T1R2+T1R3-independent chemosensory inputs contributing to behavioral discrimination of sugars in mice

Abstract

Simple sugars are thought to elicit a unitary sensation, principally via the “sweet” taste receptor type 1 taste receptor (T1R)2+T1R3, yet we previously found that rats with experience consuming two metabolically distinct sugars, glucose and fructose, subsequently licked more for glucose than fructose, even when postingestive influences were abated. The results pointed to the existence of an orosensory receptor that binds one sugar but not the other and whose signal is channeled into neural circuits that motivate ingestion. Here we sought to determine the chemosensory nature of this signal. First, we assessed whether T1R2 and/or T1R3 are necessary to acquire this behavioral discrimination, replicating our rat study in T1R2+T1R3 double-knockout (KO) mice and their wild-type counterparts as well as in two common mouse strains that vary in their sensitivity to sweeteners [C57BL/6 (B6) and 129X1/SvJ (129)]. These studies showed that extensive exposure to multiple concentrations of glucose and fructose in daily one-bottle 30-min sessions enhanced lick responses for glucose over fructose in brief-access tests. This was true even for KO mice that lacked the canonical “sweet” taste receptor. Surgical disconnection of olfactory inputs to the forebrain (bulbotomy) in B6 mice severely disrupted the ability to express this experience-dependent sugar discrimination. Importantly, these bulbotomized B6 mice exhibited severely blunted responsiveness to both sugars relative to water in brief-access lick tests, despite the fact that they have intact T1R2+T1R3 receptors. The results highlight the importance of other sources of chemosensory and postingestive inputs in shaping and maintaining “hardwired” responses to sugar.

INTRODUCTION

The discovery of the type 1 taste receptor (T1R) family nearly 20 years ago shaped the prevailing view of how the mammalian gustatory system is organized, at least with respect to “sweet” taste (1, 10, 23, 26, 27, 31, 33, 34, 39, 51). Although it was already known that a diverse array of chemicals, including simple sugars like glucose and fructose, artificial sweeteners, and some d-amino acids, elicit a certain shared quality and palatable sensation, it was unclear whether this was due to the fact that their various peripheral receptors fed into common central circuitries underlying such processes or whether they were commonly bound by a single receptor in the periphery. As it turned out, genetic deletion of one or both of the T1R proteins that comprise the heterodimeric T1R2+T1R3 receptor profoundly attenuated electrophysiological and behavioral responses to these various ligands, lending rather compelling support for the latter view (1, 7, 23, 26, 34, 47).

Nevertheless, there are still hints in the literature suggesting that alternative receptors and/or divergent signaling pathways exist for T1R ligands, especially sugars (7, 8, 13, 19–21, 35). For example, some sugars (glucose and perhaps sucrose) stimulate a cephalic-phase insulin response, even in mice that lack a functional T1R3 receptor, whereas other sugars (e.g., fructose) do not (19–21). Moreover, we recently demonstrated that when rats have had the opportunity to consume two monosaccharides, glucose and fructose, and therefore to experience their distinct postingestive effects, they subsequently respond more positively to the orosensory properties of glucose over those of fructose (41). As such, the orosensory signals generated by glucose and fructose must be differentiated by the brain. One straightforward possibility is that there is an alternative, T1R-independent, sugar taste receptor in the oral cavity whose signal is channeled into central circuits that subserve ingestive motivation. A second possibility is that taste reception of both sugars is mediated entirely via the T1Rs but because of variances in the binding affinities of each of the subunits to one or both sugars and/or the existence of functional homodimers that vary across the taste field, glucose and fructose effectively yield dissociable signals. A third possibility is that the critical alternative orosensory receptor resides outside the gustatory system. Thus here we sought to determine whether the critical source of chemosensory input needed to learn and subsequently express enhanced behavioral responsiveness for the orosensory properties of glucose over fructose is linked to the canonical sugar taste receptor, using mouse strains that are sensitive [C57BL/6 (B6)], subsensitive [129X1/SvJ (129)], or altogether insensitive [T1R2+T1R3 double knockout (KO)] to T1R ligands (experiments 1 and 2; see results for more details). A second aim of this study was to determine whether olfactory input contributes to the experience-dependent capacity to behaviorally discriminate the orosensory properties of glucose from fructose (experiment 3).

MATERIALS AND METHODS

All experimental procedures were reviewed and approved by the Florida State University Animal Care and Use Committee and conducted in accordance.

Subjects

C57BL/6 (https://www.jax.org/strain/000664) (B6) and 129X1/SvJ (https://www.jax.org/strain/000691) (129) mice were purchased from Jackson Laboratory (Bar Harbor, ME). Mice lacking both the Tas1r2 gene and the Tas1r3 gene [T1R2+T1R3 double knockout (KO)] and their wild-type (WT) counterparts were generated in-house from original breeding pairs that were homozygous-null for Tas1r2 or Tas1r3 and generously provided by Dr. Charles Zuker (Columbia University, New York, NY) as follows: Homozygous-null mice were paired with WT C57BL/6J mice to produce mice that were heterozygous for Tas1r2 or Tas1r3. Then, these mice were mated to produce heterozygous, homozygous-null, and WT mice. The resultant Tas1r2 and Tas1r3 homozygous-null mice were then paired together to generate the Tas1r2+3 homozygous-null mice for this experiment. Likewise, WT mice were paired with one another to yield the WT control mice. Single-nucleotide polymorphism scanning of the known genetic polymorphisms between the B6 and 129 strains (completed by Jackson Laboratory) revealed that the T1R2+T1R3 KO and WT mice derive ~20–30% of their genetic background from the 129X1/SvJ strain and the remaining ~70–80% from the C57BL/6 strain. At the conclusion of experiments 1 and 2, the genotypes of a subset of T1R2+T1R3 KO and WT mice from each of the homozygous breeding pairs were confirmed with real-time PCR analysis of tail samples (Transnetyx, Cordova, TN).

All mice were singly housed for at least 1 wk before the start and throughout the experiments in standard polycarbonate cages. All mice had ad libitum access to deionized water (dH₂O) and rodent chow (Purina no. 5001), except when food and/or water deprivation was instituted for experimental purposes (see Food and/or Water Deprivation Procedures and Figs. 2, 5, and 9 for details). Environmental enrichment in the form of Nestlets (Ancare) was provided in the home cage. Mice were between ~12 and 35 wk of age at the start of the experiments. Strains and sample sizes for each experiment are indicated in figure legends. Because of a vivarium-wide pinworm outbreak, T1R2+T1R3 KO and WT mice in the first experiment were maintained on Purina 5001 chow containing fenbendazole for a period of at least 14 wk; however, all mice were returned to regular chow and treatment was discontinued 12 wk before the start of the experiment.

Fig. 2.

