- Email: [email protected]
Effects of neuropeptide Y on ingestion of flavored solutions in nondeprived rats
Physiology&Behavior,Vol. 54, pp. 877-880, 1993
0031-9384/93 $6.00 + .00 Copyright© 1993 PergamonPressLtd.
Printed in the USA.
Effects of Neuropeptide Y on Ingestion of Flavored Solutions in Nondeprived Rats W E S L E Y C. L Y N C H , *~ M A R T H A
GRACE,l" C H A R L E S J. B I L L I N G T O N I " A N D A L L E N S. LEVINEI"
*Department of Psychology, Montana State University, Bozeman, M T 5971 7 and -?Research and Medicine Service, Minneapolis Veterans Administration Medical Center, Minneapolis, M N 55417 Received 23 N o v e m b e r 1992 LYNCH, W. C., M. GRACE, C. J. BILLINGTON AND A. S. LEVINE. Effectsofneuropeptide Yon ingestionofflavoredsolutions in nondeprivedrats. PHYSIOL BEHAV 54 (5) 877-880, 1993.--Recent evidence suggests that in addition to altering energy balance, neuropeptide Y (NPY) may stimulate ingestive behavior by modifying the orosensory quality of ingested substances. The present experiments investigated the effect of intracerebroventricular administration of NPY (5 #g/5 #1) on ingestion of various flavored solutions in nondeprived rats. Experiment 1 examined the effectsof NPY on ingestion of a range of concentrations of saline, sucrose, and saccharin solutions in single-bottle tests. Results indicated that NPY stimulates ingestion of both sucrose and saccharin solutions that are normally palatable. In Experiment 2, palatable sucrose solutions flavored with either orange or black cherry Kool-Aid® for separate training groups were selectively associated with NPY injection during single-bottle training sessions. Subsequent two-bottle preference tests showed a significant shift in preference toward the flavor paired with NPY during training. The results of these experiments extend previous findings by showing that NPY can stimulate ingestion of sweet solutions regardless of caloric value and may potentiate sweet taste preference via an associative mechanism. Neuropeptide Y
Feeding
Drinking
Taste preference
NEUROPEPTIDE Y (NPY) is a 36 amino acid peptide found in the brain and peripheral tissues of numerous species. Since its original characterization (13), a key focus of research has been the role NPY plays in regulation to feeding and drinking. Clark et al. (3), for instance, showed that moderate doses of NPY (2-10 #g) injected into the lateral ventricles (ICV) significantly stimulated eating behavior in rats during the light phase of the light/dark cycle, and Morley and Levine (6) found that ICV NPY in this same dose range stimulated both food and water intake in fully satiated rats. More recent work has suggested that NPY may act to preferentially stimulate carbohydrate intake via circuits linked to the medial hypothalamus (7,14) and to alter energy metabolism (1) and energy balance ( 11 ). These various effects of centrally injected NPY on food energy intake are remarkably robust and suggest that NPY produces an essential alteration in the underlying structure of ingestive behavior. Neuropeptide Y's effects on ingestive behavior could be due either to increases in perceived hunger or enhancement of the rewarding properties of food. At specific hypothalamic sites, particularly the arcuate to paraventricular nucleus projection, it appears to play a regulatory role in response to alterations in energy balance (2,5). This evidence can be interpreted as indicating that NPY signals hunger within central controlling elements. At present, this evidence is inconclusive, in part because it is ditficult to ascertain whether an animal is truly hungry or is eating for some other reason.
Flavor
Conditioning
Carbohydrate
The possibility that neuropeptide Y might alter the orosensory or rewarding consequences of ingestion, rather than prompting ingestion directly, has been less explored. Evidence that neuropeptide Y can stimulate water intake independently of deprivation or food availability (12) and that its ability to stimulate drinking depends on the orosensory properties of ingested solutions (4,8) suggests that some aspect of ingestive behavior itself may be modified by NPY. In addition, recent reports that food intake stimulated by NPY might, paradoxically, be associated with taste aversions (10) raises further interest in the possible influence of orosensory factors. The present experiments aimed to investigate the role of orosensory factors in the feeding-stimulant effects of NPY. In a pilot study we found that NPY stimulated greater intake of novelflavored sucrose solutions, on first exposure to them, than did 24-h food deprivation. Moreover, NPY appeared to potentiate intake of the solutions previously associated with NPY, under subsequent food-satiated and food-deprived testing conditions. These preliminary results suggested that NPY might enhance the gustatory evaluation of ingested solutions and perhaps promote preference for an NPY-associated flavor. The two experiments described here further examined these suggestions. Experiment 1 investigated the effects of NPY on ingestion of sucrose, saccharin, and saline solutions of various concentrations ranging from neutral (tap water) to preferred. Experiment 2 tested the hypothesis that NPY might stimulate ingestion of a novel-
Requests for reprints should be addressed to Dr. Wesley C, Lynch, Department of Psychology, 300 Traphagen Hall, Montana State University, Bozeman, MT 59717.
