Cross-Generalization Profile to Orosensory Stimuli of Rats Conditioned to Avoid a High Fat/High Sugar Diet

Cross-Generalization Profile to Orosensory Stimuli of Rats Conditioned to Avoid a High Fat/High... Abstract The orosensory characteristics of a diet play a role in its acceptance and rejection. The current study was designed to investigate the gustatory components that contribute to the intake of a palatable, high-energy diet (HE; 45% calories from fat, 17% calories from sucrose). Here, rats were conditioned to avoid HE diet by pairings with i.p. injections of LiCl to induce visceral malaise. Subsequently, the degree of generalization was tested to an array of taste compounds using a brief-access lick procedure (10-s trials, 30-min sessions). Compared to NaCl-injected controls, LiCl-injected rats suppressed licking response to 100% linoleic acid and 20% intralipid, and to a lesser extent 17% sucrose. There was more variability in the lick responses to sucrose among the LiCl-injected rats. Rats that tended to suppress licking responses to sucrose generalized this response to glucose, fructose and Na-saccharin but not to Polycose. In contrast, LiCl-injected rats did not significantly suppress lick responses to water, NaCl, citric acid, or quinine compared to controls rats. The brief access feature of this procedure, allows for behavioral measures when postingestive factors are minimized. These findings support a role for gustatory cues in the detection of high fat/high sugar diets. Furthermore, it appears that the fat component is a more salient orosensory feature of the HE diet. Introduction Upon presentation of a calorically dense diet, animals will overeat. This model provides an experimental analogy for the overconsumption of high fat/high calorie foods that lead to obesity in humans. The changes that occur upon presentation of a calorically dense diet can be characterized by an initial rapid rate of intake which can be referred to as the dynamic phase, followed a static phase characterized by a plateau in food intake and weight gain change (see Brobeck 1946). Using meal pattern analysis, we have previously shown that when presented a high-energy (HE) diet, the hyperphagia observed in rats is driven by an increase in meal size. These changes are most robust across the first several days of HE diet exposure which characterizes the dynamic phase. Over the static phase, with continual exposure to the diet, intake, and meal size decrease but remain significantly higher than those of chow-fed controls. Meal number decreases during both the dynamic and static phases (Treesukosol et al. 2014). The larger but less frequent meals raise the possibilities that orosensory stimulation which may include taste, trigeminal, and olfactory cues may be enhanced and/or inhibitory cues that signal satiety and meal termination are weakened upon presentation of a high calorie diet. It has been shown however, that the effect of satiation signals such as cholecystokinin are less effective in decreasing feeding in rats that have been maintained on a high fat diet for a longer period of time (Covasa and Ritter 1998; de Lartigue et al. 2012) suggesting increased insensitivity to satiation signals is a result of longer exposure to a high fat diet. Thus the relative contribution of postoral inhibitory influences may be less pronounced than that of orosensory changes during the hyperphagia that characterizes the dynamic phase. Upon HE diet exposure, whereas meal pattern analyses reveal a robust increase during the dynamic phase of total daily intake and meal size which then decreases with longer maintenance on the diet, a comparable spike is not observed for eating rate expressed as calories per second (Treesukosol et al. 2014). Rather, compared to eating rate in rats maintained on standard chow, rats given a HE diet show high rate of eating regardless of the day of diet presentation. Thus, it appears that more immediate signals such as those coming from the orosensory cavity rather than longer-term influences such as previous diet exposure, have a larger influence on eating rate. Further evidence in the literature support the notion that more immediate cues such as oral stimulation can influence mechanisms underlying feeding behavior. Sensory contact with oral stimuli have been shown to trigger autonomic and endocrine reflexes involved in metabolism before the influences of postingestive consequences. For example, the cephalic reflexes such as the secretion of saliva and gut responses can be elicited by taste stimuli in the oral cavity (Powley 1977; Berthoud et al. 1981). Compared to drinking water or an almond extract solution, drinking a tastant (saccharin, NaCl, or glucose) affected subsequent food intake in rats (Tordoff and Friedman 1989). Rats increased food intake during subsequent 2-h tests also following sham feeding with a corn oil emulsion or a sucrose solution, compared to a no sham-feeding condition (Tordoff and Reed 1991). Yet, when rats were maintained on a HE diet with liquid Ensure or with a diet with 45% fat and no liquid calories, sham-feeding responses to sucrose did not increase and in some cases, were lower than responses of standard chow-fed controls (Treesukosol et al. 2015). Thus under these conditions in which the stimulus was a HE diet, generalization is not observed. Given that a HE diet is a more complex stimulus than sucrose solutions or corn oil emulsions, this raises the question of what the different oral components of such a diet are. The current experiments were designed to investigate the gustatory components that contribute to the intake of a palatable, HE diet (containing 45% calories from fat and 17% calories from sucrose) which was used in previous studies (Treesukosol et al. 2014; 2015). Specifically, experiment 1 was designed to derive data to calculate concentrations of glucose, Na-saccharin Polycose, and fructose that elicit unconditioned licking responses comparable to those in response to 17% sucrose. These compounds were chosen to compare generalization to other sugars (glucose and fructose), a non-caloric sweetener (saccharin), and a palatable compound that is not considered “sweet” (Polycose). Next, based on these data, rats were conditioned to avoid a HE diet and subsequently generalization to an array of taste compounds was measured using a brief-access lick procedure. These data provide a qualitative profile of the HE diet that include the palatable stimulus array from experiment 1 as well as representatives of compounds that humans describe as sweet, sour, bitter, salty, and fatty. Together, the data from these experiments reveal the oral qualitative features of a HE diet. Materials and methods Experiment 1: determination of stimulus concentrations Subjects Eight male Sprague–Dawley rats (Harlan) with a mean body weight of 283.0 ± 1.4 g upon arrival were single-housed in hanging wire cages in a room with automatically controlled humidity, temperature, and a 12h–12h light-dark cycle. Rats had ad libitum access to chow (2018 Teklad, Harlan; 3.1 kcal/g) and water, except where noted. All procedures were approved by the Institutional Animal Care and Use Committee at The Johns Hopkins University. Procedure Brief-access taste tests were conducted during the light cycle. Brief-access taste procedure training and testing were conducted in a lickometer (Davis MS-160, DiLog Instruments, Tallahassee, FL) as described previously (Smith 2001; Glendinning et al. 2002). The rat was placed in the chamber of the testing apparatus with access to a single spout positioned ~5 mm behind a slot in the testing chamber wall. The spout was connected to a glass bottle containing a solution. To minimize potential olfactory cues from the stimuli, a small fan was positioned above the testing chamber wall to direct an air current past the spout. During training with water in the lickometer, the rats were placed on a water-restricted schedule. Access to water was removed from the home cages no more than 23 h before the test session. Water was available only during sessions in the lickometer. During the first 2 sessions in the lickometer, rats were presented with a stationary spout of water for 30 min. Total number of licks and inter-lick interval were measured. On day 3, 7 tubes of water were presented one at a time in 10-s trials across 30-min sessions. A trial was initiated when the rat licked the spout. The shutter closed at the end of each 10-s trial. During each 8-s inter-trial interval, a motorized block prepared the next spout presentation, after which the shutter reopened for the next trial. The spouts were presented in randomized blocks without replacement. Animals were able to initiate as many trials as possible during the 30-min session. At the end of training with water, ad libitum access to water resumed in the home cages for ~48 h before further testing began. Each test compound (sucrose, saccharin, glucose, Polycose, and fructose) was tested