Training groups, training and testing schedule, and stimuli for experiment 1. †Deprivation regimens are described in detail in materials and methods. ‡Single-access training stimuli were presented in randomized order without replacement across each block. This schedule resulted in five 6-day trial blocks for the glucose vs. fructose training (GvF) group and ten 3-day trial blocks for the glucose-only (Glu-Only) and sugar-naive training groups for a total of 30 sessions. *Half of the mice in the GvF group received 0.56 M glucose (G) on the very first session, and the other half received 0.56 M fructose (F). Sugars were presented in the indicated molar concentrations; corn oil emulsion (COe) was made in the indicated % (vol/vol) concentration. For brief-access testing, stimuli were serially presented in 10-s trials in a randomized order across blocks of trials (without replacement). All exposure and test sessions were run in the Davis Rigs. Dep. state, deprivation state; dH₂O, deionized water; N/A, not applicable; Sess, sessions.

Fig. 5.

Groups and schedule of exposure and testing phases for experiment 2. ^bAll training and testing sessions were conducted in the Davis Rigs. †Deprivation regimens are described in detail in materials and methods. ‡Single-access stimuli were presented in randomized order without replacement across each 6-day exposure block. *Half of the mice in the glucose vs. fructose training (GvF) group received 0.56 M glucose (G) on the very first session, whereas the other half received 0.56 M fructose (F). Sugars were presented in the indicated molar concentrations and corn oil emulsion (COe) in the indicated % (vol/vol) concentration. For brief-access testing, stimuli were serially presented in 10-s trials in a randomized order across blocks of trials (without replacement). All exposure and test sessions were conducted in the Davis Rigs. ^¤Differences in the latency to initiate glucose and fructose trials emerged on this test; therefore, to examine whether olfactory input was playing a role in the glucose vs. fructose discrimination, these mice underwent surgery to disconnect the olfactory bulb from the forebrain after this. However, because of small postsurgical sample sizes, the procedures and data were excluded. Dep. state, deprivation state; dH₂O, deionized water; N/A, not applicable; Sess, sessions.

Fig. 9.

Groups and schedule of exposure and testing phases for experiment 3. ^ƀAll training and testing sessions were conducted in the gustometers. †Deprivation regimens are described in detail in materials and methods. ‡Single-access stimuli were presented in randomized order without replacement across each 6-day exposure block. *All of the mice received 0.56 M glucose on the very first session; the schedule of sugar presentation was randomized thereafter. Sugars were presented in the indicated molar concentrations. For brief-access testing, stimuli were serially presented in 10-s trials in a randomized order across blocks of trials (without replacement). Dep. state, deprivation state; dH₂O, deionized water; F, fructose; G, glucose; N/A, not applicable; Sess, sessions.

Stimuli

Reagent-grade glucose (d-dextrose; BDH Chemicals), fructose (Sigma-Aldrich), and sucrose (Mallinckrodt) were prepared fresh with dH₂O as needed. Corn oil emulsions were made by mixing (vol/vol) 2.5%, 4.5%, or 8.9% corn oil (Winn-Dixie Brand, Jacksonville, FL) with 0.6% of an emulsifying agent (Emplex; generously donated by Caravan Ingredients, Lenexa, KS) and dH₂O in a standard kitchen blender before the training sessions, as needed, and were periodically remixed throughout the day.

Apparatuses

Single-access exposure and brief-access tests were conducted in one of two types of apparatuses: Davis Rigs (Davis MS-160; DiLog Instruments, Tallahassee, FL) for experiments 1 and 2 or the gustometer (custom made at Florida State University’s Instrumentation Shop, Tallahassee, FL; see Ref. 46 for a full description) for experiment 3.

The four identical Davis Rigs comprised a wire mesh grid floor, three Plexiglas walls, and a stainless steel front wall. The sipper tube access slot was located in the center of the front wall (~5 cm above the grid floor). Access to the sipper tube was granted through the opening and closing of a computer-controlled shutter. A motorized table was positioned just on the opposite side of the front wall. This table held up to 16 bottles with sipper tubes connected to a contact lickometer. Licks to the sipper spout were registered through a high-frequency AC circuit, time-stamped, and saved for subsequent analyses. During single-access exposure sessions, one sipper tube (i.e., solution) was available for 30 min. During brief-access tests, a series of different solutions were presented to the mouse in randomized blocks, without replacement, in short (i.e., 10 s) trials. Before each trial, the designated sipper tube was positioned in front of the access slot, the shutter opened, and the trial commenced when the mouse made contact with the sipper spout (i.e., the first lick). After 10 s elapsed, the shutter closed and the table repositioned to place a different tube in front of the access slot. The time between each trial was 7.5 s. The brief-access test sessions lasted 30 min in total, during which time the mice could initiate as many trials as possible. A small fan was located above the center access slot to minimize odor cues.

Because of equipment availability, each of six identical gustometers was used for sugar exposure and brief-access tests in experiment 3 (see results). These comprised a modified operant chamber with three equally spaced access slots on the front panel (left, center, right) and an interfacing computer-controlled fluid stimulus delivery system and lick response measurement system. For these experiments, the left and right access slots were permanently closed. Each gustometer was equipped with a turret, which held up to 14 separate stimulus delivery tubes. Proximate to this turret and just behind the center access slot of the chamber was a borosilicate glass “sample” ball, mounted on a mechanical arm by a horizontal axle. For single-exposure sessions, the mechanical arm positioned the sample ball between the turret and the center access slot and the turret was oriented so that a single tube was in line with the ball. Each lick at the sample ball was registered by a load cell and operated a pump to dispense ~1 µl/lick onto the ball. Licks were time-stamped and saved for subsequent analyses. Brief-access test sessions were run similarly, except that each trial commenced when the mouse licked at the sample ball twice within 250 ms and ended 10 s thereafter. Between each trial, the mechanical arm retracted the sample ball into wash (dH₂O) and dry (pressurized air) station before returning to it to the access position. The turret aligned a different tube (and hence solution) to the sample ball for the next trial. The time between each trial was 7.5 s. Each test lasted a total of 30 min, during which time the mouse was free to initiate as many trials as possible. Stimuli were presented in randomized blocks without replacement. A vacuum duct was located just over the center slot that continuously drew air out of the area to preclude odor cues.

Behavioral Training and Testing Procedures

Specific training and testing schedules are provided in Figs. 2, 5, and 9. Detailed descriptions of the deprivation procedures employed are found below (see Food and/or Water Deprivation Procedures).

Food and/or Water Deprivation Procedures

Four types of deprivation regimens were used to motivate mice to sample from the sipper spout and initiate trials in single-access exposure phases and brief-access tests. Each is described in turn. The particular deprivation procedures used are specified in the respective experimental schedules in Figs. 2, 5, and 9. For water deprivation, home cage water bottles were removed and fluid access was limited to the 30 min per day that the mouse was in the Davis Rig or gustometer. Mice that fell below 85% of their ad libitum body weight were provided supplemental dH₂O (1–2 ml) in the home cage after the daily session. Mice that fell below 80% for 2 consecutive days were given their home cage water bottle back until the weight was recovered. Food deprivation was either acute or chronic. Acute deprivation consisted of removing chow from the home cage ~22 h before the test session. Chronic deprivation consisted of gradually reducing mice to 85% of their ad libitum body weight by rationing chow. Once mice were at the target weight, single-access exposure sessions or testing commenced. The daily ration was provided ~30 min after the access/test session. In both food deprivation regimens, chow was returned to the home cage ~30 min after the last exposure/test session. Partial food and water deprivation was used for some phases. With this method, water bottles and chow were removed from the home cage and limited amounts of chow (1 g) and dH₂O (2 ml) were given ~30 min after the daily session. Albeit rare, mice that dropped below 85% of their ad libitum body weight were given extra chow and/or dH₂O supplements as needed. Ad libitum chow and water bottles were returned to the home cage ~30 min after the last exposure/test session.