877
878
I.YNCtt E1 Ai
flavored sucrose solution by potentiating a learned flavor preference. METHOD
Subjects Subjects were 84 male Sprague-Dawley albino rats (Biolab, St. Paul, MN) weighing 225-340 g at the start of testing. Sixty animals were used in Experiment 1 and 24 were used in Experiment 2. Animals were maintained in individual wire mesh home cages on a 12/12 h light/dark cycle with lights on daily at 0700 h. Animals had ad lib access to Purina chow pellets and tap water except as noted.
Surgery and Histology Under Nembutal anesthesia (50 mg/kg, IP), unilateral stainless steel guide cannulas (23 ga) were implanted sterotaxically into either the left or right lateral ventricles. Stereotaxic coordinates were 1 mm posterior and 1.5 mm lateral to bregma with the incisor bar adjusted 3.5 mm below the interaural line. Guide cannula tips extended 3.5 mm below skull surface. Animals were allowed at least 7 days for recovery following surgery. Injections of drug or vehicle solutions were made manually with a 10-t4 Hamilton syringe attached to a 30-ga stainless steel injector that extended 1 mm beyond the guide cannula tip. Cannula placements were verified by injecting 5 #1 of India ink following death by carbon dioxide overdose and subsequent decapitation. Data from rats with incorrect cannula placements were excluded from data analyses.
Drugs Pomine-derived NPY (Peninsula Laboratories, Belmont, CA) was mixed fresh daily and injected at a single dose (5 #g/5 #1, ICV) in both experiments. This close was selected on the basis of previous food intake studies and our own preliminary work indicating reliable stimulation of flavored solution intake in nondeprived rats (4).
Procedure All training and testing took place in home cages beginning 0900-1000 h daily. At the start of testing, preweighed bottles of ambient temperature tap water or test solutions were placed on each cage. In two-bottle preference tests left/right bottle positions were randomized. Experiment 1. Saline-, sucrose-, and saccharin-flavored solutions were offered for 4-h periods in single-bottle tests on separate test days. Animals were randomly assigned on a given test day to one of eight treatment conditions. The eight conditions formed a 2 × 4 matrix of treatments consisting of two injection types (NPY or VEH) and four solution concentrations. Concentrations of each flavor were as follows (w/v): 1) NaCI: 0%, 0.4%, 1.2%, 1.6%, 2) sucrose: 0%, 3%, 5%, 10%, and 3) Nasaccharin: 0%, 0.005%, 0.05%, 0.15%. Solutions were mixed fresh weekly with tap water, were refrigerated between test sessions, and were warmed to room temperature before each session. Each animal was tested a total of four times with 8 days separating tests. All animals were tested once with saline, once with sucrose, and twice with saccharin, in that order. The saccharin tests were repeated because NPY failed to significantly stimulate ingestion in the first test. Only data for the second saccharin test are reported here. Food and water were removed from cages during testing periods. Experiment 2. This experiment tested the hypothesis that NPY might facilitate ingestion by reinforcing a novel flavor
preference. Prior to testing, animals were randomly assigned to one of two training groups (n -- 12). Novel-flavored sucrose solutions were made by adding a standard package of unswemened Kool-Aid® (0.18 oz. artificial orange flavor or 0.13 oz. artificial black cherry flavor) to 41 of 10% (w/v) sucrose, Standard packages of Kool-Aid® flavors (rather than equal concentrations) were used to obtain approximately equal flavor intensities. Prior to the first day of training, baseline preference for the orangeand black cherry-flavored solutions was assessed in an overnight ( 17 h) two-bottle test. Three days later, rats in each group received single-bottle training with one of the novel-flavored solutions. Training, which was repeated on 2 consecutive days, consisted of offering the training flavor immediately following injection of NPY. On both training days, animals in group O received orange-flavored sucrose following NPY injection and those in group BC received black cherry-flavored sucrose. Bottles remained on the home cages for a 2-h period during training, and the amount consumed was measured at 30, 60, and 120 rain. Food and water were removed from cages during training and testing periods. On the day following training, all animals were injected with vehicle (5 M distilled water, ICV) followed immediately by a two-bottle preference test in which both orange- and black cherryflavored sucrose solutions were offered. Bottle positions were randomized and intake was measured at 1, 2, 4, and 24 h. Following 1 full day ofad lib food and water, all animals were given a second two-bottle preference test identical to the first but with bottle positions reversed. Of the original 24 animals, one died (apparently from an infection associated with cannula implantation) and five others did not drink the sucrose solutions during training. Of the remaining 18 rats for which data were analyzed, eight were in training group O and 10 were in group BC. RESULI S
