Sweet and bitter taste in the brain of awake behaving animals

Abstract

Taste is responsible for evaluating the nutritious content of food, guiding essential appetitive behaviours, preventing the ingestion of toxic substances, and helping to ensure the maintenance of a healthy diet. Sweet and bitter are two of the most salient sensory percepts for humans and other animals; sweet taste allows the identification of energy-rich nutrients whereas bitter warns against the intake of potentially noxious chemicals1. In mammals, information from taste receptor cells in the tongue is transmitted through multiple neural stations to the primary gustatory cortex in the brain2. Recent imaging studies have shown that sweet and bitter are represented in the primary gustatory cortex by neurons organized in a spatial map3,4, with each taste quality encoded by distinct cortical fields4. Here we demonstrate that by manipulating the brain fields representing sweet and bitter taste we directly control an animal’s internal representation, sensory perception, and behavioural actions. These results substantiate the segregation of taste qualities in the cortex, expose the innate nature of appetitive and aversive taste responses, and illustrate the ability of gustatory cortex to recapitulate complex behaviours in the absence of sensory input.

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Figure 1: Place preference by photostimulation of the sweet and bitter cortical fields.
Figure 2: Photostimulation of bitter and sweet cortical fields drives aversive and appetitive behaviours.
Figure 3: Go/no-go taste discrimination task in head-restrained mice.
Figure 4: Inactivation of the bitter and sweet cortical fields disrupts taste discrimination.
Figure 5: Cross-generalization between orally supplied taste stimuli and photostimulation of the sweet cortex.

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Acknowledgements

We particularly thank H. Fischman and R. Lessard for suggestions, and members of the Zuker laboratory for comments. We also thank D. Salzman, K. Scott, and R. Axel for discussions. This research was supported in part by a grant from the National Institute of Drug Abuse (DA035025) to C.S.Z., and the Intramural Research Program of the National Institutes of Health, National Institute of Dental and Craniofacial Research (to N.J.P.R.). C.S.Z. is an investigator of the Howard Hughes Medical Institute and a Senior Fellow at Janelia Farms Research Campus, Howard Hughes Medical Institute.

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Authors

Contributions

Y.P. designed the study, performed experiments, and analysed data; S.G.-S. performed animals studies, viral injections, histology and analysed data; H.J. performed c-Fos expression studies; D.T. developed the initial behavioural platforms; N.J.P.R. and C.S.Z. designed the study, analysed data, and together with Y.P. wrote the paper.

Corresponding authors

Correspondence to Nicholas J. P. Ryba or Charles S. Zuker.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Expression of ChR2 in taste cortex.

a, Samples of injection sites in the bitter and sweet cortical fields; shown are coronal sections (Fig. 1a shows a whole mount brain). ChR2–YFP expression (green), nuclei (blue; TO-PRO-3); numbers indicate position relative to bregma, and the dotted area highlight the location of the taste cortical fields (see c). b, Activation of insular neurons in sweet cortex triggers robust c-Fos expression; ChR2–YFP (green), c-Fos (red) after 10 min of in vivo photostimulation at 20 Hz, 20-ms pulses (5 s laser on, 5 s laser off, 5 mW). Dashed lines indicate the location of the stimulating cannulae/fibre. c, c-Fos (red) expression in bitter cortex (bregma 0, −0.2) after bitter tastant stimulation (10 mM quinine; see Methods for details). Note the absence of c-Fos expression in the middle (bregma +0.7) and sweet insular cortex (bregma +1.5). Importantly, specific labelling is abolished in taste blind animals (TRPM5 knockouts; middle row). The bottom row shows a diagram of the corresponding brain areas, adapted from the Allen Brain Atlas. Scale bars: 1 mm (a), 500 μm (b), 300 μm (c). PIR, piriform cortex; IC, insular cortex.

Extended Data Figure 2 Acquisition of Place preference.

a, The development of ‘place preference’ as a function of session number (each session was 30 min of training and 5 min of ‘after-training’ testing in the absence of light stimulation; n = 13 for sweet cortex, n = 15 for bitter cortex; see text and Methods for details). The average of sessions 6–8 was used in Fig. 1. Values are mean ± s.e.m. b, Representative mouse track and quantitation of preference index in control GFP-expressing mice; note no difference in preference between chambers (n = 14; Mann–Whitney U-test, P = 0.74). Values are mean ± s.e.m.