Sugar/Corn Oil Single-Access Exposure

Water-deprived mice were initially trained to lick for dH₂O in daily 30-min sessions at a single spout in the Davis Rig or gustometer. Once the mice were behaving competently, the dH₂O was replaced with 4.5% corn oil emulsion to accustom the mice to receiving calories during these 30-min sessions. Training sessions were conducted identically to these sessions, except that mice were only given access to their assigned training stimuli. KO and WT mice were divided into three training groups that were given access to 1) three glucose and fructose concentrations in separate sessions (GvF), 2) only the glucose solutions (Glu-Only), or 3) three equicaloric concentrations of corn oil emulsion (sugar naive). The stimulus concentrations were presented in randomized order without replacement in session blocks, except that the middle stimulus concentration was presented on the first session of the first block. For the GvF group, half the mice started with 0.56 M glucose and the other half started with 0.56 M fructose. In follow-up experiments, KO, B6, and 129 mice were given GvF for this exposure phase. In some cases, after initial training under water-restricted conditions mice were switched to either chronic food deprivation, in which they were maintained at 85% of their ad libitum body weight with chow rations provided after the daily training session, or partial food and water deprivation for additional training (see Figs. 2, 5, and 9). In general, these deprivation state switches took 1–5 days.

Brief-Access Training and Testing

Changes in orosensory-guided behavior as a function of training were periodically assessed with brief-access tests, which were also conducted in the Davis Rigs or gustometers. In these tests, an array of taste stimuli was presented (i.e., 7 different solutions/concentrations) in randomized blocks of 10-s trials across the 30-min session (see Figs. 2, 5, and 9). Before the first brief-access test, all mice were water restricted and accustomed to the trial structure with dH₂O for several sessions. Thereafter, sugar solutions were presented in place of dH₂O. Some tests were conducted under acute (~22–24 h) or chronic food deprivation. In general, 1–3 days was interposed between each test.

Surgery

B6 mice in experiment 3 were bulbotomized under isoflurane anesthesia (~5% induction rate; ~1.5–3% maintenance rate). A midline incision was made in the scalp, exposing the coronal suture. Bilateral burr holes were drilled just anterolaterally to the intersection of the sagittal and coronal sutures, with care taken to avoid the underlying vasculature. A blunt 26-gauge needle was used to transect the tissue between the olfactory bulb and forebrain. Gel foam was used to suppress bleeding. The incision was closed with sterile 3-0 silk suture. Sham surgeries were done identically, except that no transection was made. Mice were treated perisurgically and on the day after surgery with buprenorphine (0.1 mg/kg sc), carprofen (5 mg/kg sc), and gentamicin (8 mg/kg sc). Carprofen and gentamicin were given for an additional 2 days to aid recovery. Food intake, water intake, and nesting activity were monitored for at least 10 days after surgery. Mice that showed signs of stress after surgery were given additional buprenorphine, as needed.

Behavioral and Histological Verification of Bulbotomies

Before surgery and at the conclusion of the experiment, mice were provided a 0.5 g of a Pringles Original potato crisp in the home cage overnight to acquaint them with its orosensory and nutritive properties. Then, mice were gradually reduced to 85% of their free-feeding body weight and given a buried crisp test (BCT) to assess olfactory function. In this test (50), each mouse was transferred into a clean polycarbonate mouse cage with ~2-cm-deep clean bedding. After a 10-min acclimation period, the mouse was placed in an identical test cage. A 0.5-g Pringles Original crisp was buried 2 cm below the bedding surface in one of the four quadrants of the cage (placement of the crisp varied across tests). Latency to find the crisp (i.e., make contact with the crisp) was recorded. Any mouse that did not find the crisp within 15 min was immediately transferred into a third cage, in which 0.5 g of Pringles Original potato crisp was placed on the surface of the bedding (i.e., visual crisp test). Latency to find the crisp was recorded (maximum time limit = 15 min); this was done to ensure that failure to find the buried crisp was not simply due to a lack of motivation. Bulbotomized mice that took 1.5 times the interquartile range of the median B6 sham-operated (SHAM) group latency to find the buried crisp were considered olfactory impaired (referred to as the BULB group).

At the end of the experiments, SHAM and BULB mice were euthanized (lethal overdose with 1.5 ml/kg of Euthasol solution ip) and transcardially perfused with cold heparinized saline followed by formalin. Skulls were removed and postfixed in formalin for ~5 days. The skulls were then slowly decalcified by incubation in 10% EDTA in 0.1 M PBS (solution refreshed every 2–4 days). After the skull was completely removed, the brain was postfixed in 12% sucrose in 4% paraformaldehyde overnight, blocked, snap frozen, and then stored at −80 ^◦C until sectioning. Serial 60-µm sagittal sections were taken through the entire brain. Sections were mounted on chromium-aluminum double-subbed slides and Nissl stained for histological inspection by an experimenter who was blind to the surgical, training, and genotype conditions of the mice.

Standardization of Stimulus Licks

To compare relative licking response toward the various solutions presented in the brief-access test, the number of licks/10-s trial was standardized against water baseline as follows: lick score = mean licks to stimulus − mean licks to dH₂O.

Data Analyses

For all statistical analyses, P values of ≤0.05 were considered significant. Licks per exposure session were collapsed across concentration and compared across exposure block for each genotype/strain or surgical condition with repeated-measures ANOVAs. Licks to the various test stimuli in the brief-access tests were first standardized against water (see Standardization of Stimulus Licks). These lick scores were analyzed as a function of concentration within each exposure group or surgical condition with repeated-measures ANOVAs. Only mice that completed at least one full trial block were included in the brief-access test lick score analyses. Post hoc ANOVAs and t-tests were used, where appropriate, to break down overall significant main effects and interactions. Latency to initiate glucose versus fructose brief-access trials was analyzed with Wilcoxon matched-pairs tests at each concentration for each strain or group. Bonferroni corrections were made for multiple comparisons. Wilcoxon signed-rank tests were used to determine whether the latency to initiate fructose trials was different from 0 for SHAM and BULB groups separately. All conditions/groups included roughly equal numbers of males and females, but because of a lack of power, sex was not included as a factor in any of the analyses. Statistical outcomes are presented in figures or figure legends.

RESULTS

Experiment 1

We previously found that rats exposed to glucose and fructose (GvF) in separate daily 30-min sessions subsequently licked more for glucose than fructose, even when the postingestive effects of the two sugars were minimized in brief-access taste tests (41). Importantly, rats kept naive to sugar or given the same amount of experience with either sugar, but not both, licked comparably to glucose and fructose in these same brief-access tests. The implication is that experience with the divergent postingestive effects of glucose and fructose fostered a capacity to behaviorally discriminate them on the basis of their orosensory properties. The first aim of experiment 1 was to determine whether these same phenomena are expressed in a different species, the WT mouse. Furthermore, given that glucose and fructose are only known to bind to the same taste receptor, T1R2+T1R3, the second aim of this experiment was to determine whether this “sweet” taste receptor provides the source of input necessary to acquire and express an avidity for the orosensory properties of glucose over fructose. Mice lacking both of the subunits of the T1R2+T1R3 (KO) were thus compared with their WT counterparts.