in daily 30-min sessions across 2 consecutive days (Table 1). Concentrations of sucrose (0.01, 0.03, 0.06, 0.1, 0.3, 1.0 M; Sigma Aldrich, St. Louis, MO), saccharin (0.1, 0.5, 1, 5, 10, 50 mM; Sigma Aldrich), glucose (0.06, 0.125, 0.25, 0.5, 1.0 M; Sigma Aldrich), Polycose (1, 2, 4, 8, 16, 32%; Abbott Laboratories, Columbus, OH), and fructose (0.06, 0.125, 0.25, 0.5, 1.0, 2.0 M; Sigma Aldrich) were chosen to span the dynamic range. Concentrations were presented in randomized blocks without replacement. As with the training sessions, a trial was initiated when the rat licked the spout and an animal could initiate as many trials as possible during the daily sessions. Table 1. Experimental design for experiment 1 (determination of stimulus concentrations) Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of sucrose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of saccharin   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of glucose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of Polycose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of fructose  Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of sucrose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of saccharin   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of glucose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of Polycose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of fructose  View Large Data analysis The average number of licks per trial at a given concentration was divided by that animal’s estimated maximal lick rate (licks/10 s) yielding a standardized lick ratio. The maximal lick rate was derived from the reciprocal of the mean of the inter-lick interval (ILI) values (in ms) that was measured during the last day of licking to a stationary spout of water. Only ILI values between 50 and 200 ms were used (Allison and Castellan 1970; Davis and Smith 1990, 1992) and this value was multiplied by 10 000 (Equation 1). In this way, the Standardized Lick Ratio accounts for individual differences in local lick rate.  Estimated maximum licks=10 000 ms×1(ILI (ms)) Curves were fit to mean data for each group by using the following logistic function:  y=a(1+10(x−c)b) where x = log10 stimulus concentration, a = asymptotic lick response, b = slope, and c = log10 concentration at the inflection point. Experiment 2: generalization of conditioned aversion to HE diet Subjects A separate group of male Sprague-Dawley rats (Harlan) with a mean body weight of 281.7 ± 1.3 g (cohort 1) and 286.5 ± 1.5 g (cohort 2) upon arrival were single-housed in a room where the temperature, humidity, and lighting (12h–12h, light-dark) were automatically controls. Rats had ad libitum access to powdered chow (2018 Teklad, Harlan) and water, except where noted. All procedures were approved by the Institutional Animal Care and Use Committee at The Johns Hopkins University. Procedure Animals were trained and tested for the brief-access taste procedure in a lickometer (Davis MS-160, DiLog Instruments, Tallahassee, FL) as described in experiment 1. The rats were trained and tested in the lickometer while on a water-restricted schedule. Access to water was removed from the home cages no more than 23 h before the first session in the lickometer and water was available only during the training and testing sessions. The first 2 sessions in the lickometer involved presenting the rats with a stationary spout of water for 30 min. On day 3, 7 tubes of water were presented in randomized blocks without replacement, one at a time in 10-s trials across 30-min sessions. Animals were able to initiate as many trials as possible during each 30-min session. At the end of day 3, ad libitum water access in the home cages resumed (Table 2). Table 2. Experimental design for cohort 1 and cohort 2 of experiment 2 (generalization of conditioned aversion to HE diet) Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Food restriction  8  High energy diet across conditioning trials   Water restricted  1  Multiple 10-s sessions with water presentation   Water restricted  1  Multiple 10-s s trials with stimulus array  Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Food restriction  8  High energy diet across conditioning trials   Water restricted  1  Multiple 10-s sessions with water presentation   Water restricted  1  Multiple 10-s s trials with stimulus array  View Large After at least 1 day of ad libitum access to water and chow, animals were assigned to 1 of 2 groups [LiCl (n = 8) or NaCl (n = 7–8)] such that there were no significant group differences in body weight, total licks to a stationary spout of water, ILI values, or number of trials initiated to water. Two cohorts of animals were tested. For cohort 1, the HE diet was presented while animals had ad libitum access to standard chow but the remaining conditioning trials and all conditioning trials for cohort 2, were conducted while animals were on a food-restricted schedule. Conditioning trials were conducted such that all animals were provided access to a powdered HE-diet (4.73 kcal/g; calories from protein 20%, calories from fat 45%, calories from carbohydrate 35%; D12451, Research Diets) for 1 h in the morning during the light-cycle followed by an intraperitoneal injection of 0.15 M LiCl (Sigma Aldrich; 1.33 mL/100 g body weight) to induce visceral malaise, or 0.15 M NaCl (Sigma Aldrich; 1.33 mL/100 g body weight) to serve as a control. In the afternoon, animals were presented access to the powdered standard chow diet (3.1 kcal/g, calories from protein 24%, calories from fat 18%, calories from carbohydrate 58%; 2018 Teklad, Harlan) to allow for refeeding. Of the fat source in the HE diet, ~6% of the calories is from soy bean oil and ~39% of the calories is from lard. Of the carbohydrate source in the HE diet, ~7% is from corn starch, ~10% from maltodextrin 10, and ~17% from sucrose. In contrast, the standard chow does not contain animal fat or added complex carbohydrates. Each conditioning trial was separated by 1 day of restricted access to the powdered standard chow diet (1-h access in the morning and 1-h access in the afternoon). During this period, animals had ad libitum access to water (Table 3). Table 3. Schedule for conditioning trials for cohort 1 and cohort 2 of experiment 2 (Generalization of conditioned aversion to HE diet) Day  AM  PM  1  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  2    Food restricted  3  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  4    Food restricted  5  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  6    Food restricted  7  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow  Day  AM  PM  1  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  2    Food restricted  3  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  4    Food restricted  5  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  6    Food restricted  7  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow  Cohort 1 began conditioning with ad libitum access to standard chow. Cohort 2 was food restricted for 23 h before conditioning. Other conditions as outlined in Table 3 were the same for both cohorts. View Large After the fourth conditioning trial, animals resumed ad libitum access to standard chow. After at least 1 day of ad libitum access to food and water, water access was removed and the next day animals were tested in the lickometer for 2 days. On the first day, animals were re-habituated with the apparatus and session structure with 10-s trials to water during a 30-min session. On the second day, rats were tested in the lickometer to a panel of 7 stimuli presented in randomized blocks without replacement in 10-s trials. A 10-s water rinse was presented between each stimulus presentation during these sessions. For cohort 1, the stimuli were: water, 100% linoleic acid (Sigma Aldrich), 20% intralipid (Sigma Aldrich), 17% sucrose (Sigma Aldrich), 0.376 M NaCl (Sigma Aldrich), 0.131 mM quinine (Sigma Aldrich), and 10.4 mM citric acid (Sigma Aldrich). For cohort 2, the stimuli used were: 17% sucrose (Sigma Aldrich), 5.2% glucose (Sigma Aldrich), 5.5% fructose (Sigma Aldrich), 0.17% Na-saccharin (Sigma Aldrich), 2.8% Polycose (Abbott Laboratories, Columbus, OH), and 10.4 mM citric acid (Sigma Aldrich). The stimuli used for testing cohort 2 did not elicit strong suppression scores as with cohort 1. To ensure that the weak suppression scores were stimulus array specific and not cohort specific, an additional test day was conducted to serve as a control with cohort 2, during which 5.5% fructose was replaced with 20% intralipid. Even though testing was during extinction, these rats showed strong suppression scores to 20% intralipid on the additional test day providing evidence that the lower suppression scores were stimuli specific. As with the water training sessions, a trial was initiated when the rat licked the spout and animals could initiate as many trials as possible during the 30-min sessions (Table 2). A