Capsaicin-induced visceral deafferentation does not attenuate flavor conditioning by intragastric fat infusions in mice

Abstract

The postoral actions of sugar and fat can rapidly stimulate the intake of and preference for flavors associated with these nutrients via a process known as appetition. Prior findings revealed that postoral glucose appetition is not attenuated following capsaicin-induced visceral deafferentation. The present experiment determined if capsaicin treatment altered fat appetition in C57BL/6 mice. Following capsaicin (Cap) or control (Con) treatment, mice were fitted with chronic intragastric (IG) catheters. They were then given 1-h sessions with a flavored saccharin solution (CS−) paired with IG water infusion or a different flavor (CS+) paired with IG 6.4% fat infusion. IG fat stimulated CS+ intakes in both Cap and Con mice, and the groups displayed similar preferences for CS+ over CS− in two-choice tests. These results confirm prior reports of normal fat conditioning in rats exposed to capsaicin or vagal deafferentation surgery. In contrast, other recent findings indicate that total or selective vagotomy alters the preference of mice for dilute vs. concentrated fat sources.

1. Introduction

There is extensive evidence that gut nutrient sensing can condition food preferences and, in some cases, simulate food intake. This postoral nutrient conditioning process has been referred to as appetition to distinguish it from the nutrient satiation process that suppresses food intake [8]. Mouse studies have implicated intestinal glucose transporters/sensors (SGLT1, SGLT3, GLUT2) and fatty acid sensors (GPR40, GPR120) in postoral carbohydrate and fat appetition, respectively, although other pre- and post-absorptive sensing mechanisms may be involved [12,15,21]. The gut-brain pathways that mediate postoral nutrient appetition remain to be identified but rat studies indicate that vagal afferent fibers are not essential for carbohydrate or fat appetition [5,11,13,16]. Recent findings also revealed that intact vagal innervation is not required for glucose appetition in mice [7,20], although one study indicated that, in contrast to rats, vagal signaling may be essential for postoral fat conditioning in mice [7]. This study involved total subdiaphragmatic vagotomy (TVX) and it possible that disruption in gut motor function impaired fat conditioning. The present experiment determined if visceral deafferentation produced by systemic capsaicin treatment impairs flavor conditioning by gastric fat infusions. In prior studies capsaicin treatment did not block carbohydrate or fat conditioning in rats or glucose conditioning in mice [5,20], but its effect on fat-conditioned flavor preferences has not been investigated.

2. Materials and methods

2.1. Animals

Male C57BL/6J (B6) mice (8 wk old) purchased from Jackson Laboratories (Bar Harbor, MA) were singly housed in plastic tub cages kept in a test room maintained at 22°C with a 12:12-h light-dark cycle. The mice were maintained on chow (5001; PMI Nutrition International, Brentwood, MO) prior to food restriction. During testing they were fed fixed-size chow pellets (0.5 or 1 g, cat. nos. F0171, F0173; Bio-Serv, Frenchtown, NJ), which allowed for precise adjustment of the daily food ration. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Brooklyn College and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

At 10 wk of age mice were treated with systemic capsaicin (n=13, 50 mg/kg; Sigma, St. Louis, MO) or vehicle (n=11) according to a previously published procedure [19] except that the mice were initially anesthetized with isoflurane (2%). The efficacy of the capsaicin treatment was evaluated at 9 days after treatment and at the end of behavioral testing by using a corneal chemosensory test that involved monitoring the eye-wiping reflex after ocular administration of a 7.5 μl drop of 0.2% NH4OH solution [19,20].

At 12 wk of age the mice were anesthetized with isoflurane and fitted with a gastric catheter, as described previously (48). Two weeks later they were briefly (5 min) anesthetized with isoflurane and fitted with an infusion harness and spring tether (model CIH62; Instech Laboratories, Plymouth Meeting, PA). Plastic tubing connected the gastric catheter to an infusion swivel mounted on a counterbalanced lever (Instech Laboratories). Each animal was then returned to its home cage and the swivel counterbalanced lever was attached above the cage.