Single-sugar exposure sessions.

WT and KO were subdivided into three exposure groups. One group (glucose vs. fructose, GvF) was exposed to three equimolar concentrations of glucose and fructose in separate daily 30-min sessions to effectively provide extensive experience with the oral and postoral properties of these two sugars. A second group of WT and KO mice were given the same amount of exposure to one sugar (glucose, Glu-Only). A third group of WT and KO mice were kept naive to sugar (sugar naive) and instead were provided with equimolar corn oil emulsions during these exposure sessions. All mice underwent these exposure sessions while water deprived. This was simply done to motivate ample intake and exposure, especially in the KO mice, who do not generally consume much sugar without reason. Furthermore, three different concentrations of each stimulus were used to preclude WT and KO mice from simply learning about intensity and/or viscosity cues during this phase. Exposure session intake data are collapsed across concentration in Fig. 1.

Fig. 1.

Experiment 1. A and C: mean ± SE total licks for glucose vs. fructose (GvF), collapsed across concentration for each 6-day exposure block for wild-type (WT, A) and T1R2+T1R3 knockout (KO, C) mice. WT (n = 8): sugar effect: F(1,7) = 31.49, P = 0.0008; block effect: F(4,28) = 9.71, P = 0.00005; sugar × block: F(4,28) = 2.86, P = 0.04. KO (n = 8): sugar effect: F(1,7) = 27.36, P = 0.03; block effect: F(4,28) = 1.46, P = 0.24; sugar × block: F(4,28) = 4.49, P = 0.006. Note that because of computer malfunction the data were lost for 1 session for 1 mouse in the GvF KO group, precluding its inclusion in the repeated-measures statistics and Bonferroni corrections. *Bonferroni-corrected significant pairwise differences in glucose and fructose intake. B and D: intake of glucose and fructose, collapsed across concentration, on the final exposure block (block 5) for each individual GvF WT (B) or GvF KO (D) mouse. E: mean ± SE total licks for glucose, collapsed across concentration, across ten 3-day exposure blocks for T1R2+T1R3 KO (n = 8) and WT (n = 8) mice given access to 1 sugar (Glu-Only). F: mean ± SE total licks for corn oil emulsion, collapsed across concentration, across ten 3-day exposure blocks for the T1R2+T1R3 KO (n = 7) and WT (n = 8) mice kept sugar naive.

The GvF KO mice, like the GvF WT mice, initially consumed comparable amounts of the two sugars. Again, this was not surprising, because the mice were water deprived to promote intake. However, over time both WT and KO mice selectively reduced their consumption of fructose relative to glucose (Fig. 1, A and C). As seen in Fig. 1, B and D, on the final exposure block all WT and KO mice in the GvF-exposed condition consumed more glucose relative to fructose.

Brief-access test sessions.

Because 30-min intake can be influenced by a myriad of factors, including early postingestive events, we then sought to determine whether the GvF exposure rendered an avidity for glucose over fructose, when the postingestive influences of the two sugars were abated, in a brief-access test. The WT and KO mice from each exposure condition were subjected to a series of these tests, in which the three concentrations of glucose and fructose were presented within the same session, in short (10 s) successive trials (order randomized) (details in materials and methods and Fig. 2).

WT mice that were kept sugar naive or provided extensive experience with only glucose (Glu-Only) licked in concentration-dependent fashions for both sugars in these brief-access tests, resembling our previous findings in rats (41) (Fig. 3, A and B and E and F). Sugar-naive KO mice were virtually unresponsive to the two sugars (Fig. 3, C and D). Stimulus-dependent lick responses were not rescued in Glu-Only KO mice, even though they had a lot of experience with that sugar (Fig. 3, G and H). Further replicating our initial findings in rats (41), WT mice exposed to both sugars (GvF) during a prior exposure phase licked selectively more for glucose, across all three concentrations, in these brief-access trials (Fig. 3, I and J). Importantly, GvF KO mice also licked significantly more for glucose, relative to fructose, in the brief-access test, despite the absence of functional T1R2 and T1R3 receptor subunits (Fig. 3, K and L). When the concentration ranges were downshifted in a subsequent test, GvF WT and KO mice continued to respond more for glucose, although the overall lick difference was noticeably smaller, just missing statistical significance in the GvF WT group (Fig. 4).

Fig. 3.

Experiment 1. A, C, E, G, I, and K: mean ± SE lick score for glucose and fructose, presented in randomized order of brief-access trials for wild-type (WT) and knockout (KO) mice kept sugar naive (A and C), previously exposed to glucose only (Glu-Only; E and G), or previously exposed to both sugars, glucose and fructose [glucose vs. fructose (GvF); I and K]. Data are collapsed across 2 tests conducted under food deprivation (tests 2 and 3). Test 1 was conducted under water deprivation; those data are presented in Fig. 7. Median (semi-interquartile range) latencies to initiate glucose (G) vs. fructose (F) trials are displayed below the respective concentrations in each panel. Bold italic numbers indicate Bonferroni-corrected significant pairwise differences in latency to initiate glucose and fructose trials. Sugar-naive WT (n = 8): sugar effect: F(1,7) = 0.13, P = 0.73; concentration effect: F(2,14) = 31.08, P = 0.000007; sugar × concentration: F(2,14) =9.74, P = 0.002; cumulative trials over 2 tests: 96.25 ± 8.02. Sugar-naive KO (n = 7): sugar effect: F(1,6) = 4.61, P = 0.08; concentration effect: F(2,12) = 0.05, P = 0.95; sugar × concentration: F(2,12) = 0.62, P = 0.56; cumulative trials over 2 tests: 79.38 ± 16.52. Glu-Only WT (n = 8): sugar effect: F(1,7) = 15.46, P = 0.006; concentration effect: F(2,14) = 109.76, P < 0.0000001; sugar × concentration: F(2,14) =25.81, P = 0.00002; cumulative trials over 2 tests: 100.63 ± 5.29. Glu-Only KO (n = 7): sugar effect: F(1,6) = 1.04, P = 0.35, concentration effect: F(2,12) = 1.15, P = 0.34; sugar × concentration: F(2,12) = 3.72, P = 0.06; cumulative trials over 2 tests: 72.88 ± 13.30. GvF WT (n = 8): sugar effect: F(1,7) = 23.06, P = 0.002; concentration effect: F(2,14) = 2.58, P = 0.11; sugar × concentration: F(2,14) = 2.68, P = 0.10; cumulative trials over 2 tests: 95.00 ± 14.35. GvF KO (n = 5): sugar effect: F(1,4) = 12.30, P = 0.02; concentration effect: F(2,8) = 0.36, P = 0.71; sugar × concentration: F(2,8) = 2.93, P = 0.11; cumulative trials over 2 tests: 81.50 ± 16.08. *Bonferroni-corrected significant pairwise differences in lick score between glucose and fructose. B, D, F, H, J, and L: lick scores for glucose and fructose, averaged across concentration, for each individual mouse in the sugar-naive (WT, B; KO, D), Glu-Only (WT, F; KO, H), and GvF (WT, J; KO, L) groups.