Experiment 1 Table 1 summarizes the results of Experiment 1. Mean 4-h intake volumes (+SEM) following injection of NPY and VEH are shown for all concentrations of the three solutions. Numbers of animals included in statistical analyses are shown in brackets. Data from outliers (more than two standard deviations from the mean) were not included. Data for each solution type were analyzed separately by two-way (solution concentration x drug treatment) ANOVAs. Post hoc tests were performed using the Fisher Protected LSD test. In tests with saline there was a significant main effect of drug treatment, F (1,47) = 7.09, p < 0.05, and saline concentration, F (3,47) = 4.26, p < 0.05, but no significant interaction, Post hoc tests indicated that drug treatment significantly affected intake of only the 1.2% NaC1 concentration (p < 0.05). This effect, however, was due mainly to an unanticipated decrease in baseline intake and apparently not to NPY stimulation of saline drinking. In the sucrose tests there was also a significant main effect of drug treatment, F (3,48) = 24.72, p = 0.001, and concentration, F (3,48) = 3.94, p = 0.01, as well as a significant treatment X concentration interaction, F (3,48) = 4,133, p = 0.01. Post hoc tests indicated that NPY stimulated ingestion of all three sucrose concentrations (p < 0.05) but did not significantly increase the intake of tap water. In the saccharin tests there was again a significant main effect of drug treatment, F (1,47) = 7.13, p = 0.01, and solution concentration, F (3,47) = 4.75, p = 0.006, but no significant treatment X concentration interaction. In the case of saccharin, post hoc tests showed that NPY significantly increased intake of only the most preferred (0.05%) concentration (p < 0.05). Although mean intake of other saccharin concen-
NPY A N D T A S T E
879
trations was also increased by NPY, these increases were not statistically reliable.
Experiment 2 Figure 1 shows the relative preference for the orange-flavored sucrose solution [(volume of O / v o l u m e O + BC) × 100] for each group during the 17-h test given prior to training (baseline) and during two 24-h tests beginning 24 h (day 1) and 72 h (day 3) following training. Note that both groups showed a slight preference (approximately 5%) for the orange over the black cherry flavor during the baseline test. For the BC-trained group, relative preference for the orange flavor decreased from 52.2% on the baseline day to 31.9% by day 3. This was a shift of 20.4% toward the trained (BC) flavor. For the O-trained group, preference for the orange flavor increased from 56.2% on the baseline day to 86.0% on day 3. This was a shift of 29.8%, also in the direction of the trained (O) flavor. A two-way A N O V A (treatment × day, with day as a repeated factor) showed a significant main effect of treatment, F (1,15) = 17.23, p < 0.001, as well as a significant treatment × day interaction, F (2,30) = 3.41, p < 0.05. Between groups t -tests used to assess the relative preference for the orange flavor (where O + BC preferences = 100%) showed no significant difference between groups on the baseline day (t = 0.16, p = 0.436) but a significant difference between training groups on both day 1 (t = 2.24, p = 0.020) and day 3 (t = 4.87, p < 0.001) posttraining. Paired t -tests were used to determine whether the shifts in preference toward the trained flavor were significant. Results of this analysis indicated that shifts from baseline to day 1, for the two training groups as a whole, fell short of statistical reliability (t = !.28, p = 0.110), whereas shifts from baseline to day 3 were highly significant (t = 3.39, p < 0.002). DISCUSSION The results of Experiment l demonstrate that N P Y can stimulate ingestion of both caloric and noncaloric sweet solutions in fully sated rats. Whether this effect is secondary to an increased drive for calories or to a more specific increase in orosensory
100 ee O
80 60
¢.7 ¢lJ L
40 20 0
I
Baseline
I Day 1 Day of Testing
FIG. 1. Group mean relative preference (+SEM) [(volume O/volume O + BC) × 100%] during one 17-h test session before training (baseline) and two 24-h test sessions after training (day 1 and day 3). During two intervening training sessions, NPY injection (5 zg/5 zl, ICV) was followed by 2-h access to a single, novel-flavored sucrose solution: group O (O) was trained with the orange flavor, group BC (A) was trained with the black cherry flavor. See text for further details.