similar conditioning schedule has also been successfully used previously in mice (Eylam et al. 2003; Dotson and Spector 2007; Treesukosol et al. 2011) and avoidance of fat-containing diets have been conditioned previously in rats (Larue 1978). Concentrations for sweeteners were matched to the diet composition of 17% sucrose using data from Experiment 1 and concentrations for citric acid, NaCl, and quinine were derived from comparable response curves from published data sets (Grobe and Spector 2008). At the end of the experiment, animals were given ad libitum access to food and water for at least 1 day and then all animals were presented HE diet in a food-restricted state for 1-h and intake was recorded to assess the maintenance of the conditioned avoidance. Data analysis To assess the acquisition of the avoidance, HE diet intake was compared between each LiCl group and its respective NaCl control group across each of the 4 conditioning trials using t-tests. For responses to the panel of stimuli, a suppression score for each LiCl-injected rats was calculated for each of the test stimuli (TS) during the test session (Nowlis et al. 1980; Eylam et al. 2003). For a given TS and LiCl-injected rat, this value was derived by the equation:  Suppression score=1−Rat Licks TSLiClGroup Licks TSNaCl where Rat Licks TSLiCl = the number of licks to a given TS of an individual animal in the LiCl-injected group, and Group Licks TSNaCl = the group mean for licks to the TS for the NaCl-injected group. This allows suppression scores for each LiCl-injected rat to each of the test stimuli to be obtained. A score of 0 indicates equal licking responses to the taste stimulus between the LiCl- and NaCl-injected groups (i.e. no licking suppression). A suppression score of 1.0 indicates complete suppression of licking to the taste stimulus by the LiCl-injected rat. Results Experiment 1: determination of stimulus concentrations Results from experiment 1 are shown in Figure 1. Note that in contrast to increasing licking behavior in response to ascending concentrations of some natural sugars such as sucrose, response to saccharin in rats has previously been shown to yield an inverted U-shape function peaking at approximately 8 mM (Smith 2000). To determine concentrations that elicit comparable licking, the concentration values (x) were solved by using: Figure 1. View largeDownload slide Mean ± SE standardized lick ratio values across concentrations of sucrose, Na-saccharin, glucose, Polycose, and fructose derived from a brief-access paradigm (experiment 1) to assess concentration-dependent changes in licking behavior. These data were used to calculate concentrations of other stimuli that elicit unconditioned licking responses comparable to those in response to 17% sucrose. Figure 1. View largeDownload slide Mean ± SE standardized lick ratio values across concentrations of sucrose, Na-saccharin, glucose, Polycose, and fructose derived from a brief-access paradigm (experiment 1) to assess concentration-dependent changes in licking behavior. These data were used to calculate concentrations of other stimuli that elicit unconditioned licking responses comparable to those in response to 17% sucrose.  x=log10(ay−1)b+c Based on the response curves from experiment 1, 17% sucrose corresponded to 0.85 of the maximal licking rate. Next, the concentrations that corresponded to 0.85 of the maximal licking rate for glucose, Na-saccharin, Polycose, and fructose were calculated. Concentrations for citric acid, NaCl, and quinine were derived from comparable response curves from published data sets (Dotson et al. 2005). Experiment 2: generalization of conditioned aversion to HE diet In both cohorts, LiCl-injected rats showed evidence of avoidance acquisition to the HE diet across the conditioning trials (Figure 2). For cohort 1, there was no significant group difference in intake during the first pairing trial (t(15) = −0.364, P = 0.721), but the LiCl group ate significantly less than controls from the second trial onwards (t(15) > 3.576, P < 0.003). Similarly for cohort 2, HE intake of the LiCl- and NaCl-injected groups was similar in the first pairing trial (t(14) = 1.045, P = 0.314) but LiCl-injected rats suppressed HE diet intake in subsequent sessions (t(14) > 4.698, P < 0.001). These data indicate there was no significant difference in intake between the groups at baseline and confirm effectiveness of the conditioning procedures. Figure 2. View largeDownload slide Intake of HE diet during 1-h conditioning trials for NaCl-injected (black bars) and LiCl-injected (white bars) rats in cohort 1 (left panel) and cohort 2 (right panel) in experiment 2. Note, Cohort 1 began conditioning with ad libitum access to standard chow (Pairing trial 1). Cohort 2 was food restricted for 23 h before conditioning. Conditions for pairing trial 2 onwards were the same for both cohorts. *Denotes significant group differences. Figure 2. View largeDownload slide Intake of HE diet during 1-h conditioning trials for NaCl-injected (black bars) and LiCl-injected (white bars) rats in cohort 1 (left panel) and cohort 2 (right panel) in experiment 2. Note, Cohort 1 began conditioning with ad libitum access to standard chow (Pairing trial 1). Cohort 2 was food restricted for 23 h before conditioning. Conditions for pairing trial 2 onwards were the same for both cohorts. *Denotes significant group differences. Brief-access testing Rats conditioned to avoid the HE diet displayed cross-generalization to 100% linoleic acid, 20% intralipid, and 17% sucrose. Suppression ratio scores were significantly higher than 0 for 100% linoleic acid (t(9) = 3.122, P = 0.014), 20% intralipid (t(9) = 11.092, P < 0.001), and 17% sucrose (t(9) = 2.861, P = 0.021) but the LiCl-injected rats did not generalize conditioned avoidance to the other stimuli presented (Figure 3). There was more variability in licking responses to sucrose in the LiCl group compared to the NaCl group. Thus in cohort 2, licking responses to 17% sucrose as well as other sweeteners and Polycose were measured. Here, rats conditioned to avoid the HE diet did not display cross-generalization to sucrose or any of the compounds tested (Figure 3). Within the LiCl group, animals that tended to suppress lick responses to 17% sucrose also did so to 5.2% glucose, 5.5% fructose, and 0.17% Na-saccharin. In contrast, significant correlations were not observed between responses to sucrose with Polycose, citric acid, or water (Figure 4). Furthermore, significant correlations were not revealed between lick responses between sucrose and intralipid (r = −0.177, P = 0.649) nor sucrose and linoleic acid (r = 0.257, P = 0.505) in cohort 1. Nor were significant correlations revealed between HE diet intake during the second pairing and lick responses to sucrose (cohorts 1, 2), linoleic acid, or intralipid (cohort 1) (r > −0.613, P > 0.790). In cohort 1, a significant correlation between suppression lick scores to sucrose and NaCl was found (r = 0.890, P = 0.001). Also, animals that tended to suppress responses to sucrose, also did so to citric acid in cohort 1 (r = 0.634, P = 0.067) and cohort 2 (r = 0.682, P = 0.062) but these comparisons did not reach statistical significance. Figure 3. View largeDownload slide Suppression ratio scores for rats conditioned to avoid the HE diet indicate degree of generalization to water (W), 100% linoleic acid (LA), 20% intralipid (IL), 17% sucrose (S), 0.376 M NaCl (N), 0.131 mM quinine (Q), 10.4 mM citric acid (CA), 5.2% glucose (G), 5.5% fructose (F), 0.17% Na-saccharin (SA) and 2.8% Polycose (P) for cohort 1 (A), and cohort 2 (B). Suppression ratio scores significantly higher than 0 (denoted by *) is evident for 100% linoleic acid, 20% intralipid and 17% sucrose for cohort 1. Figure 3. View largeDownload slide Suppression ratio scores for rats conditioned to avoid the HE diet indicate degree of generalization to water (W), 100% linoleic acid (LA), 20% intralipid (IL), 17% sucrose (S), 0.376 M NaCl (N), 0.131 mM quinine (Q), 10.4 mM citric acid (CA), 5.2% glucose (G), 5.5% fructose (F), 0.17% Na-saccharin (SA) and 2.8% Polycose (P) for cohort 1 (A), and cohort 2 (B). Suppression ratio scores significantly higher than 0 (denoted by *) is evident for 100% linoleic acid, 20% intralipid and 17% sucrose for cohort 1. Figure 4. View largeDownload slide Comparisons of suppression ratio scores for sucrose and other test stimuli in rats of cohort 2 conditioned to avoid the HE diet. Figure 4. View largeDownload slide Comparisons of suppression ratio scores for sucrose and other test stimuli in rats of cohort 2 conditioned to avoid the HE diet. After brief-access testing, avoidance of the HE diet was still evident in the LiCl-injected rats (Figure 2; “after test”). In the second cohort, lick responses to 20% intralipid in 10-s trials were measured in a subsequent brief-access test, suppression ratio scores were significantly higher than 0 (t(7) = 41.469, P < 0.001). These data confirm