Fig. 4.

Experiment 1. A and C: mean ± SE lick scores for glucose and fructose in a lower concentration range than used for training, presented in randomized order in brief-access trials of wild-type (WT, A) and knockout (KO, C) mice that were previously exposed to glucose and fructose (GvF). GvF WT (n = 6): sugar effect: F(1,5) = 10.33, P = 0.02; concentration effect: F(2,10) = 3.59, P = 0.07. sugar × concentration: F(2,10) = 0.50, P = 0.62. GvF KO (n = 5): sugar effect: F(1,4) = 5.83, P = 0.07; concentration effect: F(2,8) = 1.86, P = 0.22; sugar × concentration: F(2,8) = 3.01, P = 0.11]. WT mice initiated a mean ± SE of 37.71 ± 27.23 trials, whereas KO mice initiated a mean ± SE of 31.14 ± 17.77 trials on this lower-concentration test. B and D: lick scores for glucose and fructose, averaged across concentration, for each individual WT(B) or KO (D) mouse in the GvF-exposed groups.

Together these data provide strong evidence that mice can distinguish the orosensory properties of glucose and fructose provided they have had prior ingestive experience with them both and that this capacity is not dependent on input from the T1R2+T1R3 receptor.

Experiment 2

To further probe the source of sensory input enabling this phenomenon, naive T1R2+T1R3 KO as well as B6 and 129 control mice were given exposure to glucose and fructose (GvF) in a series of 30-min intake sessions in experiment 2 (just as in experiment 1; full schedule in Fig. 5). B6 and 129 are the background strains for the KO mice and have different alleles on the Tas1r3 gene that are associated with differences in their sensitivity and preferences for T1R3-dependent ligands (2, 6, 16, 18). Under certain, but not all, conditions B6 mice are more responsive to sugars and other sweet substances than 129 mice (3, 11, 14, 18, 24, 37).

Single-sugar exposure sessions.

As in experiment 1, the exposure phase was initially conducted while mice were water restricted (blocks 1–5) to promote sampling, especially in the KO and 129 mice. Interestingly, under these conditions all three strains consumed roughly equal amounts of the glucose and fructose over successive blocks (Fig. 6). Nevertheless, B6 mice did display significantly elevated glucose intake in the fifth exposure block (Fig. 6, A and B). Because water deprivation may have been driving fluid intake to a ceiling and/or obscuring metabolic differences between the two sugars, all mice were shifted to food deprivation after block 5. Indeed, on the very first food-deprived exposure block (block 6) B6, 129, and KO mice all consumed significantly more glucose than fructose (Fig. 6).

Fig. 6.

Experiment 2. A, C, and E: mean ± SE total licks for glucose (red) or fructose (blue), collapsed across concentration for each 6-day exposure block for C57BL/6 (B6) (n = 16; A), 129X1/SvJ (129) (n = 16; C), and T1R2+T1R3 knockout (KO) (n = 15; E) mice. Exposure phase was conducted under 2 different deprivation conditions: water deprivation (wd) for blocks 1–5 and chronic food deprivation (fd) for blocks 6–10. The switch is demarcated by the vertical dashed line. Arrowheads on the x-axis indicate when brief-access probe tests were conducted. Blocks 1–5: B6 sugar effect: F(1,15) =24.12, P = 0.0002; block effect: F(4,60) = 20.18, P ≤ 0.00001; sugar × block: F(4,60) = 3.71, P = 0.009; 129 sugar effect: F(1,15) = 0.25, P = 0.63; block effect: F(4,60) = 0.88, P = 0.48; sugar × block: F(4,60) = 0.68, P = 0.61; KO sugar effect: F(1,15) = 22.54, P = 0.0003; block effect: F(4,60) = 13.81, P ≤ 0.000001; sugar × block: F(4,60) = 1.97, P = 0.11. Blocks 6–10: B6 sugar effect: F(1,15) = 29.56, P ≤ 0.000001; block effect: F(4, 60) = 11.83, P ≤ 0.00001; sugar × block: F(4,60) = 25.73, P ≤ 0.000001; 129 sugar effect: F(1,15) =210.32, P ≤ 0.000001; block effect: F(4,60) = 5.10, P = 0.001; sugar × block: F(4,60) = 11.78, P ≤ 0.000001; KO sugar effect: F(1,14) = 92.32, P ≤ 0.000001; block: F(4,54) = 5.30, P = 0.001; sugar × block: F(4,56) = 27.56, P ≤ 0.000001. *Significant Bonferroni-corrected pairwise comparisons. B, D, and F: intake of glucose and fructose, collapsed across concentration, on the final water deprivation exposure block (block 5), the first food deprivation exposure block (block 6), and the last food deprivation block (block 10) for each individual B6 (B), 129 (D), or KO (F) mouse.

Brief-access test sessions.

After some additional exposure, these mice were tested in a series of three brief-access tests to probe whether such exposure conferred increased acceptability for glucose when the postingestive influences of glucose and fructose were removed (conducted after exposure block 7). Tests 1 and 3 of this series were conducted after overnight food deprivation; test 2 was conducted after overnight water deprivation.

B6 mice showed robust enhancement of licking for glucose relative to fructose in this initial series of brief-access tests (test 1 and 2 data shown in Fig. 7, test 3 in Fig. 8, A and B). Interestingly, food-deprived KO and 129 mice also displayed an enhanced avidity for the orosensory properties of glucose in (test 1 in Fig. 7; test 3 in Fig. 8, E and F and I and J), but the lick differences were relatively weak in these two strains. Indeed, close inspection of the individual 129 and KO mice in Fig. 8, F and J, respectively, shows that some mice were not exhibiting any overall lick differences in these initial brief-access tests.

Fig. 7.

Outcomes of additional brief-access tests for experiments 1 and 2. Shaded subcolumns represent glucose lick scores; adjacent nonshaded subcolumns represent fructose lick scores. Significant effects or differences are indicated in italicized text. 129, 129X1/SvJ; B6, C57BL/6; Glu-Only, glucose-only training group; GvF, glucose vs. fructose training group; KO, knockout; WT, wild type.

Fig. 8.

