Strain differences in sucrose- and fructose-conditioned flavor preferences in mice

Abstract

Genetic factors strongly influence the intake and preference for sugar and saccharin solutions in inbred mouse strains. The present study determined if genetic variance also influences the learned preferences for flavors added to sugar solutions. Conditioned flavor preferences (CFPs) are produced in rodents by adding a flavor (CS+) to a sugar solution and a different flavor (CS−) to a saccharin solution (CS−) in one-bottle training trials; the CS+ is subsequently preferred to the CS− when both are presented in saccharin solutions in two-bottle tests. With some sugars (e.g., sucrose), flavor preferences are reinforced by both sweet taste and post-oral nutrient effects, whereas with other sugars (e.g., fructose), sweet taste is the primary reinforcer. Sucrose and fructose were used in three experiments to condition flavor preferences in one outbred (CD-1) and eight inbred strains which have “sensitive” (SWR/J, SJL/J, C57BL/10J, C57BL/6J) or “sub-sensitive” (DBA/2J, BALB/cJ, C3H/HeJ, 129P3/J) sweet taste receptors (T1R2/T1R3). Food-restricted mice of each strain were trained (1 h/day) to drink flavored 16% sucrose (CS+ 16S, Experiment 1), 16% fructose (CS+ 16F, Experiment 2) or 8% fructose + 0.2% saccharin (CS+ 8F, Experiment 3) solutions on five alternate days and a differently flavored saccharin solution (0.05% or 0.2%, CS−) on the other five alternating days. The CS+ and CS− flavors were presented in 0.2% saccharin for two-bottle testing over six days. All strains preferred the CS+ 16S to CS− although there were significant strain differences in the magnitude and persistence of the sucrose preference. The strains also differed in the magnitude and persistence of preferences for the CS+ 16F and CS+ 8F flavors over the CS− with two strains failing to prefer the fructose-paired flavors. Sucrose conditioned stronger preferences than did fructose which is attributed to differences in the taste and post-oral actions of the sugars. These differential training intakes may not have influenced the sucrose-CFP because of the post-oral reinforcing actions of sucrose. Overall, sweet sensitive and sub-sensitive mice did not differ in sucrose-CFP, but unexpectedly, the sub-sensitive mice displayed stronger fructose-CFP. This may be related to differential training intakes of CS+ and CS− solutions: sweet sensitive mice consumed more CS− than CS+ during training while sub-sensitive mice consumed more CS+.

Introduction

Systematic analyses of inbred mouse strain differences have emerged as important sources of information regarding the genetic control of all aspects of ingestive behavior, including those involving sweet taste preferences (see reviews: [12], [33], [44]). Mouse strain differences have been observed for intake and sensitivity to both sugars and artificial sweeteners in many studies [5], [11], [22], [28], [30], [32], [34], [37], [43]. Studies of these strain differences led to the discovery of two genes (Tas1r2, Tas1r3) that code for the T1R2 and T1R3 proteins that form the sweet taste receptor [4]. Furthermore, mouse strain differences in sweetener preference were found to reflect different alleles of the Tas1r3 gene which resulted in “sensitive” and “subsensitive” forms of the T1R2/T1R3 sweet taste receptor [4].

Allelic variation of the Tas1r3 gene, however, does not account for all of the differences in sweetener intake, and other genes are implicated [14]. In addition, experiential factors can greatly influence sugar intake and preference of inbred mouse strains [37]. For example, naïve 129P3/J (129) mice, which have the sub-sensitive sweet receptor, display weaker preferences for dilute sucrose solutions than do sweet sensitive C57BL/6J (B6) mice, but after experience with concentrated sugar solutions, the sugar preferences of the two strains are indistinguishable [37]. This experiential influence was attributed to the post-oral actions of sucrose enhancing sweet taste preference. Consistent with this interpretation, [41] reported that intragastric (IG) sucrose infusions conditioned preferences for flavored saccharin solutions in both B6 and 129 strains.

Conditioned flavor preference (CFP) produced by sucrose has also been reported in rats that are trained with an arbitrary flavor mixed directly into the sugar solution [31]. In this case, however, the sweet taste of sucrose as well as its post-oral effects can reinforce the flavor preference. The potency of sweet taste alone to produce CFP was first demonstrated in an early study in which rats were trained with one flavor (the conditioned stimulus, CS+) added to a concentrated saccharin solution and a different flavor (the CS−) added to a less preferred dilute saccharin solution [27]. In subsequent choice tests, the rats preferred the CS+ to the CS− flavor when both were presented in solutions containing the same amount of saccharin. This form of learning is referred to as flavor–flavor (or flavor–taste) conditioning to distinguish it from the flavor–nutrient conditioning produced exclusively by the post-oral effects of sugars [36]. A special case of flavor–flavor learning was reported by [39] in which rats acquired a preference for a CS+ flavor added to an 8% fructose solution over a CS− flavor added to a less preferred saccharin solution. Although a sugar with post-oral nutrient effects, fructose, unlike sucrose or glucose does not condition flavor preferences in rats when infused IG during short-term training sessions [3], [40]. Similarly, B6 mice fail to acquire a preference for a CS+ flavor paired with IG fructose infusion although they display strong preferences for a CS+ flavor paired with IG sucrose or glucose infusion ([41]; Sclafani, unpublished data). Thus, the preference for a flavor mixed into a fructose solution is attributed to the sweet taste rather than the post-oral action of the sugar [1], [2], [6], [18], [26], [39].

While mouse strain differences in sweetener preference have been extensively investigated, learned preferences for flavors added to sweetener solutions have not been examined. The present study, therefore, investigated sugar-CFP in one outbred strain (CD-1) and eight inbred strains, four of which have the sensitive sweet taste receptor (C57BL/6J, C57BL/10J, SJL/J, SWR/J) and four of which have the sub-sensitive receptor (BALB/cJ, C3H/HeJ, DBA/2J, 129P3/J). These strains are a subset of 12 mouse strains investigated in our laboratories for 24-h sugar and fat preferences [28], [29], and were selected because they consumed a criterion level of sucrose (1 ml/1 h) in pharmacological studies performed in food-restricted mice [19], [20]. This criterion is important for the present study in that these strains display a level of drinking necessary to demonstrate a significant preference in short-term (1 h) tests, and minimize the possibility of “floor effects” associated with minimal intake. Flavor conditioning was investigated in three experiments using sucrose, fructose and saccharin. In the first experiment, mice were trained with CS+ flavored 16% sucrose and CS− flavored 0.05% saccharin solutions, and preferences were evaluated in two-bottle tests with both flavors presented in 0.2% saccharin solutions. A similar design was used in Experiment 2 except that the sugar was 16% fructose. Both the oral (sweet taste) and post-oral actions of sucrose contribute to the sugar's conditioning effects, whereas fructose conditioning is based primarily on the oral properties of this sugar [39], [40]. It was predicted that strain differences in fructose-CFP would be more closely associated with the sweet taster status of the inbred strains than sucrose-CFP. A third experiment was conducted with mice trained with a combined 8% fructose + 0.2% saccharin CS+ solution relative to a 0.2% saccharin CS− solution in a paradigm which has been extensively used in flavor–flavor preference studies conducted with rats [7], [10], [17], [26], [39]. A comparison of the results obtained in Experiments 2 and 3 would reveal which conditioning procedure was more effective in producing fructose-based CFP in mice.