the effectiveness and maintenance of the conditioned avoidance and reliability of the cross-generalization to 20% intralipid even following an extinction trial. Discussion Rats conditioned to avoid the HE diet (45% fat, 17% sucrose) generalized avoidance to intralipid and linoleic acid but there was more variability in cross-generalization to sucrose. These findings show that orally, the “fat” component of the HE diet is more salient than the sucrose component. This is consistent with prior reports in which rats conditioned to avoid corn oil generalized avoidance to a sucrose + corn oil mixture, more so than rats conditioned to avoid sucrose (Smith et al. 2000). Similarly rats conditioned to avoid a glucose + saccharin + corn oil emulsion suppressed licking only to stimuli that contained corn oil (Smith 2004). Furthermore, in regard to how the orosensory properties of fats drive behavior, when rats were given only food and water or food plus a glucose and saccharin solution over 6 weeks, rats regulated caloric intake such that they gained weight at comparable rates. In contrast, in a group for which corn oil was added (i.e. food, glucose + saccharin + fat), rats increased caloric intake and body weight (Smith 2004). Using sham-feeding techniques which involve orosensory stimulation but minimal postingestive stimulation it was shown that orosensory properties of fats is sufficient to drive sham feeding in rats (Greenberg et al. 1996). Taken together it appears that the “fat” component is a salient feature of an oral stimulus. Here, there was more variability in responses to sucrose in rats conditioned to avoid the diet, compared to those of NaCl-injected controls. Polysaccharides are glucose polymers of varying chain lengths. Polycose has been the most studied polysaccharide in taste experiments and contains a small amount of glucose and maltose but primarily consists of glucose polymers of 3 or more glucose units (Kennedy et al. 1995). In studies in which taste aversion generalization procedures were employed, rats, mice and gerbils conditioned to avoid sucrose also avoid other compounds that humans describe as sweet such as fructose and glucose (Nowlis et al. 1980; Dugas du Villard et al. 1981; Jakinovich 1982; Ninomiya et al. 1984; Nissenbaum and Sclafani 1987; Sako et al. 1994). However, rats conditioned to avoid Polycose or sucrose show no or very weak cross-generalization (Nissenbaum and Sclafani 1987; Ramirez 1991; Sako et al. 1994) suggesting these 2 carbohydrate solutions have different taste qualities. Similarly, hamsters trained to avoid Polycose, sucrose, or a mixture of the 2 compounds display some degree of cross-generalization, but Polycose and sucrose, nonetheless, appear to have characteristics that make the compounds distinct from one another (Formaker et al. 1998). It has been shown that the T1R2 and T1R3 proteins are unnecessary for mice to express normal affective responses to Polycose yet mice missing one or both of these proteins display severely blunted responses to saccharin and sucrose (Treesukosol et al. 2009; Zukerman et al. 2009). Furthermore, mice missing the T1R2 or T1R3 subunit show severely impaired taste detection performance to compounds humans label as sweet including sucrose and glucose but are competent in the detection task when the stimulus is Polycose (Treesukosol et al. 2012). In the current study, rats in the LiCl group that suppressed responses to sucrose tended to also suppress responses to fructose, glucose, and Na-saccharin but not to Polycose. This suggests there is a “sweet” component to the HE diet that is separate from polysaccharide taste. These findings are also consistent with evidence that polysaccharides (such as Polycose) and stimuli that humans describes as sweet, (such as sucrose) are separate taste qualities (Sclafani 1987). In classical conditioning, latent inhibition refers to the observation that compared to a novel stimulus, it is more difficult to form a new association with a more familiar stimulus. A taste compound repeatedly presented without aversive consequences requires more pairings with negative consequences to elicit conditioned taste aversion of comparable intensity (Best 1975; Albert and Ayres 1989). In the current experiments, rats conditioned to avoid the HE diet showed strong generalization to “fatty” stimuli and more varied generalization to the “sweet” stimuli raising the possibility that latent inhibition contributed to these differential responses. In other words, the strong generalization to linoleic acid is driven by novelty to the stimulus and the weaker generalization to sucrose is driven by some familiarity to the stimulus. The fat and carbohydrate sources of the HE diet used in the conditioning trials and that of the chow diet that the animals were familiar are not similar. Of particular interest, the chow diet has no added sucrose or complex carbohydrates. Thus, it is unlikely that familiarity with some components of the HE diet explains the differential responses to the “fat” and “sweet” components observed in the generalization profiles. In response to the presentation of a palatable HE diet, rats (and other animals) will overeat. The hyperphagia induced by the availability of a HE diet may be driven by enhanced orosensory cues and/or dampened inhibitory feedback yet we have previously demonstrated that these alterations in signaling do not generalize to increased responsiveness to other palatable stimuli as measured by sham-feeding intake of sucrose (Treesukosol et al. 2015). Here in some animals, conditioned avoidance of the HE diet cross-generalizes to sucrose and other “sweet” compounds but not to Polycose. Thus it appears that calorically dense diet induced hyperphagia does not necessarily generalize to increased responses to other palatable stimuli. In the psychophysical literature, features of chemosensory mixtures are measured by comparing responses to single components of the mixture. A number of models have been proposed to describe these perceptual experiences (Frijters 1987). When conditioned to avoid single component stimuli and then tested with mixtures that include the conditioned stimulus, rats (Smith and Theodore 1984), and hamsters (Frank et al. 1989) avoid mixtures containing the conditioned stimulus in proportion to its concentration in the mixture. It has been shown that conditioned aversions to binary (Frank and Nowlis 1989) or ternary mixtures (Frank et al. 2003) more readily generalize to single components than vice versa. A possible explanation for this asymmetry in generalization profiles is that it is adaptive for an organism conditioned to avoid a complex stimulus to then avoid each identified component due to uncertainty as to which component of the mixture is associated with negative consequences. In contrast, an organism conditioned to avoid a single component may generalize less so to a mixture containing the conditioned stimulus because the other components provide a safe context (Taylor and Boakes 2002; Frank et al. 2003). Thus in the current study, by examining the degree to which conditioned avoidance of a HE diet (a stimulus of multiple components) generalizes to more simple components provides a qualitative profile of the HE diet. The concentrations of the stimuli in the cross-generalization test were chosen to match the components of the HE diet. The diet contains 17% sucrose thus this concentration was chosen. Next, concentrations of glucose, fructose, Na-saccharin, and Polycose that elicit comparable unconditioned lick responses as 17% sucrose were chosen. Findings from previous studies in which solutions (which allow for more accurate concentration matching) were used for both conditioning and testing of generalization, showed that corn oil was a more salient component than glucose or saccharin (Smith 2004). This is consistent with the current data that the “fat” component is the more salient feature. Here, we chose to focus on the qualitative aspects of the stimuli but the data also raises the question of whether less variability in the responses to the sweeteners would be observed if a higher concentration range were tested. This procedure could be employed to conduct studies using varying concentrations of test stimuli to address the question of relative intensity of the diet components. The brief-access feature of the procedure employed here allows for behavioral measures in which the influence of postingestive factors are minimized. These findings support a role for gustatory cues in the responsiveness towards high fat/high sugar diets. Furthermore, it appears that the fat component is a salient orosensory feature of the palatable, HE diet. Funding This work was supported by the National Institutes of Health (grant number DK019302 to T.H.M). 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Cross-Generalization Profile to Orosensory Stimuli of Rats Conditioned to Avoid a High Fat/High Sugar Diet