Experiment 2. A, E, I and C, G, K: mean ± SE lick scores for glucose and fructose, presented in randomized order of brief-access trials, on 3 separate tests that were conducted relatively early in the exposure phase (test 3; A, E, I) and after additional successive blocks of sugar exposure (test 4; C, G, K). Test 1 and 2 data are in Fig. 7. Median (semi-interquartile range) latencies to initiate glucose (G) vs. fructose (F) trials are displayed in text below the respective concentrations in each panel. Bold italic numbers indicate Bonferroni-corrected significant pairwise differences in latency to initiate glucose and fructose trials. Lick scores and trials on test 3: C57BL/6 (B6) (n = 16) sugar effect: F(1,15) = 100.14, P ≤ 0.000001; F(2,30) = 25.71, P ≤ 0.000001; sugar × concentration: F(2,30) = 3.08, P = 0.06; trials: 48.18 ± 3.49; 129X1/SvJ (129) (n = 15) sugar effect: F(1, 14) = 7.26, P = 0.02; concentration effect: F(2,28) = 29.52, P ≤ 0.000001; sugar × concentration: F(2,28) = 0.68, P = 0.52; trials: 30.13 ± 4.12; knockout (KO, n = 14) sugar effect: F(1,13) = 23.45, P = 0.0003; concentration effect: F(2,26) = 6.59, P = 0.005; sugar × concentration: F(2,26) = 8.21, P = 0.002; trials: 28.19 ± 4.06. B, F, and J: lick scores for glucose and fructose, averaged across concentration, on test 3 for each individual wild-type (WT, B), 129 (F), or KO (J) mouse. Test 4: B6 (n = 16) sugar effect: F(1,15) = 527.78, P ≤ 0.000001; concentration effect: F(2,30) =36.81, P ≤ 0.000001; sugar × concentration: F(2,30) = 10.18, P = 0.0004; trials: 49.44 ±3.16; 129 (n = 16) sugar effect: F(1,15) =45.46, P = 0.000007; concentration effect: F(2,30) = 72.15, P ≤ 0.0000001; sugar × concentration: F(2,30) = 8.63, P = 0.001; trials: 38.5 ± 3.79; KO (n = 14) sugar effect: F(1,13) = 53.54, P = 0.000006; concentration effect: F(2,26) = 10.10, P = 0.0006; sugar × concentration: F(2,26) = 8.65, P = 0.001; trials 26.73 ± 2.43. D, H, and L: lick scores for glucose and fructose, averaged across concentration, on test 4 for each individual WT (D), 129 (H), or KO (L) mouse. *Bonferroni-corrected significant pairwise differences in lick scores.

To see if more robust responding to glucose would develop with additional exposure, particularly in the 129 and KO mice, all mice were given three more blocks of single-access GvF exposure (blocks 8–10 in Fig. 6) while food deprived and then retested for their lick responses to glucose and fructose in a final food-deprived brief-access test (test 4). Indeed, all strains licked significantly more for glucose than fructose at each of the three concentrations tested in this final test (Fig. 8, C and D; G and H; K and L).

It became clear, however, that after such extensive exposure to the two sugars (i.e., by test 4) some mice were attending to cues other than taste. This was apparent in the longer latency to initiate fructose trials relative to glucose trials for B6 and KO mice. The median (semi-interquartile range) latencies to initiate glucose and fructose trials for each of the three test concentrations are shown in tables below the corresponding panels in Fig. 8. The differences were small and rather variable, but the general tendency for latencies to be longer on fructose trials than on glucose trials was statistically significant at select concentrations on test 4 for B6 and KO mice (Fig. 8, C and K). No such systematic latency difference was seen for the prior probe test for B6 mice (test 3; table below Fig. 8A). KO mice took statistically longer to initiate fructose trials than glucose trials only at the middle concentration on test 3 (table below Fig. 8I).

It is important to note that because of the randomized schedule of stimulus presentation mice were not able to predict which stimulus would be available on a given trial in the brief-access test. Moreover, the apparatus is designed such that visual cues are obscured. Thus we reasoned that the mice that were showing latency differences were possibly using olfactory cues, despite our best efforts to minimize such signals with fans. Previous work has shown that olfactory stimuli associated with increasing concentrations of sucrose, NaCl, and other taste solutions can come to influence the inclination to initiate licking (32, 38). Glucose and fructose solutions do not have any known volatiles or distinguishing olfactory characteristics, but it is possible that extensive exposure could sensitize these subjects to otherwise subtle olfactory features. Interestingly, 129 mice initiated glucose and fructose trials with comparable latencies, both very rapidly, but nevertheless licked significantly more for glucose than fructose in these brief-access tests. Thus, at least in that case, latency was not correlated with licking behavior (Fig. 8, E and G). Examination of the trial latencies in experiment 1 revealed that naive mice and those exposed to Glu-Only initiated glucose and fructose trials with comparable latencies, as did the GvF-exposed KO mice (Fig, 3, A, C, E, G, and K). GvF-exposed WT mice from experiment 1 took statistically longer to initiate fructose trials than glucose trials at the middle concentration only, but this difference was largely driven by one mouse. Having said that, neither the lack of effect on latency in 129 mice nor the lack of differential latencies for other mice with less exposure (experiment 2, test 3; experiment 1, tests 2 and 3) precludes the possibility that the mice were also using orthonasal and/or retronasal cues to guide licking behavior; it could be that olfactory cues simply did not impact the speed with which the mice were initiating trials (see discussion).

Experiment 3

To assess the possibility that olfactory input plays a role in the behavioral discrimination of oral glucose and fructose, these B6, 129, and KO mice underwent surgery to disconnect the olfactory bulb from the rest of the brain (bilateral bulbotomy) and then were tested for their ability to express preferential licking for glucose in a brief-access test. Although postoperative tests indicated that surgical disconnection between the olfactory bulb and forebrain abolished preferential licking for glucose, analyses were ultimately severely underpowered because of age- and surgery-related attrition (data not shown). Therefore, we performed bulbotomy or sham surgeries in a new cohort of B6 mice and then gave them exposure to glucose and fructose in daily 30-min single access sessions (full schedule of experiment 3 in Fig. 9). Olfactory dysfunction was assessed by a test of latency to locate a buried potato crisp (BCT) conducted at the conclusion of the experiment. Mice were categorized as olfactory impaired if they were statistically slower to find the crisp than the SHAM B6 group (see materials and methods for the statistical criterion); these mice are designated as the BULB group. Only BULB mice that were significantly impaired on this BCT were included in the single-exposure session and brief-access test analyses. Figure 10 shows representative thionin-stained bilateral sagittal sections from one SHAM mouse and three BULB mice; the three BULB mice shown were all statistically impaired on the BCT, but to varying degrees (see Fig. 10).

Fig. 10.

Representative Nissl-stained 30-µm sagittal sections spanning from the right (R; top) to the left (L; bottom) hemispheres of a mouse that underwent sham surgery (m188; left) and 3 mice that underwent bulbotomy surgeries and subsequently failed the buried crisp test (BULB) from experiment 3. BULB mice m181 and m190 both failed to find the crisp within the allotted time (900 s) but were able to locate the crisp on the visual test with a latency of 16.96 and 25.42 s, respectively. BULB mouse m167 was also significantly impaired on the postsurgical buried crisp test but eventually located it with a latency of 118.80 s.