appetite remains unclear. The fact that N P Y stimulates intake of a palatable noncaloric saccharin solution would seem to favor an orosensory interpretation, although a shift in energy balance caused by NPY, and resulting in an increase in preference for tastes previously associated with hunger, cannot be ruled out. In the case of sucrose, NPY stimulated intake of all three concentrations within the normally preferred range (9). Thus, regardless of caloric value, N P Y appears to stimulate ingestion of normally palatable sweet-tasting solutions. The effect of NPY on saline intake is weak at best. Although intake of all three solution concentrations was somewhat increased by NPY, the only statistically significant effect was due
TABLE 1 EFFECT OF NPY ON INTAKE (g/4 h) OF VARIOUS SOLUTION CONCENTRATIONS Saline Solution Concentrations
NPY VEH
0%
0.4%
1.2%
1.6%
1.000 + 0.141 [7] 1.243 + 0.293 [7]
1.433 + 0.211 [6] 0.983 -+ 0.174 [6]
1.562 -+ 0.269 [8]* 0.487 + 0.095 [8]
2.443 _+ 0.506 [7] 1.500 _ 0.442 [6]
Sucrose Solution Concentrations
NPY VEH
0%
2%
5%
10%
1.829 _+0.478 [7] 0.917 _+ 0.221 [6]
11.500 _+ 5.152 [8]* 0.900 _+ 0.175 [7]
26.157 _+ 6.151 [7]* 0.657 _+ 0.247 [7]
13.129 _+ 4.700 [7]* 1.329 _+ 0.475 [7]
Saccharin Solution Concentrations
NPY VEH
I Day 3
0%
0.005%
0.05%
0.15%
2.329 + 0.252 [8] 1.688 -+ 0.236 [7]
1.671 _+ 0.519 [6] 1.217 _+ 0.717 [6]
6.267 -+ 1.762 [7]* 1.875 -+ 0.578 [8]
4.333 _+ 1.430 [6] 3.400 _+ 0.768 [7]
Values are mean _+ SEM. Number of animals included in statistical analyses are shown in brackets.
880
LYN(~H ET AI,
to an unexpected decrease in baseline intake of the 1.2% solution. The cause of this decrease is unknown. In an independent pilot study, we found that NPY produced a small, but again nonsignificant, increase in intake of a 0.8% concentration of saline. Thus, it appears that any stimulatory effect of NPY on saline ingestion in nondeprived animals is weak at best, limited to a narrow range of concentrations, and probably of limited physiological significance. Water intake was not significantly stimulated by NPY in any of the three tests in Experiment 1. The reason for this failure to stimulate water drinking is uncertain. Morley et al. (7) reported increases in water drinking following ICV NPY, but their animals had access to tbod at the time of testing. However, Stanley and Leibowitz (12) reported that NPY injected directly into the paraventricular hypothalamic nucleus stimulates water intake independently of food availability. Based on the present results, it appears that NPY injected into the lateral ventricles does not significantly stimulate water intake in the absence of food intake. The difference between the present results and those of Stanley and Leibowitz may be due to the greater effectiveness or more selective nature of site-specific hypothalamic injections. Results from Experiment 2 show that associating NPY with one of two equally preferred novel-flavored sucrose solutions results in a shift in preference toward the NPY-associated flavor. This suggests that NPY may promote ingestion of an otherwise palatable solution by strengthening a gustatory preference. Whether this shift is due to a positively reinforcing effect of NPY or is secondary to ingestion stimulated by NPY remains to be determined. We noted that preference for the NPY-associated flavor appeared to increase over the 3 days following training. Over this period, intake of the flavor associated with NPY increased relative to intake of the nonassociated flavor. As a result, relative preference for the NPY-associated flavor increased with additional drinking time. One possible explanation for this apparent increase in preference from day I to day 3 is that the choice of the preferred flavor developed gradually. Consistent with this idea, we found that overall preference for the trained flavor increased from 56.4% at the end of the first hour to 65. 1% by 24 h on test day 1. Thus, animals seemed to sample both flavors during the early hours of test day 1 before making a firm choice in favor of the trained flavor.
The results of Experiment 2, showing a preference tbr the NPY-associated flavor, are clearly at variance with a recent report of NPY-conditioned taste aversion by Sipols el al. (10). Two differences between these studies seem potentially important. In the Sipols study the tastants used were unsweetened Kool-Aid~,R., solutions. Since unsweetened Kool-Aid~) is sour to humans, it may have been aversive to rats. In addition, food was apparently available during conditioning trials in the Sipols study. The failure to restrict food intake during conditioning is potentially important because NPY may have stimulated excessive amounts of food intake, resulting in subsequent gastric distress or nausea. Thus, the aversion to flavored solutions reported by Sipols et al. may not have been due directly to NPY-flavor association, but instead either to the aversive quality of the tastants or the aversive consequences of excess ingestion. Taken together, the results of the present experiments extend previous findings in at least two ways. First, the results of Experiment I suggest that, in addition to its ability to stimulate carbohydrate intake (7), NPY has the ability to stimulate ingestion via an orosensory mechanism involving sweet taste. The stimulation of saccharin intake in nondeprived animals suggests either that NPY facilitates a previously learned association between sweet taste and the carbohydrate quality or that it stimulates an orosensory appetite for sweet. Whether NPY's stimulant effect on saccharin intake might extinguish with repeated testing, as the former hypothesis suggests, is unknown. Second, the results of Experiment 2 suggest that NPY is capable of enhancing intake by strengthening an association between a novel flavor and the carbohydrate or caloric quality of an ingested solution. How important this mechanism might be to the effect of NPY on the ingestion of food under ad lib feeding conditions remains to be determined. In general, the results of these studies suggest that centrally injected NPY is capable of stimulating intake of sweet solutions on the basis of the oral sensor3.' qualities of the ingested substances and independently of caloric content. ACKNOWLEDGEMENTS This work was supported by the Department of Veterans Affairs Medical Center and NIDA ROI-DAO3999. The authors wish to acknowledge the technical assistance of Ms. Wendy Welch.