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Abstract

Abstract The orosensory characteristics of a diet play a role in its acceptance and rejection. The current study was designed to investigate the gustatory components that contribute to the intake of a palatable, high-energy diet (HE; 45% calories from fat, 17% calories from sucrose). Here, rats were conditioned to avoid HE diet by pairings with i.p. injections of LiCl to induce visceral malaise. Subsequently, the degree of generalization was tested to an array of taste compounds using a brief-access lick procedure (10-s trials, 30-min sessions). Compared to NaCl-injected controls, LiCl-injected rats suppressed licking response to 100% linoleic acid and 20% intralipid, and to a lesser extent 17% sucrose. There was more variability in the lick responses to sucrose among the LiCl-injected rats. Rats that tended to suppress licking responses to sucrose generalized this response to glucose, fructose and Na-saccharin but not to Polycose. In contrast, LiCl-injected rats did not significantly suppress lick responses to water, NaCl, citric acid, or quinine compared to controls rats. The brief access feature of this procedure, allows for behavioral measures when postingestive factors are minimized. These findings support a role for gustatory cues in the detection of high fat/high sugar diets. Furthermore, it appears that the fat component is a more salient orosensory feature of the HE diet. Introduction Upon presentation of a calorically dense diet, animals will overeat. This model provides an experimental analogy for the overconsumption of high fat/high calorie foods that lead to obesity in humans. The changes that occur upon presentation of a calorically dense diet can be characterized by an initial rapid rate of intake which can be referred to as the dynamic phase, followed a static phase characterized by a plateau in food intake and weight gain change (see Brobeck 1946). Using meal pattern analysis, we have previously shown that when presented a high-energy (HE) diet, the hyperphagia observed in rats is driven by an increase in meal size. These changes are most robust across the first several days of HE diet exposure which characterizes the dynamic phase. Over the static phase, with continual exposure to the diet, intake, and meal size decrease but remain significantly higher than those of chow-fed controls. Meal number decreases during both the dynamic and static phases (Treesukosol et al. 2014). The larger but less frequent meals raise the possibilities that orosensory stimulation which may include taste, trigeminal, and olfactory cues may be enhanced and/or inhibitory cues that signal satiety and meal termination are weakened upon presentation of a high calorie diet. It has been shown however, that the effect of satiation signals such as cholecystokinin are less effective in decreasing feeding in rats that have been maintained on a high fat diet for a longer period of time (Covasa and Ritter 1998; de Lartigue et al. 2012) suggesting increased insensitivity to satiation signals is a result of longer exposure to a high fat diet. Thus the relative contribution of postoral inhibitory influences may be less pronounced than that of orosensory changes during the hyperphagia that characterizes the dynamic phase. Upon HE diet exposure, whereas meal pattern analyses reveal a robust increase during the dynamic phase of total daily intake and meal size which then decreases with longer maintenance on the diet, a comparable spike is not observed for eating rate expressed as calories per second (Treesukosol et al. 2014). Rather, compared to eating rate in rats maintained on standard chow, rats given a HE diet show high rate of eating regardless of the day of diet presentation. Thus, it appears that more immediate signals such as those coming from the orosensory cavity rather than longer-term influences such as previous diet exposure, have a larger influence on eating rate. Further evidence in the literature support the notion that more immediate cues such as oral stimulation can influence mechanisms underlying feeding behavior. Sensory contact with oral stimuli have been shown to trigger autonomic and endocrine reflexes involved in metabolism before the influences of postingestive consequences. For example, the cephalic reflexes such as the secretion of saliva and gut responses can be elicited by taste stimuli in the oral cavity (Powley 1977; Berthoud et al. 1981). Compared to drinking water or an almond extract solution, drinking a tastant (saccharin, NaCl, or glucose) affected subsequent food intake in rats (Tordoff and Friedman 1989). Rats increased food intake during subsequent 2-h tests also following sham feeding with a corn oil emulsion or a sucrose solution, compared to a no sham-feeding condition (Tordoff and Reed 1991). Yet, when rats were maintained on a HE diet with liquid Ensure or with a diet with 45% fat and no liquid calories, sham-feeding responses to sucrose did not increase and in some cases, were lower than responses of standard chow-fed controls (Treesukosol et al. 2015). Thus under these conditions in which the stimulus was a HE diet, generalization is not observed. Given that a HE diet is a more complex stimulus than sucrose solutions or corn oil emulsions, this raises the question of what the different oral components of such a diet are. The current experiments were designed to investigate the gustatory components that contribute to the intake of a palatable, HE diet (containing 45% calories from fat and 17% calories from sucrose) which was used in previous studies (Treesukosol et al. 2014; 2015). Specifically, experiment 1 was designed to derive data to calculate concentrations of glucose, Na-saccharin Polycose, and fructose that elicit unconditioned licking responses comparable to those in response to 17% sucrose. These compounds were chosen to compare generalization to other sugars (glucose and fructose), a non-caloric sweetener (saccharin), and a palatable compound that is not considered “sweet” (Polycose). Next, based on these data, rats were conditioned to avoid a HE diet and subsequently generalization to an array of taste compounds was measured using a brief-access lick procedure. These data provide a qualitative profile of the HE diet that include the palatable stimulus array from experiment 1 as well as representatives of compounds that humans describe as sweet, sour, bitter, salty, and fatty. Together, the data from these experiments reveal the oral qualitative features of a HE diet. Materials and methods Experiment 1: determination of stimulus concentrations Subjects Eight male Sprague–Dawley rats (Harlan) with a mean body weight of 283.0 ± 1.4 g upon arrival were single-housed in hanging wire cages in a room with automatically controlled humidity, temperature, and a 12h–12h light-dark cycle. Rats had ad libitum access to chow (2018 Teklad, Harlan; 3.1 kcal/g) and water, except where noted. All procedures were approved by the Institutional Animal Care and Use Committee at The Johns Hopkins University. Procedure Brief-access taste tests were conducted during the light cycle. Brief-access taste procedure training and testing were conducted in a lickometer (Davis MS-160, DiLog Instruments, Tallahassee, FL) as described previously (Smith 2001; Glendinning et al. 2002). The rat was placed in the chamber of the testing apparatus with access to a single spout positioned ~5 mm behind a slot in the testing chamber wall. The spout was connected to a glass bottle containing a solution. To minimize potential olfactory cues from the stimuli, a small fan was positioned above the testing chamber wall to direct an air current past the spout. During training with water in the lickometer, the rats were placed on a water-restricted schedule. Access to water was removed from the home cages no more than 23 h before the test session. Water was available only during sessions in the lickometer. During the first 2 sessions in the lickometer, rats were presented with a stationary spout of water for 30 min. Total number of licks and inter-lick interval were measured. On day 3, 7 tubes of water were presented one at a time in 10-s trials across 30-min sessions. A trial was initiated when the rat licked the spout. The shutter closed at the end of each 10-s trial. During each 8-s inter-trial interval, a motorized block prepared the next spout presentation, after which the shutter reopened for the next trial. The spouts were presented in randomized blocks without replacement. Animals were able to initiate as many trials as possible during the 30-min session. At the end of training with water, ad libitum access to water resumed in the home cages for ~48 h before further testing began. Each test compound (sucrose, saccharin, glucose, Polycose, and fructose) was tested in daily 30-min sessions across 2 consecutive days (Table 1). Concentrations of sucrose (0.01, 0.03, 0.06, 0.1, 0.3, 1.0 M; Sigma Aldrich, St. Louis, MO), saccharin (0.1, 0.5, 1, 5, 10, 50 mM; Sigma Aldrich), glucose (0.06, 0.125, 0.25, 0.5, 1.0 M; Sigma Aldrich), Polycose (1, 2, 4, 8, 16, 32%; Abbott Laboratories, Columbus, OH), and fructose (0.06, 0.125, 0.25, 0.5, 1.0, 2.0 M; Sigma Aldrich) were chosen to span the dynamic range. Concentrations were presented in randomized blocks without replacement. As with the training sessions, a trial was initiated when the rat licked the spout and an animal could initiate as many trials as possible during the daily sessions. Table 1. Experimental design for experiment 1 (determination of stimulus concentrations) Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of sucrose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of saccharin   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of glucose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of Polycose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of fructose  Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of sucrose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of saccharin   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of glucose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of Polycose   Ad lib food and water  2  Multiple 10-s trials with varying concentrations of fructose  View Large Data analysis The average number of licks per trial at a given concentration was divided by that animal’s estimated maximal lick rate (licks/10 s) yielding a standardized lick ratio. The maximal lick rate was derived from the reciprocal of the mean of the inter-lick interval (ILI) values (in ms) that was measured during the last day of licking to a stationary spout of water. Only ILI values between 50 and 200 ms were used (Allison and Castellan 1970; Davis and Smith 1990, 1992) and this value was multiplied by 10 000 (Equation 1). In this way, the Standardized Lick Ratio accounts for individual differences in local lick rate.  Estimated maximum licks=10 000 ms×1(ILI (ms)) Curves were fit to mean data for each group by using the following logistic function:  y=a(1+10(x−c)b) where x = log10 stimulus concentration, a = asymptotic lick response, b = slope, and c = log10 concentration at the inflection point. Experiment 2: generalization of conditioned aversion to HE diet Subjects A separate group of male Sprague-Dawley rats (Harlan) with a mean body weight of 281.7 ± 1.3 g (cohort 1) and 286.5 ± 1.5 g (cohort 2) upon arrival were single-housed in a room where the temperature, humidity, and lighting (12h–12h, light-dark) were automatically controls. Rats had ad libitum access to powdered chow (2018 Teklad, Harlan) and water, except where noted. All procedures were approved by the Institutional Animal Care and Use Committee at The Johns Hopkins University. Procedure Animals were trained and tested for the brief-access taste procedure in a lickometer (Davis MS-160, DiLog Instruments, Tallahassee, FL) as described in experiment 1. The rats were trained and tested in the lickometer while on a water-restricted schedule. Access to water was removed from the home cages no more than 23 h before the first session in the lickometer and water was available only during the training and testing sessions. The first 2 sessions in the lickometer involved presenting the rats with a stationary spout of water for 30 min. On day 3, 7 tubes of water were presented in randomized blocks without replacement, one at a time in 10-s trials across 30-min sessions. Animals were able to initiate as many trials as possible during each 30-min session. At the end of day 3, ad libitum water access in the home cages resumed (Table 2). Table 2. Experimental design for cohort 1 and cohort 2 of experiment 2 (generalization of conditioned aversion to HE diet) Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Food restriction  8  High energy diet across conditioning trials   Water restricted  1  Multiple 10-s sessions with water presentation   Water restricted  1  Multiple 10-s s trials with stimulus array  Condition  Days  Stimulus  Water restricted  2  Stationary water  Water restricted  1  Multiple 10-s trials with water presentation  Two days hydration, no testing   Food restriction  8  High energy diet across conditioning trials   Water restricted  1  Multiple 10-s sessions with water presentation   Water restricted  1  Multiple 10-s s trials with stimulus array  View Large After at least 1 day of ad libitum access to water and chow, animals were assigned to 1 of 2 groups [LiCl (n = 8) or NaCl (n = 7–8)] such that there were no significant group differences in body weight, total licks to a stationary spout of water, ILI values, or number of trials initiated to water. Two cohorts of animals were tested. For cohort 1, the HE diet was presented while animals had ad libitum access to standard chow but the remaining conditioning trials and all conditioning trials for cohort 2, were conducted while animals were on a food-restricted schedule. Conditioning trials were conducted such that all animals were provided access to a powdered HE-diet (4.73 kcal/g; calories from protein 20%, calories from fat 45%, calories from carbohydrate 35%; D12451, Research Diets) for 1 h in the morning during the light-cycle followed by an intraperitoneal injection of 0.15 M LiCl (Sigma Aldrich; 1.33 mL/100 g body weight) to induce visceral malaise, or 0.15 M NaCl (Sigma Aldrich; 1.33 mL/100 g body weight) to serve as a control. In the afternoon, animals were presented access to the powdered standard chow diet (3.1 kcal/g, calories from protein 24%, calories from fat 18%, calories from carbohydrate 58%; 2018 Teklad, Harlan) to allow for refeeding. Of the fat source in the HE diet, ~6% of the calories is from soy bean oil and ~39% of the calories is from lard. Of the carbohydrate source in the HE diet, ~7% is from corn starch, ~10% from maltodextrin 10, and ~17% from sucrose. In contrast, the standard chow does not contain animal fat or added complex carbohydrates. Each conditioning trial was separated by 1 day of restricted access to the powdered standard chow diet (1-h access in the morning and 1-h access in the afternoon). During this period, animals had ad libitum access to water (Table 3). Table 3. Schedule for conditioning trials for cohort 1 and cohort 2 of experiment 2 (Generalization of conditioned aversion to HE diet) Day  AM  PM  1  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  2    Food restricted  3  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  4    Food restricted  5  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  6    Food restricted  7  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow  Day  AM  PM  1  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  2    Food restricted  3  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  4    Food restricted  5  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow until following day  6    Food restricted  7  High energy diet (1 h) followed by LiCl or NaCl injection  Presentation of ad libitum access to standard chow  Cohort 1 began conditioning with ad libitum access to standard chow. Cohort 2 was food restricted for 23 h before conditioning. Other conditions as outlined in Table 3 were the same for both cohorts. View Large After the fourth conditioning trial, animals resumed ad libitum access to standard chow. After at least 1 day of ad libitum access to food and water, water access was removed and the next day animals were tested in the lickometer for 2 days. On the first day, animals were re-habituated with the apparatus and session structure with 10-s trials to water during a 30-min session. On the second day, rats were tested in the lickometer to a panel of 7 stimuli presented in randomized blocks without replacement in 10-s trials. A 10-s water rinse was presented between each stimulus presentation during these sessions. For cohort 1, the stimuli were: water, 100% linoleic acid (Sigma Aldrich), 20% intralipid (Sigma Aldrich), 17% sucrose (Sigma Aldrich), 0.376 M NaCl (Sigma Aldrich), 0.131 mM quinine (Sigma Aldrich), and 10.4 mM citric acid (Sigma Aldrich). For cohort 2, the stimuli used were: 17% sucrose (Sigma Aldrich), 5.2% glucose (Sigma Aldrich), 5.5% fructose (Sigma Aldrich), 0.17% Na-saccharin (Sigma Aldrich), 2.8% Polycose (Abbott Laboratories, Columbus, OH), and 10.4 mM citric acid (Sigma Aldrich). The stimuli used for testing cohort 2 did not elicit strong suppression scores as with cohort 1. To ensure that the weak suppression scores were stimulus array specific and not cohort specific, an additional test day was conducted to serve as a control with cohort 2, during which 5.5% fructose was replaced with 20% intralipid. Even though testing was during extinction, these rats showed strong suppression scores to 20% intralipid on the additional test day providing evidence that the lower suppression scores were stimuli specific. As with the water training sessions, a trial was initiated when the rat licked the spout and animals could initiate as many trials