Both SHAM and BULB mice consumed more glucose than fructose in single-access 30-min sessions across the exposure phase (Fig. 11). When subsequently tested in the brief-access test (chronically food deprived), SHAM mice displayed robust preferential licking for glucose over fructose, particularly at the 0.316 and 0.56 M concentrations (Fig. 12, A and B). Although BULB mice consumed significantly more glucose in the longer term (30-min intake sessions), these mice displayed noticeably weak avidity for glucose in the brief access test, when the influence of postoral signals were abated (Fig. 12, C and D). Neither the SHAM nor the BULB mice displayed significant differences in latency to initiate glucose and fructose trials on the brief-access tests (latencies shown in Fig. 12, A and C). Moreover, considering there was substantial variability in performance on the BCT in the BULB mice, we examined whether lick score differences, especially at the low concentration, were related to latency to initiate trials on the brief-access test (Fig. 12, E and F) and latency to find the crisp in the BCT (Fig. 12G). No such associations were evident. In fact, one mouse that failed to find the buried crisp showed the greatest difference in licking for glucose over fructose.

Fig. 11.

Experiment 3. A and C: mean ± SE total licks for glucose or fructose, collapsed across concentration, across four 6-day exposure blocks for sham-operated (SHAM, A) and individual bulbotomized (BULB, C) C57BL/6 (B6) mice. SHAM (n = 6): sugar effect: F(1,5) = 57.83, P = 0.0006; block effect: F(3,15) = 1.46, P = 0.27; sugar × block: F(3,15) = 11.07, P = 0.0004. BULB (n = 6): sugar effect: F(1,5) = 36.48, P = 0.002; block effect: F(3,15) = 0.28, P = 0.84; sugar × block: F(3,15) = 13.64, P = 0.0001. B and D: intakes of glucose and fructose, collapsed across concentration, on the final 30-min single-access continuous-exposure block (block 4) for each individual SHAM (B) or BULB (D) mouse. A final exposure block was conducted after block 4, in which each of the sugar solutions was presented in the 10-s brief-access trial format for a 30-min single session, order randomized (block 5). This was done to prepare the mice for the upcoming brief-access test sessions. Two mice (1 from each surgical condition) were excluded from analyses because they received only 5 of the 6 possible sugar concentrations in this phase [SHAM (n = 5): t(5) = 3.83, P = 0.02; BULB (n = 5): t(4) = 4.09, P = 0.02]. *Significant Bonferroni-corrected pairwise comparisons.

Fig. 12.

A and C: mean ± SE lick scores for 3 concentrations of glucose and 3 concentrations of fructose presented in randomized order in post-exposure-phase brief-access trials (lick scores per stimulus collapsed across 3 tests) for sham-operated (SHAM, A) and bulbotomized (BULB, C) mice. SHAM (n = 6): sugar effect: F(1,5) = 21.70, P = 0.006; concentration effect: F(2,10) = 1.76, P = 0.22; sugar × concentration: F(2,10) = 1.43, P = 0.28; mean ± SE cumulative trials across 3 tests: 188.2 ± 11.34. BULB (n = 6): sugar effect F(1,5) = 12.33, P = 0.02; concentration effect: F(2,10) = 10.08, P = 0.04; sugar × concentration: F(2,10) = 0.58, P = 0.57; means ± SE cumulative trials over 3 tests: 154.7 ± 20.7. Median (semi-interquartile range) latencies to initiate glucose (G) vs. fructose (F) trials are displayed in text below the respective concentrations in each panel. *Significant post hoc pairwise differences in lick scores, after Bonferroni correction. B and D: lick scores for glucose and fructose, averaged across concentration, for each individual SHAM (B) or BULB (D) mouse. E: median difference in latency (glucose minus fructose) to initiate trials on glucose vs. fructose trials was not different from 0 in SHAM [W(−19), P = 0.063] or BULB [W(9), P = 0.44] mice; each data point represents an individual mouse. F: median difference in latency to initiate trials at each concentration for each individual BULB mouse. G: mean difference in lick scores collapsed across concentration and plotted as a function of latency to find the buried crisp in BULB mice. The median (solid teal vertical line) and range (dashed teal vertical lines) of the SHAM group’s latency to find the crisp in the postsurgical test are plotted on the x-axis for reference. The solid vertical gray line on the x-axis indicates the buried crisp test time limit. Mice that failed to find the crisp within 900 s are plotted on that line.

DISCUSSION

Experience with consuming glucose and fructose, two metabolically distinct sugars, fosters significant overconsumption of glucose relative to fructose in mice, including those that lack the functional primary taste receptor for both sugars (T1R2+T1R3). Under naive conditions, glucose and fructose stimulate ingestion through their primary and mutual activation of T1R2+T1R3 in the oral cavity. Consistent with this, sugar-naive WT mice responded in a comparable concentration-dependent fashion to the two sugars when the sugars were presented in a brief-access test; an important feature of the brief-access test is that the trial duration and structure preclude the postingestive signals generated by the sugars from differentially influencing licking behaviors. Without a functional T1R2+T1R3 heterodimer, naive KO mice were virtually unresponsive to the two sugars. Interestingly, experience with a single sugar, glucose (Glu-Only), did not appear to change these basic response profiles in WT or KO mice, perhaps because these mice were not faced with a metabolic or physiological impetus to recognize the advantages of glucose. Experience with both sugars (GvF), on the other hand, profoundly changed the brief-access licking response profile. GvF-exposed WT and KO mice (experiment 1) licked substantially more for glucose than fructose during these same postexposure brief-access tests, as did B6 and 129 mice (experiment 2). Together, these results not only replicate our previous findings in rats (41) but importantly extend them to show that T1R2+T1R3 is not necessary to acquire or express the experience-dependent avidity for the orosensory properties of glucose. A T1R2+T1R3-independent receptor subserving oral sugar sensing must therefore exist and be capable of relaying critical signals to the central circuits involved in ingestive motivation. Indeed, experience with the distinct postingestive consequences of initially similar-tasting substances confers an alternative source of chemosensory input with a motivational potency capable of eclipsing that arising from the prototypical “sweet” sensor.

Taste vs. Olfactory Inputs

The emergence of differences in latency to initiate fructose trials versus glucose trials after prolonged sugar exposure in experiment 2, albeit minor, was surprising; glucose and fructose are low-vapor stimuli that do not, to the best of our knowledge, emit distinguishing odor properties. The 129 mice with equivalent exposure to the two sugars never exhibited a difference in initiating glucose and fructose trials, despite showing significant lick differences in favor of glucose. Of course, we cannot rule out the possibility that experienced B6, WT, and KO mice were able to detect distinguishing features associated with contaminants in the reagent-grade chemicals. Nor can we rule out the possibility that the mice that failed to show any clear latency differences in experiments 1–3 were not using olfactory cues to guide responses (e.g., retronasally).

Surgical disruption of olfactory inputs to forebrain profoundly disrupted, but did not completely abolish, the ability to acquire and/or express an orosensory-based enhancement in licking for glucose after the typical single-access exposure in experiment 3. Although this might point to the straightforward conclusion that odor cues contribute to this rapid sugar discrimination, it is important to bear in mind that disruptions to the olfactory system produce behavioral phenotypes that have been collectively described as depression-like, characterized by a lack of responsivity to stimuli that normally drive an adaptive behavioral response (e.g., Refs. 25, 45). The fact of the matter is that surgeries that physically disconnect the olfactory system from the rest of the brain result in significant alterations, either directly or through secondary degeneration, of cortical and limbic structures, which happen to be critical for a number of sensory-reward-dependent behaviors (9, 49), Recent studies have found that taste and olfactory signals converge upon single cells within the piriform and gustatory cortices (e.g., Refs. 29, 30, 40). Although these cells might be contributing to integrative sensations such as flavor, given the overlap between taste and olfactory projections in the brain we cannot dismiss the possibility that damage produced by the surgery affected normal responsivity to taste stimuli even independent of a primary effect on olfactory function.

