High salt recruits aversive taste pathways

Abstract

In the tongue, distinct classes of taste receptor cells detect the five basic tastes; sweet, sour, bitter, sodium salt and umami. Among these qualities, bitter and sour stimuli are innately aversive, whereas sweet and umami are appetitive and generally attractive to animals. By contrast, salty taste is unique in that increasing salt concentration fundamentally transforms an innately appetitive stimulus into a powerfully aversive one. This appetitive-aversive balance helps to maintain appropriate salt consumption, and represents an important part of fluid and electrolyte homeostasis. We have shown previously that the appetitive responses to NaCl are mediated by taste receptor cells expressing the epithelial sodium channel, ENaC, but the cellular substrate for salt aversion was unknown. Here we examine the cellular and molecular basis for the rejection of high concentrations of salts. We show that high salt recruits the two primary aversive taste pathways by activating the sour- and bitter-taste-sensing cells. We also demonstrate that genetic silencing of these pathways abolishes behavioural aversion to concentrated salt, without impairing salt attraction. Notably, mice devoid of salt-aversion pathways show unimpeded, continuous attraction even to very high concentrations of NaCl. We propose that the 'co-opting' of sour and bitter neural pathways evolved as a means to ensure that high levels of salt reliably trigger robust behavioural rejection, thus preventing its potentially detrimental effects on health.

Figure 1. Bitter receptor cells mediate high-salt taste responses

Allyl isothiocyanate (AITC) acts as a selective inhibitor of bitter and high-salt taste responses. (a) Shown are integrated chorda tympani responses to taste stimuli (see methods for details) before (−) and after (+) application of AITC; amiloride was used to selectively eliminate the contribution of the ENaC-dependent, low-salt pathways. AITC completely inhibited bitter responses (0.1 mM cycloheximide) and significantly suppressed high-salt (250 or 500 mM NaCl + amiloride and KCl) responses (highlighted in red) but did not affect responses to low salt (60 mM NaCl) or other taste qualities; representative responses from multiple animals are shown. See Supplementary Figure 1 for quantitation. (b) Calcium imaging of taste cell responses confirmed that T2R32-Sapphire positive taste cells respond to bitter stimuli (mixture of 1 mM cycloheximide, 1 mM quinine, and 10 mM denatonium) and high-salt (500 mM KCl) but not to sour stimuli (100 mM citric acid). Shown is a taste bud overlaid with Sapphire fluorescence (dotted circle, left) and pseudo-colored images depicting taste responses to high-salt, bitter and sour stimuli (right panels); scale bar, 10 µm. Below the imaging panels are representative ΔF/F traces for these tastants from three additional Sapphire-positive cells. In total, 15 and 12 Sapphire-positive cells were activated by bitter and KCl respectively; among these, 11 cells were activated by both compounds, but not by sour stimuli (see Supplementary Figure 4)

Figure 2. High-salt responses in bitter cells are TRPM5/PLCβ2 dependent

(a) Representative chorda tympani responses from control (WT), TRPM5-KO, PLCβ2-KO (PLC KO) and T2R32-PLCβ2 (T2R-PLC) rescue mice before (−) and after (+) application of AITC. Note that both TRPM5-KO and PLCβ2-KO mice lose bitter responses and significant part of their response to high-salt together with all sensitivity to AITC. Expressing PLCβ2 in just the bitter cells of PLCβ2-KO mice (T2R-PLC rescue) fully restores normal bitter and high-salt responses as well as AITC sensitivity of these responses (denoted by red traces); note responses to NaCl and amiloride were from different animals from the other responses shown. (b) Quantification of normalized responses, before (open bars) and after (red bars) application of AITC (mean ± s.e.m, n ≥ 3 animals, see methods for normalization). AITC treatment almost completely suppressed responses to 0.1 mM cycloheximide and reduced by half the responses to 500 mM KCl and 500 mM NaCl in the presence of 10 µM amiloride in control and T2R-PLC rescue animals (Student’s t-test, P < 0.05).

Figure 3. PKD2L1-expressing cells mediate the residual TRPM5/PLCβ2-independent high-salt responses

Integrated chorda tympani recordings show that (a) silencing PKD2L1 sour-cells affects high-salt taste responses. PKD2L1-TeNT mice have severe deficits in their responses to high-salt while TRPM5-KO / PKD2L1-TeNT double mutant animals completely lose all amiloride insensitive NaCl (high-salt) responses (highlighted as red traces). (b) Quantification demonstrates that TRPM5-KO / PKD2L1-TeNT (Double, shown as red bars) mice exhibit normal responses to low salt, but (c) lack responses to high-salt (Student’s t-test, P< 0.001). (d) The double mutant animals also fail to respond to sweet, bitter, sour, umami as well as non-sodium salts. Data (b–d) were normalized to the response of 60 mM NaCl and are means ± s.e.m, n ≥ 3 animals.

Figure 4. TRPM5-KO / PKD2L1-TeNT mice exhibit no taste aversion to high-salt

Immediate lick assays were used to measure behavioral responses to KCl (aversive, panel a) and NaCl (attractive, panel b). (a) Control mice (WT, solid black line) exhibit robust dose dependent behavioral aversion to increasing concentrations of KCl. In contrast, TRPM5-KO / PKD2L1-TeNT double mutant animals (red line) do not avoid high-salt stimuli; single mutants (dotted lines) behave as control animals. Two-way ANOVA with post hoc test for individual concentrations revealed significant differences at 500 mM KCl between the double mutants and other genotypes (P< 0.001), and at 250 mM KCl between the double mutants and control or PKD2L1-TeNT mice (P<0.001). (b) After sodium depletion, control mice (black line) exhibit powerful attractive responses to NaCl (see also Supplementary Figure 7) but the attraction is considerably reduced at higher concentration (500 mM). In contrast, double mutant animals (red line) show a continuous increase in attraction even at concentrations as high as 500 mM NaCl (two-way ANOVA with post hoc test, P< 0.001). Values are means ± s.e.m., n ≥ 6 mice.