Three animals died during surgery (1 capsaicin (Cap), 2 control (Con) mice). Nine of the remaining capsaicin-treatment mice showed little or no eye wiping during the two tests compared to the vehicle-treated controls (1.7 vs. 13.1, t(16) = 8.4, P < 0.001). Three other capsaicin mice showed eye wipes within the range of the control mice during the second test and were discarded from the experiment.

2.2. Test solutions

Flavored nonnutritive solutions were prepared using 0.025% sodium saccharin (Sigma Chemical Co., St. Louis, MO), 0.02% ethyl acetate or propyl acetate (Sigma) and deionized water. An unflavored 0.025% saccharin solution was used for preliminary training. For half of the subjects, the ethyl acetate solution (the CS−) was paired with IG infusion of water while the propyl acetate solution (the CS+) was paired with IG fat infusions; the CS− and CS+ flavors were reversed for the remaining animals. The fat infusions contained 6.4% soybean oil prepared by diluting 20% Intralipid (Baxter, Deerfield, IL) with deionized water. The IG infusions (0.5 ml/min) were automatically delivered via a syringe pump controlled by a computer that detected the mouse’s licking response to the sipper tube [2]. The volume infused matched the amount of saccharin solution consumed.

2.3. Procedure

Three weeks postsurgery, the mice were water restricted and trained to drink unflavored saccharin paired with IG water infusion for two 1 h/day sessions in infusion cages described in detail elsewhere [2]. The mice were then food restricted, maintained at 85–90% of ad libitum body weight, and given four daily 1-h sessions with unflavored saccharin paired with IG water. In these and subsequent one-bottle training sessions with flavored solutions the left-right position of the sipper spouts was alternated daily.

Following these preliminary sessions, the mice were given three daily 1-h test sessions with the CS− solution paired with IG water infusions followed by three sessions with the CS+ solution paired with IG fat infusions. They were next given four alternating 1 h/day sessions with the CS−, CS+, CS− and CS+, in that order, with each solution paired with its respective infusion. In the last of these CS− and CS+ sessions the mice were given a second sipper tube containing water not paired with IG infusions to familiarize them to the presence of two sipper tubes in the subsequent two-bottle test. The two-bottle test, with the CS+ and CS− solutions no longer paired with IG infusions, was conducted over four 1 h/day sessions. The positions of the CS+ and CS− bottles alternated daily during two-bottle testing.

2.4. Data analysis

CS− licks and total intakes (oral + IG infusion) during the second and third 1 h/day sessions were averaged. The average data of these two-session, referred to as Test 0, and the licks and intakes during the first three CS+ sessions (Tests 1–3) were analyzed using a mixed model analysis of variance (ANOVA) with a group factor and a repeated measures factor (Tests 0–3). The mean licks of CS− and CS+ during the subsequent alternating sessions were compared in a separate ANOVA. Additional analyses are described in the results.

3. Results

The Cap and Con mice increased their 1-h licks when shifted from the CS− (Test 0) to the CS+ (Tests 1-3) [F(3,48) = 57.8, P < 0.001] (Fig. 1A). The groups differed somewhat in their licking response to the CS+ solution [Group x Test interaction [F(3,48) = 2.9, P < 0.05], but there were no significant group differences in Tests 0 to 3 licks. The Cap mice increased their 1-h licks by 51% to 93%, and the Con mice from 31% to 93% in Tests 1 to 3 relative to Test 0. Both groups also increased their total fluid intakes (CS + IG) from Test 0 to 3 [F(3,48) = 26.1, P < 0.001]; intake increased in the Cap mice from 2.7 to 3.9 g/h, and in the Con mice from 3.1 to 4.5 g/h. In the four alternating CS+ and CS− sessions, the Con mice tended to lick more for the CS+ than CS− (2141 vs. 1919 licks/h) while the Cap mice showed the opposite pattern (1940 vs. 1986 licks/h) but there were no Group or Group × Test differences.