Extended Data Figure 3 Photostimulation of insular cortical fields overcomes natural taste valence.

a, Quantitation of licking responses in mice expressing ChR2 in the bitter cortical fields (n = 13, analysis of variance (ANOVA) test, Tukey’s honest significant difference post hoc test). Photostimulation of the bitter cortical fields significantly suppress the natural attraction of the sweet tastant (4 mM AceK). b, Quantitation of licking responses in mice expressing ChR2 in the sweet cortical fields (n = 14, ANOVA test, Tukey’s honest significant difference post hoc test). Photostimulation of the sweet cortical fields significantly overcomes the natural aversion of the bitter tastant (1 mM quinine). In both experiments, mice were water-restrained (but exhibited an average of not more than 30 licks per 5-s water trial) such that they were motivated to drink the bitter while showing attraction to sweet. Values are mean ± s.e.m.

Extended Data Figure 4 TRPM5 knockout mice do not taste sweet and bitter.

Taste preference was tested in the head-restrained assay for wild type and TRPM5 homozygous mutants. Tastants were randomly delivered for a 5-s window (ten trials each). No significant difference was observed between water and sweet/bitter tastants in TRPM5 knockouts (ANOVA test, P = 0.62, n = 10; see ref. 10 for more details); circles indicate individual animals; bar graphs show mean ± s.e.m.

Extended Data Figure 5 Inactivation of the bitter cortical fields in animals trained to go to bitter and no-go to sweet.

a, Quantitation of performance ratios before and after bilateral silencing of the bitter cortical fields (NBQX, 5 mg ml−1; n = 7) in animals trained to go to bitter and no-go to sweet. Note the impact in bitter taste discrimination, but no significant effect in sweet taste (Mann–Whitney U-test, P <0.002). After washout of the drug, the animal’s ability to recognize bitter is restored (Mann–Whitney U-test, P <0.005). b, Quantitation of performance ratios with saline (0.9%) control in the bitter cortical fields (n = 6, Mann–Whitney U-test, P = 0.56). In both experiments, mice were trained with quinine and AceK, and tested with two pairs of sweet/bitter tastants (0.1 mM quinine and 2 mM AceK, 2 μM cycloheximide and 50 mM sucrose; see Methods for details).

Extended Data Figure 6 Sweet and low salt are appetitive tastants.

Taste preference was tested during a 10-min window using the head-restrained assay (see Methods for details). Four tastants were randomly delivered to animals for 5 s each (ten trials per tastant). Note that animals show significant attraction to sweet (AceK) and low salt (NaCl), but strong aversion to bitter (n = 11, ANOVA test, Tukey’s honest significant difference post hoc test); circles indicate individual animals; bar graphs show mean ± s.e.m. These conditions were used in the experiments described in Fig. 5 and Extended Data Fig. 7.

Extended Data Figure 7 Cross-generalization between orally supplied taste stimuli and photostimulation of the bitter cortex.

a, Representative histograms illustrating cross-generalization between taste stimulation and photostimulation of the bitter cortical field. The mouse was trained to go to bitter (0.5 mM quinine) and no-go to sweet (4 mM AceK) and low salt (20 mM NaCl). b, Quantitation of the responses from individual animals to quinine, AceK, salt and salt + light (n = 8, Mann–Whitney U-test, P <0.002). See also Fig. 4.

Supplementary information

Supplementary Table

This file contains Supplementary Table 1, coordinates of the injection and cannulation sites. (PDF 83 kb)

Behavioural responses to bitter cortex stimulation.

A mouse expressing ChR2-YFP in the bitter cortex before and after photostimulation. First trial, control experiment with the animal robustly drinking water (yellow circle indicates trial-initiation). Second trial shows prototypical orofacial responses (normally triggered by oral presentation of bitter tastants), now elicited by direct stimulation of bitter cortex (5-10 mW). Third trial shows gaping responses, and attempts to clean the mouth of the "fictive" bitter taste following strong stimulation (10-20 mW); note that the animal does not even sample the water drop; under these stimulating conditions 30% of the animals exhibit gagging behaviour. Stimulation of sweet cortex never induced such behaviour. (MOV 3993 kb)

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Peng, Y., Gillis-Smith, S., Jin, H. et al. Sweet and bitter taste in the brain of awake behaving animals. Nature 527, 512–515 (2015). https://doi.org/10.1038/nature15763

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