as possible during the 30-min sessions (Table 2). A similar conditioning schedule has also been successfully used previously in mice (Eylam et al. 2003; Dotson and Spector 2007; Treesukosol et al. 2011) and avoidance of fat-containing diets have been conditioned previously in rats (Larue 1978). Concentrations for sweeteners were matched to the diet composition of 17% sucrose using data from Experiment 1 and concentrations for citric acid, NaCl, and quinine were derived from comparable response curves from published data sets (Grobe and Spector 2008). At the end of the experiment, animals were given ad libitum access to food and water for at least 1 day and then all animals were presented HE diet in a food-restricted state for 1-h and intake was recorded to assess the maintenance of the conditioned avoidance. Data analysis To assess the acquisition of the avoidance, HE diet intake was compared between each LiCl group and its respective NaCl control group across each of the 4 conditioning trials using t-tests. For responses to the panel of stimuli, a suppression score for each LiCl-injected rats was calculated for each of the test stimuli (TS) during the test session (Nowlis et al. 1980; Eylam et al. 2003). For a given TS and LiCl-injected rat, this value was derived by the equation:  Suppression score=1−Rat Licks TSLiClGroup Licks TSNaCl where Rat Licks TSLiCl = the number of licks to a given TS of an individual animal in the LiCl-injected group, and Group Licks TSNaCl = the group mean for licks to the TS for the NaCl-injected group. This allows suppression scores for each LiCl-injected rat to each of the test stimuli to be obtained. A score of 0 indicates equal licking responses to the taste stimulus between the LiCl- and NaCl-injected groups (i.e. no licking suppression). A suppression score of 1.0 indicates complete suppression of licking to the taste stimulus by the LiCl-injected rat. Results Experiment 1: determination of stimulus concentrations Results from experiment 1 are shown in Figure 1. Note that in contrast to increasing licking behavior in response to ascending concentrations of some natural sugars such as sucrose, response to saccharin in rats has previously been shown to yield an inverted U-shape function peaking at approximately 8 mM (Smith 2000). To determine concentrations that elicit comparable licking, the concentration values (x) were solved by using: Figure 1. View largeDownload slide Mean ± SE standardized lick ratio values across concentrations of sucrose, Na-saccharin, glucose, Polycose, and fructose derived from a brief-access paradigm (experiment 1) to assess concentration-dependent changes in licking behavior. These data were used to calculate concentrations of other stimuli that elicit unconditioned licking responses comparable to those in response to 17% sucrose. Figure 1. View largeDownload slide Mean ± SE standardized lick ratio values across concentrations of sucrose, Na-saccharin, glucose, Polycose, and fructose derived from a brief-access paradigm (experiment 1) to assess concentration-dependent changes in licking behavior. These data were used to calculate concentrations of other stimuli that elicit unconditioned licking responses comparable to those in response to 17% sucrose.  x=log10(ay−1)b+c Based on the response curves from experiment 1, 17% sucrose corresponded to 0.85 of the maximal licking rate. Next, the concentrations that corresponded to 0.85 of the maximal licking rate for glucose, Na-saccharin, Polycose, and fructose were calculated. Concentrations for citric acid, NaCl, and quinine were derived from comparable response curves from published data sets (Dotson et al. 2005). Experiment 2: generalization of conditioned aversion to HE diet In both cohorts, LiCl-injected rats showed evidence of avoidance acquisition to the HE diet across the conditioning trials (Figure 2). For cohort 1, there was no significant group difference in intake during the first pairing trial (t(15) = −0.364, P = 0.721), but the LiCl group ate significantly less than controls from the second trial onwards (t(15) > 3.576, P < 0.003). Similarly for cohort 2, HE intake of the LiCl- and NaCl-injected groups was similar in the first pairing trial (t(14) = 1.045, P = 0.314) but LiCl-injected rats suppressed HE diet intake in subsequent sessions (t(14) > 4.698, P < 0.001). These data indicate there was no significant difference in intake between the groups at baseline and confirm effectiveness of the conditioning procedures. Figure 2. View largeDownload slide Intake of HE diet during 1-h conditioning trials for NaCl-injected (black bars) and LiCl-injected (white bars) rats in cohort 1 (left panel) and cohort 2 (right panel) in experiment 2. Note, Cohort 1 began conditioning with ad libitum access to standard chow (Pairing trial 1). Cohort 2 was food restricted for 23 h before conditioning. Conditions for pairing trial 2 onwards were the same for both cohorts. *Denotes significant group differences. Figure 2. View largeDownload slide Intake of HE diet during 1-h conditioning trials for NaCl-injected (black bars) and LiCl-injected (white bars) rats in cohort 1 (left panel) and cohort 2 (right panel) in experiment 2. Note, Cohort 1 began conditioning with ad libitum access to standard chow (Pairing trial 1). Cohort 2 was food restricted for 23 h before conditioning. Conditions for pairing trial 2 onwards were the same for both cohorts. *Denotes significant group differences. Brief-access testing Rats conditioned to avoid the HE diet displayed cross-generalization to 100% linoleic acid, 20% intralipid, and 17% sucrose. Suppression ratio scores were significantly higher than 0 for 100% linoleic acid (t(9) = 3.122, P = 0.014), 20% intralipid (t(9) = 11.092, P < 0.001), and 17% sucrose (t(9) = 2.861, P = 0.021) but the LiCl-injected rats did not generalize conditioned avoidance to the other stimuli presented (Figure 3). There was more variability in licking responses to sucrose in the LiCl group compared to the NaCl group. Thus in cohort 2, licking responses to 17% sucrose as well as other sweeteners and Polycose were measured. Here, rats conditioned to avoid the HE diet did not display cross-generalization to sucrose or any of the compounds tested (Figure 3). Within the LiCl group, animals that tended to suppress lick responses to 17% sucrose also did so to 5.2% glucose, 5.5% fructose, and 0.17% Na-saccharin. In contrast, significant correlations were not observed between responses to sucrose with Polycose, citric acid, or water (Figure 4). Furthermore, significant correlations were not revealed between lick responses between sucrose and intralipid (r = −0.177, P = 0.649) nor sucrose and linoleic acid (r = 0.257, P = 0.505) in cohort 1. Nor were significant correlations revealed between HE diet intake during the second pairing and lick responses to sucrose (cohorts 1, 2), linoleic acid, or intralipid (cohort 1) (r > −0.613, P > 0.790). In cohort 1, a significant correlation between suppression lick scores to sucrose and NaCl was found (r = 0.890, P = 0.001). Also, animals that tended to suppress responses to sucrose, also did so to citric acid in cohort 1 (r = 0.634, P = 0.067) and cohort 2 (r = 0.682, P = 0.062) but these comparisons did not reach statistical significance. Figure 3. View largeDownload slide Suppression ratio scores for rats conditioned to avoid the HE diet indicate degree of generalization to water (W), 100% linoleic acid (LA), 20% intralipid (IL), 17% sucrose (S), 0.376 M NaCl (N), 0.131 mM quinine (Q), 10.4 mM citric acid (CA), 5.2% glucose (G), 5.5% fructose (F), 0.17% Na-saccharin (SA) and 2.8% Polycose (P) for cohort 1 (A), and cohort 2 (B). Suppression ratio scores significantly higher than 0 (denoted by *) is evident for 100% linoleic acid, 20% intralipid and 17% sucrose for cohort 1. Figure 3. View largeDownload slide Suppression ratio scores for rats conditioned to avoid the HE diet indicate degree of generalization to water (W), 100% linoleic acid (LA), 20% intralipid (IL), 17% sucrose (S), 0.376 M NaCl (N), 0.131 mM quinine (Q), 10.4 mM citric acid (CA), 5.2% glucose (G), 5.5% fructose (F), 0.17% Na-saccharin (SA) and 2.8% Polycose (P) for cohort 1 (A), and cohort 2 (B). Suppression ratio scores significantly higher than 0 (denoted by *) is evident for 100% linoleic acid, 20% intralipid and 17% sucrose for cohort 1. Figure 4. View largeDownload slide Comparisons of suppression ratio scores for sucrose and other test stimuli in rats of cohort 2 conditioned to avoid the HE diet. Figure 4. View largeDownload slide Comparisons of suppression ratio scores for sucrose and other test stimuli in rats of cohort 2 conditioned to avoid the HE diet. After brief-access testing, avoidance of the HE diet was still evident in the LiCl-injected rats (Figure 2; “after test”). In the second cohort, lick responses to 20% intralipid in 10-s trials were measured in a subsequent brief-access test, suppression ratio scores were significantly higher