REFERENCES 1. Billington, C. J.; Briggs, J. E.; Grace, M.; Levine, A. S. Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am. J. Physiol. 260:R321-R327; 1991. 2. Brady, L. S.; Smith, M. A.; Gold, P. W,; Herkenham, M. Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinol 52:441-447; 1990. 3. Clark, J. T.; Kalra, P. S.; Crowley, W. R.; Kalra, S. P. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115:427-429; 1984. 4. Grace, M.; Welch, W.; Billington, C. J.; Levine, A. S. The effect of neuropeptide Y on intake of flavored solutions. Soc. Neurosci. Abstr. 15:894; 1989. 5. Kalra, S. P.; Dube, M. G.; Sahu, A.; Phelps, C. P.; Kalra, P. S. Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc. Natl. Acad. Sci. USA 88:10931-10935; 1991. 6. Levine, A. S.; Morley, J. E. Neuropeptide Y: A potent inducer of consummatory behavior in rats. Peptides 5:1025-1029; 1984. 7. Morley, J. E.; Levine, A. S.; Gosnell, B. A.; Kneip, J.; Grace, M. Effect of neuropeptide Y on ingestive behaviors in the rat. Am. J. Physiol. 252:R599-R609; 1987.
8. Paez, X.; Nyce, J. W.; Myers, R. D. Differential feeding responses evoked in the rat by NPY and NPY~_27 injected intracerebroventricularly. Pharmacol. Biochem. Behav. 38:379-384; 1991. 9. Pfaffmann, C.; Norgren, R.; Grill, H. J. Sensory affect and motivation. In: Wenzel, B. M.; Zeigler, H. P., eds. Tonic functions in sensory systems. New York: New York Academy of Sciences; 1977:18-34. 10. Sipols, A. J.; Brief, D. J.; Ginter, K. L.; Saghafi, S.; Woods, S. C. Neuropeptide Y paradoxically increases food intake yet causes conditioned flavor aversions. Physiol. Behav. 51(6): 1257-1260; 1992. 11. Stanley, B.; Daniel, D.; Chin, A.; Leibowitz, S. F. Paraventricular nucleus injections ofpeptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion. Peptides 6:1205-1211 ; 1985. 12. Stanley, B. G.; Leibowitz, S. F. Neuropeptide Y: Stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci. 35:2635-2642; 1984. 13. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296:659-660; 1982. 14. Tempel, D. L.; Leibowitz, S. F. Neuropeptide Y effect on feeding behavior: Relationship to norepinephrine and the circadian system. Ann. NY Acad. Sci. 611:529-530; 1990.
0031-9384/93 $6.00 + .00 Copyright© 1993 PergamonPressLtd.
Printed in the USA.
