium lactate, NaCl, and MgCl2. Our results suggest that CaCl2, calcium lactate, NaCl, and MgCl2 taste more palatable to rats when they are calcium-deprived than replete, and this may be responsible for the increased intake of these solutions following calcium deprivation.

Magnesium appetite in the rat.

Rats modify their ingestive behaviour to correct deficiencies of minerals such as sodium and calcium. Here, we examined the effect of magnesium deprivation on the ingestion of MgCl2 and other solutions. Male Sprague-Dawley rats were fed a nutritionally complete or magnesium-deficient diet and were then given 3.2, 10, 32, or 100mM MgCl2, 32mM CaCl2, 32mM NaCl, 10mM HCl, or 2.5mM saccharin, and their intake was measured for 24h in a two-bottle choice test with water. Within the first 5 min, magnesium-deprived subjects given 3.2, 32, or 100mM MgCl2 or 32mM CaCl2 drank significantly more of these solutions than did replete rats. In a separate study, rats fed replete, magnesium-deficient, or calcium-deficient diets were given a three-bottle choice between water, 32mM MgCl2, and 32mM CaCl2. The deprived rats preferred the solution that ameliorated their deficiency; for example, during the first 1h, the magnesium-deprived rats drank 3.1 +/- 0.5ml MgCl2 and 1.1 +/- 0.4ml CaCl2, whereas the calcium-deprived rats drank 1.8 +/- 0.5ml MgCl2 and 3.9 +/- 0.4ml CaCl2. Thus, magnesium deprivation leads to a compensatory appetite for magnesium, and the appetites for magnesium and calcium are distinct and specific. The rapid expression of magnesium appetite suggests that it depends in part on innate, gustatory factors.

Calcium-deprived rats sham-drink CaCl2 and NaCl.

Calcium-deprived rats are often thought to increase their calcium intake as a result of learning, but recent studies indicate that there is also an unlearned component to the appetite. They also ingest large amounts of some non-calcium minerals, including sodium. We examined the contribution of post-ingestive feedback to drinking using calcium-deprived and replete rats that could sham-drink CaCl2 and NaCl. Rats fitted with gastric cannulae in order to allow ingested fluids to drain freely drank 0.3 M NaCl in six 1-h sessions with their cannulae open (sham), followed by two sessions with their cannulae closed. Their intake of 0.03 M CaCl2 was then measured in a similar series of tests (six with cannula open followed by two with it closed). Ingestion of both NaCl and CaCl2 was significantly greater in calcium-deprived than in replete subjects under both open and closed conditions. These differences reached significance within 15 min after the onset of drinking during the first test with NaCl, and within 5 min in subsequent tests. The differences in CaCl2 intake generally reached significance within 5 min, including during the first test. Because there was minimal opportunity for post-ingestive NaCl or CaCl2 to mediate learning, the results provide additional support that the appetite for CaCl2 and NaCl in calcium-deprived rats can be driven solely by orosensory factors.

Lesions of the subfornical organ decrease the calcium appetite of calcium-deprived rats.

There are several indications that neurons in the rats subfornical organ (SFO) are sensitive to internal calcium status. We investigated the role of the SFO in regulating calcium intake by comparing the consumption of 30 mM CaCl2 by rats with (a) lesions of >90% of the SFO, (b) lesions that left the SFO mostly intact but disconnected its rostroventral stalk, (c) misplaced lesions that spared most of the SFO, or (d) a sham lesion procedure. In one experiment involving calcium-replete rats, these four groups had similar CaCl2 intakes. In another experiment involving calcium-deprived rats, those with lesions of the SFO or its rostroventral stalk consumed less CaCl2 than did those with missed or sham lesions. The SFO therefore appears to play a role in the calcium appetite that accompanies calcium deprivation in rats, most likely through its rostroventral efferents, but it is not important for need-free calcium intake.

COMPARISON OF DIFFERENCES BETWEEN PWD/PhJ AND C57BL/6J MICE IN CALCIUM SOLUTION PREFERENCES AND CHORDA TYMPANI NERVE RESPONSES

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.

Licking for taste solutions by potassium-deprived rats: specificity and mechanisms.

There has been little work on the specificity and mechanisms underlying the appetite of potassium (K(+)) deprived rats, and there are conflicting results. To investigate the contribution of oral factors to changes in intake induced by K(+) deficiency, we conducted two experiments using 20-s brief access tests. In Experiment 1, K(+)-deprived rats licked less for water than did replete rats. After adjusting for this difference, K(+)-deprived rats exhibited increased licking for 100 mM CaCl(2), 100 mM MgCl(2), and 100 mM FeCl(2) compared with K(+)-replete rats. In Experiment 2, which used larger rats, the K(+)-deprived and replete groups licked equally for water, 500 mM Na.Gluconate, 350 mM KCl, 500 mM KHCO(3), and 1 mM quinine.HCl, but the K(+)-deprived rats licked more for 500 mM KCl, 500 mM CsCl, and 500 mM NaCl than did the replete rats. Licking was unaffected by addition to NaCl of 200 muM amiloride, an epithelial Na(+) channel (ENaC) blocker, or 100 muM ruthenium red, a vanilloid receptor 1 (VR-1) antagonist, or by addition to KCl of 50 muM 4-aminopyridine, a K(+) channel blocker. These findings suggest that K(+)-deprivation produces a non-specific appetite that is guided by oral factors. We found no evidence that this response was mediated by ENaC, VR-1, or K(+) channels in taste receptor cells.

Experience with Sugar Modifies Behavioral but not Taste-Evoked Medullary Responses to Sweeteners in Mice

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).