Fig. 1.

A. Mean (+sem) 1-h total licks are plotted for one-bottle Tests 0–3 in capsaicin-treated (CAP, n=9) and control (CON, n=9) mice. The mice drank (1 h/day) a CS− flavored saccharin solution paired with IG water self-infusions in Test 0 before being switched to a CS+ flavored saccharin solution paired with IG 6.4% Intralipid self-infusions in Tests 1–3. Significant differences (P < 0.05) between Test 0 and Tests 1–3 licks are indicated by an asterisk. B. Mean (+sem) 1-h total licks are plotted for CS+ and CS− flavored saccharin solutions during the two-bottle preference test for capsaicin-treated and control mice. CS+ and CS− intakes were not paired with IG infusions during the test. Number atop bar represents mean percent preference for the CS+ solution. Significant differences (P < 0.05) between CS+ and CS− licks are indicated by an asterisk.

In the two-bottle choice test, the Cap and Con mice licked substantially more for the CS+ than the CS− [F(1,16) = 47.6, P < 0.001] and the groups did not differ in their CS licks or CS+ percent preference (82% vs. 86%, respectively) (Fig. 1B). The groups also consumed significantly more CS+ than CS− solution in the test (Cap: 2.6 vs. 0.9 g/h; Con: 2.9 vs. 0.7 g/h) [F(1,16) = 55.7, P < 0.001].

4. Discussion

The present findings revealed that capsaicin-induced visceral deafferentation did not attenuate postoral fat appetition in B6 mice. IG fat infusions stimulated the licking and intake of a CS+ solution and conditioned a CS+ flavor preference to similar degrees in capsaicin-treated and control mice. Consistent with prior findings [2,20], the 6.4% fat infusions increased licking of the CS+ solution in the first test session and nearly doubled CS+ licking by Test 3 relative to the CS− intake in Test 0. The significant fat conditioned CS+ preferences displayed by the Cap and Con groups are consistent with findings obtained with capsaicin-treated and control rats [5]. Whereas the fat infusions stimulated CS+ test intakes in the present study, they decreased CS+ intakes, relative to CS− intakes in the prior study even though the rats were infused with 3.5% rather than 6.5% fat emulsions. However, duodenal rather than gastric infusions were used in the rat experiment which is important because duodenal fat infusions are more satiating that gastric infusions [6].

The fat-conditioned CS+ preference displayed by capsaicin mice contrasts with the report that postoral fat conditioning is “disrupted” in mice with total subdiaphragmatic vagotomy [7]. Conceivably, systemic capsaicin treatment may spare some critical vagal afferents that are destroyed by TVX. Alternatively, gastrointestinal motor disruption produced by TVX may attenuate flavor conditioning by altering fat processing in the gut. Compatible with this view, we observed that selective surgical afferent vagotomy (SDA), which spares many vagal efferents, did not block postoral fat conditioning in rats [11]. On the other hand, Han et al. [3] reported disrupted fat conditioning in mice with highly selective afferent vagotomy (HSDA) produced by neurochemical lesions of CCK-responsive afferents in the right vagal branch. However, comparisons of these various studies is complicated by the different conditioning paradigms used. In this and our prior fat conditioning studies, animals were trained to associate CS+ and CS− flavors with postoral infusions of fat emulsions and water, respectively [2,5,11,15]. In contrast, Qu et al. [7] assessed fat conditioning by having mice orally consume dilute and concentrated fat emulsions. In particular, the mice were given an initial 10-min two-bottle choice test with 7.5% vs. 30% fat (Intralipid) followed by a 24-h choice test and then another 10-min test with the same fat emulsions. In the first 10-min test, control mice showed no preference for the two emulsions, whereas in the second 10-min test, they consumed more 7.5% than 30% fat (in ml) which was attributed to their learning about the postoral actions of the two emulsions in the intervening 24-h test. In contrast, the TVX mice equally preferred the two fat emulsions in the first and second 10-min tests which was taken as evidence for “a deficit in lipid post-ingestive signaling.” The control mice may have developed a preference for the 7.5% fat because the satiating actions of the 30% fat counteracted its postoral appetition actions [9,14,17,18]. Conceivably, the TVX mice in the Qu et al. [7] study may not have preferred 7.5% to 30% fat because vagotomy reduced the satiating and therefore the appetition-limiting actions of the concentrated fat.