Effects of Neuropeptide Y on Ingestion of Flavored Solutions in Nondeprived Rats W E S L E Y C. L Y N C H , *~ M A R T H A
GRACE,l" C H A R L E S J. B I L L I N G T O N I " A N D A L L E N S. LEVINEI"
*Department of Psychology, Montana State University, Bozeman, M T 5971 7 and -?Research and Medicine Service, Minneapolis Veterans Administration Medical Center, Minneapolis, M N 55417 Received 23 N o v e m b e r 1992 LYNCH, W. C., M. GRACE, C. J. BILLINGTON AND A. S. LEVINE. Effectsofneuropeptide Yon ingestionofflavoredsolutions in nondeprivedrats. PHYSIOL BEHAV 54 (5) 877-880, 1993.--Recent evidence suggests that in addition to altering energy balance, neuropeptide Y (NPY) may stimulate ingestive behavior by modifying the orosensory quality of ingested substances. The present experiments investigated the effect of intracerebroventricular administration of NPY (5 #g/5 #1) on ingestion of various flavored solutions in nondeprived rats. Experiment 1 examined the effectsof NPY on ingestion of a range of concentrations of saline, sucrose, and saccharin solutions in single-bottle tests. Results indicated that NPY stimulates ingestion of both sucrose and saccharin solutions that are normally palatable. In Experiment 2, palatable sucrose solutions flavored with either orange or black cherry Kool-Aid® for separate training groups were selectively associated with NPY injection during single-bottle training sessions. Subsequent two-bottle preference tests showed a significant shift in preference toward the flavor paired with NPY during training. The results of these experiments extend previous findings by showing that NPY can stimulate ingestion of sweet solutions regardless of caloric value and may potentiate sweet taste preference via an associative mechanism. Neuropeptide Y
Feeding
Drinking
Taste preference
NEUROPEPTIDE Y (NPY) is a 36 amino acid peptide found in the brain and peripheral tissues of numerous species. Since its original characterization (13), a key focus of research has been the role NPY plays in regulation to feeding and drinking. Clark et al. (3), for instance, showed that moderate doses of NPY (2-10 #g) injected into the lateral ventricles (ICV) significantly stimulated eating behavior in rats during the light phase of the light/dark cycle, and Morley and Levine (6) found that ICV NPY in this same dose range stimulated both food and water intake in fully satiated rats. More recent work has suggested that NPY may act to preferentially stimulate carbohydrate intake via circuits linked to the medial hypothalamus (7,14) and to alter energy metabolism (1) and energy balance ( 11 ). These various effects of centrally injected NPY on food energy intake are remarkably robust and suggest that NPY produces an essential alteration in the underlying structure of ingestive behavior. Neuropeptide Y's effects on ingestive behavior could be due either to increases in perceived hunger or enhancement of the rewarding properties of food. At specific hypothalamic sites, particularly the arcuate to paraventricular nucleus projection, it appears to play a regulatory role in response to alterations in energy balance (2,5). This evidence can be interpreted as indicating that NPY signals hunger within central controlling elements. At present, this evidence is inconclusive, in part because it is ditficult to ascertain whether an animal is truly hungry or is eating for some other reason.
Flavor
Conditioning
Carbohydrate
The possibility that neuropeptide Y might alter the orosensory or rewarding consequences of ingestion, rather than prompting ingestion directly, has been less explored. Evidence that neuropeptide Y can stimulate water intake independently of deprivation or food availability (12) and that its ability to stimulate drinking depends on the orosensory properties of ingested solutions (4,8) suggests that some aspect of ingestive behavior itself may be modified by NPY. In addition, recent reports that food intake stimulated by NPY might, paradoxically, be associated with taste aversions (10) raises further interest in the possible influence of orosensory factors. The present experiments aimed to investigate the role of orosensory factors in the feeding-stimulant effects of NPY. In a pilot study we found that NPY stimulated greater intake of novelflavored sucrose solutions, on first exposure to them, than did 24-h food deprivation. Moreover, NPY appeared to potentiate intake of the solutions previously associated with NPY, under subsequent food-satiated and food-deprived testing conditions. These preliminary results suggested that NPY might enhance the gustatory evaluation of ingested solutions and perhaps promote preference for an NPY-associated flavor. The two experiments described here further examined these suggestions. Experiment 1 investigated the effects of NPY on ingestion of sucrose, saccharin, and saline solutions of various concentrations ranging from neutral (tap water) to preferred. Experiment 2 tested the hypothesis that NPY might stimulate ingestion of a novel-
Requests for reprints should be addressed to Dr. Wesley C, Lynch, Department of Psychology, 300 Traphagen Hall, Montana State University, Bozeman, MT 59717.
877
878
I.YNCtt E1 Ai
flavored sucrose solution by potentiating a learned flavor preference. METHOD
Subjects Subjects were 84 male Sprague-Dawley albino rats (Biolab, St. Paul, MN) weighing 225-340 g at the start of testing. Sixty animals were used in Experiment 1 and 24 were used in Experiment 2. Animals were maintained in individual wire mesh home cages on a 12/12 h light/dark cycle with lights on daily at 0700 h. Animals had ad lib access to Purina chow pellets and tap water except as noted.
Surgery and Histology Under Nembutal anesthesia (50 mg/kg, IP), unilateral stainless steel guide cannulas (23 ga) were implanted sterotaxically into either the left or right lateral ventricles. Stereotaxic coordinates were 1 mm posterior and 1.5 mm lateral to bregma with the incisor bar adjusted 3.5 mm below the interaural line. Guide cannula tips extended 3.5 mm below skull surface. Animals were allowed at least 7 days for recovery following surgery. Injections of drug or vehicle solutions were made manually with a 10-t4 Hamilton syringe attached to a 30-ga stainless steel injector that extended 1 mm beyond the guide cannula tip. Cannula placements were verified by injecting 5 #1 of India ink following death by carbon dioxide overdose and subsequent decapitation. Data from rats with incorrect cannula placements were excluded from data analyses.