than 0 (t(7) = 41.469, P < 0.001). These data confirm the effectiveness and maintenance of the conditioned avoidance and reliability of the cross-generalization to 20% intralipid even following an extinction trial. Discussion Rats conditioned to avoid the HE diet (45% fat, 17% sucrose) generalized avoidance to intralipid and linoleic acid but there was more variability in cross-generalization to sucrose. These findings show that orally, the “fat” component of the HE diet is more salient than the sucrose component. This is consistent with prior reports in which rats conditioned to avoid corn oil generalized avoidance to a sucrose + corn oil mixture, more so than rats conditioned to avoid sucrose (Smith et al. 2000). Similarly rats conditioned to avoid a glucose + saccharin + corn oil emulsion suppressed licking only to stimuli that contained corn oil (Smith 2004). Furthermore, in regard to how the orosensory properties of fats drive behavior, when rats were given only food and water or food plus a glucose and saccharin solution over 6 weeks, rats regulated caloric intake such that they gained weight at comparable rates. In contrast, in a group for which corn oil was added (i.e. food, glucose + saccharin + fat), rats increased caloric intake and body weight (Smith 2004). Using sham-feeding techniques which involve orosensory stimulation but minimal postingestive stimulation it was shown that orosensory properties of fats is sufficient to drive sham feeding in rats (Greenberg et al. 1996). Taken together it appears that the “fat” component is a salient feature of an oral stimulus. Here, there was more variability in responses to sucrose in rats conditioned to avoid the diet, compared to those of NaCl-injected controls. Polysaccharides are glucose polymers of varying chain lengths. Polycose has been the most studied polysaccharide in taste experiments and contains a small amount of glucose and maltose but primarily consists of glucose polymers of 3 or more glucose units (Kennedy et al. 1995). In studies in which taste aversion generalization procedures were employed, rats, mice and gerbils conditioned to avoid sucrose also avoid other compounds that humans describe as sweet such as fructose and glucose (Nowlis et al. 1980; Dugas du Villard et al. 1981; Jakinovich 1982; Ninomiya et al. 1984; Nissenbaum and Sclafani 1987; Sako et al. 1994). However, rats conditioned to avoid Polycose or sucrose show no or very weak cross-generalization (Nissenbaum and Sclafani 1987; Ramirez 1991; Sako et al. 1994) suggesting these 2 carbohydrate solutions have different taste qualities. Similarly, hamsters trained to avoid Polycose, sucrose, or a mixture of the 2 compounds display some degree of cross-generalization, but Polycose and sucrose, nonetheless, appear to have characteristics that make the compounds distinct from one another (Formaker et al. 1998). It has been shown that the T1R2 and T1R3 proteins are unnecessary for mice to express normal affective responses to Polycose yet mice missing one or both of these proteins display severely blunted responses to saccharin and sucrose (Treesukosol et al. 2009; Zukerman et al. 2009). Furthermore, mice missing the T1R2 or T1R3 subunit show severely impaired taste detection performance to compounds humans label as sweet including sucrose and glucose but are competent in the detection task when the stimulus is Polycose (Treesukosol et al. 2012). In the current study, rats in the LiCl group that suppressed responses to sucrose tended to also suppress responses to fructose, glucose, and Na-saccharin but not to Polycose. This suggests there is a “sweet” component to the HE diet that is separate from polysaccharide taste. These findings are also consistent with evidence that polysaccharides (such as Polycose) and stimuli that humans describes as sweet, (such as sucrose) are separate taste qualities (Sclafani 1987). In classical conditioning, latent inhibition refers to the observation that compared to a novel stimulus, it is more difficult to form a new association with a more familiar stimulus. A taste compound repeatedly presented without aversive consequences requires more pairings with negative consequences to elicit conditioned taste aversion of comparable intensity (Best 1975; Albert and Ayres 1989). In the current experiments, rats conditioned to avoid the HE diet showed strong generalization to “fatty” stimuli and more varied generalization to the “sweet” stimuli raising the possibility that latent inhibition contributed to these differential responses. In other words, the strong generalization to linoleic acid is driven by novelty to the stimulus and the weaker generalization to sucrose is driven by some familiarity to the stimulus. The fat and carbohydrate sources of the HE diet used in the conditioning trials and that of the chow diet that the animals were familiar are not similar. Of particular interest, the chow diet has no added sucrose or complex carbohydrates. Thus, it is unlikely that familiarity with some components of the HE diet explains the differential responses to the “fat” and “sweet” components observed in the generalization profiles. In response to the presentation of a palatable HE diet, rats (and other animals) will overeat. The hyperphagia induced by the availability of a HE diet may be driven by enhanced orosensory cues and/or dampened inhibitory feedback yet we have previously demonstrated that these alterations in signaling do not generalize to increased responsiveness to other palatable stimuli as measured by sham-feeding intake of sucrose (Treesukosol et al. 2015). Here in some animals, conditioned avoidance of the HE diet cross-generalizes to sucrose and other “sweet” compounds but not to Polycose. Thus it appears that calorically dense diet induced hyperphagia does not necessarily generalize to increased responses to other palatable stimuli. In the psychophysical literature, features of chemosensory mixtures are measured by comparing responses to single components of the mixture. A number of models have been proposed to describe these perceptual experiences (Frijters 1987). When conditioned to avoid single component stimuli and then tested with mixtures that include the conditioned stimulus, rats (Smith and Theodore 1984), and hamsters (Frank et al. 1989) avoid mixtures containing the conditioned stimulus in proportion to its concentration in the mixture. It has been shown that conditioned aversions to binary (Frank and Nowlis 1989) or ternary mixtures (Frank et al. 2003) more readily generalize to single components than vice versa. A possible explanation for this asymmetry in generalization profiles is that it is adaptive for an organism conditioned to avoid a complex stimulus to then avoid each identified component due to uncertainty as to which component of the mixture is associated with negative consequences. In contrast, an organism conditioned to avoid a single component may generalize less so to a mixture containing the conditioned stimulus because the other components provide a safe context (Taylor and Boakes 2002; Frank et al. 2003). Thus in the current study, by examining the degree to which conditioned avoidance of a HE diet (a stimulus of multiple components) generalizes to more simple components provides a qualitative profile of the HE diet. The concentrations of the stimuli in the cross-generalization test were chosen to match the components of the HE diet. The diet contains 17% sucrose thus this concentration was chosen. Next, concentrations of glucose, fructose, Na-saccharin, and Polycose that elicit comparable unconditioned lick responses as 17% sucrose were chosen. Findings from previous studies in which solutions (which allow for more accurate concentration matching) were used for both conditioning and testing of generalization, showed that corn oil was a more salient component than glucose or saccharin (Smith 2004). This is consistent with the current data that the “fat” component is the more salient feature. Here, we chose to focus on the qualitative aspects of the stimuli but the data also raises the question of whether less variability in the responses to the sweeteners would be observed if a higher concentration range were tested. This procedure could be employed to conduct studies using varying concentrations of test stimuli to address the question of relative intensity of the diet components. The brief-access feature of the procedure employed here allows for behavioral measures in which the influence of postingestive factors are minimized. These findings support a role for gustatory cues in the responsiveness towards high fat/high sugar diets. Furthermore, it appears that the fat component is a salient orosensory feature of the palatable, HE diet. Funding This work was supported by the National Institutes of Health (grant number DK019302 to T.H.M). 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Published: Mar 1, 2018

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