Although a number of studies have demonstrated that olfactory dysfunction reduces sugar intake and/or preference (e.g., Refs. 36, 45, 53), only one study to date has attempted to interrogate whether this was related to a specific effect on taste function in short-duration licking tests (48). Rats treated with zinc sulfate to destroy the olfactory epithelium licked vigorously for sucrose solutions, like control rats; however, in that study rats were water restricted for testing and thus licking was at ceiling levels, precluding any inference regarding whether this disruption to the olfactory system affected taste-guided behavior toward sugar. Here, experiment 3 circumvented this issue with food deprivation, instead of water deprivation, providing the first assessment of the behavioral responsivity of olfactory-compromised mice to the orosensory properties of sugars.

Comparison of the brief-access test concentration-response functions from intact WT mice that did not have GvF training (Fig. 3, A and B) with those from BULB mice that received GvF training (Fig. 12, C and D) clearly shows that bulbotomized B6 mice display severely blunted responsiveness to both glucose and fructose relative to water. This strongly implicates a general orosensory-based motivational deficit toward sugars despite the fact that these B6 mice have the full benefit of the T1R2+T1R3 heterodimer. Indeed, our tests conducted after bulbotomy in GvF-exposed KO mice were suggestive of more global taste-based deficits (data not shown), findings that will be more explicitly assessed in follow-up studies. Whether these surgeries would also affect other aspects of taste function, such as taste-elicited reflexes, is unknown. It will be important for future work to more fully characterize the nature of taste dysfunctions associated with bulbotomy. Of note was the considerable variability in residual olfactory function, as measured by the BCT, that did not relate in any straightforward way to performance on the brief-access test. Recovery or partial recovery of olfactory capacity did not necessarily result in recovery of the glucose versus fructose discrimination. One possibility, bearing in mind that the BCT does not allow us to assess olfactory acuity, is that the damage to other brain areas was less readily restored than olfaction. Even assuming that olfactory input is an important component underlying the behavioral discrimination of glucose and fructose, the present results absolutely do not rule out the possibility that taste input is also necessary.

Moreover, other recent studies corroborate the existence of an alternative glucose taste receptor. Glendinning et al. (19, 20) recently showed that glucose and glucose-containing sugars unconditionally stimulate the release of insulin from an oral site of action, but through a T1R3-independent receptor; other sugars, including fructose, do not stimulate the same response. The present results certainly do not refute the possibility of an alternative taste receptor for one or both sugars that feeds into circuits controlling ingestive motivation. Given that the vapor pressures of glucose and fructose are very low, it is difficult to see the utility of a specialized olfactory receptor for this monosaccharide subserving such a critical homeostatic response.

Other Determinants of Enhanced Responsivity to Glucose

The increased motivation to lick for glucose over fructose in brief-access tests clearly depended on experience with both sugars. Naive WT mice licked comparably to both sugars, whereas naive KO mice were virtually unresponsive to the sugars in brief-access tests (experiment 1). This implicates a critical role for postingestive signals both in driving the overconsumption of glucose over fructose in 30-min sessions and in reinforcing the eventual rapid licking responses to glucose over fructose. There are a number of postingestive sensory and metabolic events associated with the two sugars that could potentially contribute to the development and expression of this phenomenon. Notably, T1R2 and T1R3 are expressed in the gastrointestinal tract, liver, brain, and other tissues, where, some have argued, they contribute to extraoral sugar reception (e.g., Refs. 12, 22, 28, but see Ref. 42). Yet the present experiments clearly show that global deletion of both T1R2 and T1R3 does not disrupt the ability to detect and/or process a critical signal for driving glucose consumption over equicaloric fructose consumption, both within 30-min intake sessions and in the conference of this motivation onto the associated orosensory features of the sugars. Previous studies have shown that food-deprived mice lacking a single T1R3 subunit also show no deficit in their consumption of glucose-containing sugars and capacity to develop a preference for a flavor paired with intragastric infusions of a glucose-containing sugar (42, 44). A series of recent studies point to glucose transporters (SGLT1/SGLT3) in the conveyance of positive postoral feedback associated with glucose (43, 52). Future work will need to explore whether this same mechanism contributes to the acquired behavioral discrimination between glucose and fructose shown here.

Because naive KO mice do not readily consume sugars, it was necessary to manipulate the deprivation state of the mice to promote intake and hence experience with the different sugars. Water deprivation was used initially to motivate ingestion in experiments 1 and 2. Intake differences between the two sugars emerged (earlier) in WT and B6 mice during these 30-min exposure phase sessions; KO mice were slower to exhibit this difference in experiment 1, and KO and 129 mice in experiment 2 failed to show such a difference while in the initial water-deprived state in experiment 2. One possibility is that the T1R2 and T1R3 deletion in the KO mice and T1R3 polymorphism in the 129 mice impaired responding to and learning about the two sugars, even if these sensitivity deficits were not absolutely critical (see above). However, a number of other differences between B6 and 129 mice may contribute to the emergence of glucose avidity. For example, clear metabolic differences have been characterized in 129 and B6 mice (5, 15, 17). Whether these influence the physiological states produced by the deprivation conditions used in the present experiments and/or glucose versus fructose responsivity, independent of T1R, remains to be determined. It could be that 129 and KO mice were learning about the differences between the two sugars early on but were sufficiently more motivated by water deprivation to consume significant amounts of both; this would need to be experimentally tested. Additionally, 129 mice have been shown to display learning and/or performance deficits compared with B6 mice, including on tasks that do not depend on food/fluid reward (4, 9).

Perspectives and Significance

Foods and fluids are complex stimuli, in terms of both their multisensory properties and their eventual biological consequences. The ability to link the earliest perceptible properties of an ingestant (e.g., taste, texture, and smell) with its specific ultimate consequences is essential for health and survival. Mice were impressively able to exploit minute orally signaled chemosensory differences between these two sugars to guide intake toward the most efficient nutrient source and do so without the benefit of the canonical “sweet” taste receptor. These sensory capacities along with underlying neural adaptations and integrations that underlie the adaptive responses are oftentimes overlooked. Our results underscore the need to better understand chemosensory function in a broader context.

GRANTS

This work was supported by grants from the National Institute of Deafness and Other Communications Disorders: R01-DC-004574 (to A. C. Spector) and F32-DC-013494 (to L. A. Schier).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.A.S. and A.C.S. conceived and designed research; L.A.S., C.I.-Y., and G.D.B. performed experiments; L.A.S. analyzed data; L.A.S. and A.C.S. interpreted results of experiments; L.A.S. prepared figures; L.A.S. drafted manuscript; L.A.S. and A.C.S. edited and revised manuscript; L.A.S., C.I.-Y., G.D.B., and A.C.S. approved final version of manuscript.