Drugs Pomine-derived NPY (Peninsula Laboratories, Belmont, CA) was mixed fresh daily and injected at a single dose (5 #g/5 #1, ICV) in both experiments. This close was selected on the basis of previous food intake studies and our own preliminary work indicating reliable stimulation of flavored solution intake in nondeprived rats (4).
Procedure All training and testing took place in home cages beginning 0900-1000 h daily. At the start of testing, preweighed bottles of ambient temperature tap water or test solutions were placed on each cage. In two-bottle preference tests left/right bottle positions were randomized. Experiment 1. Saline-, sucrose-, and saccharin-flavored solutions were offered for 4-h periods in single-bottle tests on separate test days. Animals were randomly assigned on a given test day to one of eight treatment conditions. The eight conditions formed a 2 × 4 matrix of treatments consisting of two injection types (NPY or VEH) and four solution concentrations. Concentrations of each flavor were as follows (w/v): 1) NaCI: 0%, 0.4%, 1.2%, 1.6%, 2) sucrose: 0%, 3%, 5%, 10%, and 3) Nasaccharin: 0%, 0.005%, 0.05%, 0.15%. Solutions were mixed fresh weekly with tap water, were refrigerated between test sessions, and were warmed to room temperature before each session. Each animal was tested a total of four times with 8 days separating tests. All animals were tested once with saline, once with sucrose, and twice with saccharin, in that order. The saccharin tests were repeated because NPY failed to significantly stimulate ingestion in the first test. Only data for the second saccharin test are reported here. Food and water were removed from cages during testing periods. Experiment 2. This experiment tested the hypothesis that NPY might facilitate ingestion by reinforcing a novel flavor
preference. Prior to testing, animals were randomly assigned to one of two training groups (n -- 12). Novel-flavored sucrose solutions were made by adding a standard package of unswemened Kool-Aid® (0.18 oz. artificial orange flavor or 0.13 oz. artificial black cherry flavor) to 41 of 10% (w/v) sucrose, Standard packages of Kool-Aid® flavors (rather than equal concentrations) were used to obtain approximately equal flavor intensities. Prior to the first day of training, baseline preference for the orangeand black cherry-flavored solutions was assessed in an overnight ( 17 h) two-bottle test. Three days later, rats in each group received single-bottle training with one of the novel-flavored solutions. Training, which was repeated on 2 consecutive days, consisted of offering the training flavor immediately following injection of NPY. On both training days, animals in group O received orange-flavored sucrose following NPY injection and those in group BC received black cherry-flavored sucrose. Bottles remained on the home cages for a 2-h period during training, and the amount consumed was measured at 30, 60, and 120 rain. Food and water were removed from cages during training and testing periods. On the day following training, all animals were injected with vehicle (5 M distilled water, ICV) followed immediately by a two-bottle preference test in which both orange- and black cherryflavored sucrose solutions were offered. Bottle positions were randomized and intake was measured at 1, 2, 4, and 24 h. Following 1 full day ofad lib food and water, all animals were given a second two-bottle preference test identical to the first but with bottle positions reversed. Of the original 24 animals, one died (apparently from an infection associated with cannula implantation) and five others did not drink the sucrose solutions during training. Of the remaining 18 rats for which data were analyzed, eight were in training group O and 10 were in group BC. RESULI S
Experiment 1 Table 1 summarizes the results of Experiment 1. Mean 4-h intake volumes (+SEM) following injection of NPY and VEH are shown for all concentrations of the three solutions. Numbers of animals included in statistical analyses are shown in brackets. Data from outliers (more than two standard deviations from the mean) were not included. Data for each solution type were analyzed separately by two-way (solution concentration x drug treatment) ANOVAs. Post hoc tests were performed using the Fisher Protected LSD test. In tests with saline there was a significant main effect of drug treatment, F (1,47) = 7.09, p < 0.05, and saline concentration, F (3,47) = 4.26, p < 0.05, but no significant interaction, Post hoc tests indicated that drug treatment significantly affected intake of only the 1.2% NaC1 concentration (p < 0.05). This effect, however, was due mainly to an unanticipated decrease in baseline intake and apparently not to NPY stimulation of saline drinking. In the sucrose tests there was also a significant main effect of drug treatment, F (3,48) = 24.72, p = 0.001, and concentration, F (3,48) = 3.94, p = 0.01, as well as a significant treatment X concentration interaction, F (3,48) = 4,133, p = 0.01. Post hoc tests indicated that NPY stimulated ingestion of all three sucrose concentrations (p < 0.05) but did not significantly increase the intake of tap water. In the saccharin tests there was again a significant main effect of drug treatment, F (1,47) = 7.13, p = 0.01, and solution concentration, F (3,47) = 4.75, p = 0.006, but no significant treatment X concentration interaction. In the case of saccharin, post hoc tests showed that NPY significantly increased intake of only the most preferred (0.05%) concentration (p < 0.05). Although mean intake of other saccharin concen-
NPY A N D T A S T E
879
trations was also increased by NPY, these increases were not statistically reliable.