Han et al. [3] also assessed fat conditioning by comparing the response to two different fat emulsions. In this case, mice were trained to associate one CS+ flavor with IG infusions of 5% Intralipid and a different CS+ flavor with IG infusions of 20% Intralipid. (Note, the fat emulsions were diluted to about 2.5% and 10% in the stomach by the ingested CS solutions.) In a subsequent two-choice choice test without IG infusions, the control mice preferred the CS+20% flavor to the CS+5% flavor whereas the HSDA mice equally preferred the two CS+ flavors. This was taken as evidence that HSDA interfered with postoral fat flavor learning. Yet, during one-bottle training, HSDA and control mice displayed similar increases in their CS+5% and CS+20% intakes over sessions, which is indicative of postoral fat appetition. The failure of animals to prefer one nutrient-paired CS+ over another does not, by itself, demonstrate a lack of nutrient sensing or flavor conditioning. We previously reported that rats trained to associate a CS+ flavor paired with IG infusions of a high-fat, high-calorie liquid diet and a differently flavored CS+ paired with a high-carbohydrate, low-calorie diet equally preferred to the two CS+ solutions in a direct choice test. Yet the rats significantly preferred both CS+ flavors to a CS− flavor that was paired with IG water infusions [4]. It is not known if the HSDA mice in the Han et al. [3] study would have preferred the CS+5% and CS+20% flavors to a CS− flavor since they were not trained with a CS−/IG water option. In another conditioning assay, however, Han et al. [3] reported that control mice increased their licking at a dry sipper tube which was paired with IG infusions of 20% fat (Intralipid) whereas HSDA mice did not. Taken together, their results indicate that HSDA reduced postoral fat sensing in the absence of flavor cues but don’t necessarily establish that it blocked flavor conditioning. Other findings indicate that flavor preference conditioning is a more sensitive assay of postoral nutrient sensing than is dry sipper tube licking [10]. As noted above, afferent vagotomy (SDA) did not block postoral fat conditioning in rats, nor did celiac-superior mesenteric ganglionectomy (CGX), but SDA combined with CGX blocked conditioning [11]. This suggests that vagal and sympathetic afferents are required for postoral fat appetition in rats, although a role for sympathetic efferent fibers cannot be ruled out. Future experiments should investigate if vagal and sympathetic afferents are essential for fat appetition in mice which, show stronger fat conditioning than do rats [1,20].

Zukerman et al. [20] reported that capsaicin-treated mice, like controls, acquired a preference for a flavor mixed into a glucose solution over a different flavor mixed into an initially more-preferred nonnutritive sucralose solution, which was attributed to the postoral reinforcing actions of glucose. Similarly, Qu et al. [7] observed that TVX and control mice learned to prefer glucose to sucralose after 24-h exposure to both sweeteners. Consistent with these results, capsaicin treatment, TVX or SDA surgery in rats did not attenuate CS+ flavor conditioning by postoral infusions of glucose or Polycose [5,11,13,16]. Furthermore, CGX and CGX + SDA surgery attenuated but did not block Polycose-conditioned flavor preferences [11]. In contrast, glutamate-conditioned flavor preferences were blocked by TVX in rats and mice [7,16]. Taken together, the findings indicate differential involvement of gut-brain neural pathways in postoral fat, glucose, and glutamate flavor conditioning.

HIGHLIGHTS.

• Postoral actions of fat stimulate appetite in mice.

• Intragastric self-infusions of 6.4% Intralipid conditioned flavor preferences.

• Capsaicin-induced visceral deafferentation did not attenuate fat conditioning.

• The capsaicin findings are similar to those observed with postoral glucose conditioning.