Experiment 2 Figure 1 shows the relative preference for the orange-flavored sucrose solution [(volume of O / v o l u m e O + BC) × 100] for each group during the 17-h test given prior to training (baseline) and during two 24-h tests beginning 24 h (day 1) and 72 h (day 3) following training. Note that both groups showed a slight preference (approximately 5%) for the orange over the black cherry flavor during the baseline test. For the BC-trained group, relative preference for the orange flavor decreased from 52.2% on the baseline day to 31.9% by day 3. This was a shift of 20.4% toward the trained (BC) flavor. For the O-trained group, preference for the orange flavor increased from 56.2% on the baseline day to 86.0% on day 3. This was a shift of 29.8%, also in the direction of the trained (O) flavor. A two-way A N O V A (treatment × day, with day as a repeated factor) showed a significant main effect of treatment, F (1,15) = 17.23, p < 0.001, as well as a significant treatment × day interaction, F (2,30) = 3.41, p < 0.05. Between groups t -tests used to assess the relative preference for the orange flavor (where O + BC preferences = 100%) showed no significant difference between groups on the baseline day (t = 0.16, p = 0.436) but a significant difference between training groups on both day 1 (t = 2.24, p = 0.020) and day 3 (t = 4.87, p < 0.001) posttraining. Paired t -tests were used to determine whether the shifts in preference toward the trained flavor were significant. Results of this analysis indicated that shifts from baseline to day 1, for the two training groups as a whole, fell short of statistical reliability (t = !.28, p = 0.110), whereas shifts from baseline to day 3 were highly significant (t = 3.39, p < 0.002). DISCUSSION The results of Experiment l demonstrate that N P Y can stimulate ingestion of both caloric and noncaloric sweet solutions in fully sated rats. Whether this effect is secondary to an increased drive for calories or to a more specific increase in orosensory
100 ee O
80 60
¢.7 ¢lJ L
40 20 0
I
Baseline
I Day 1 Day of Testing
FIG. 1. Group mean relative preference (+SEM) [(volume O/volume O + BC) × 100%] during one 17-h test session before training (baseline) and two 24-h test sessions after training (day 1 and day 3). During two intervening training sessions, NPY injection (5 zg/5 zl, ICV) was followed by 2-h access to a single, novel-flavored sucrose solution: group O (O) was trained with the orange flavor, group BC (A) was trained with the black cherry flavor. See text for further details.
appetite remains unclear. The fact that N P Y stimulates intake of a palatable noncaloric saccharin solution would seem to favor an orosensory interpretation, although a shift in energy balance caused by NPY, and resulting in an increase in preference for tastes previously associated with hunger, cannot be ruled out. In the case of sucrose, NPY stimulated intake of all three concentrations within the normally preferred range (9). Thus, regardless of caloric value, N P Y appears to stimulate ingestion of normally palatable sweet-tasting solutions. The effect of NPY on saline intake is weak at best. Although intake of all three solution concentrations was somewhat increased by NPY, the only statistically significant effect was due
TABLE 1 EFFECT OF NPY ON INTAKE (g/4 h) OF VARIOUS SOLUTION CONCENTRATIONS Saline Solution Concentrations
NPY VEH
0%
0.4%
1.2%
1.6%
1.000 + 0.141 [7] 1.243 + 0.293 [7]
1.433 + 0.211 [6] 0.983 -+ 0.174 [6]
1.562 -+ 0.269 [8]* 0.487 + 0.095 [8]
2.443 _+ 0.506 [7] 1.500 _ 0.442 [6]
Sucrose Solution Concentrations
NPY VEH
0%
2%
5%
10%
1.829 _+0.478 [7] 0.917 _+ 0.221 [6]
11.500 _+ 5.152 [8]* 0.900 _+ 0.175 [7]
26.157 _+ 6.151 [7]* 0.657 _+ 0.247 [7]
13.129 _+ 4.700 [7]* 1.329 _+ 0.475 [7]
Saccharin Solution Concentrations
NPY VEH
I Day 3
0%
0.005%
0.05%
0.15%
2.329 + 0.252 [8] 1.688 -+ 0.236 [7]
1.671 _+ 0.519 [6] 1.217 _+ 0.717 [6]
6.267 -+ 1.762 [7]* 1.875 -+ 0.578 [8]
4.333 _+ 1.430 [6] 3.400 _+ 0.768 [7]
Values are mean _+ SEM. Number of animals included in statistical analyses are shown in brackets.
880
LYN(~H ET AI,
to an unexpected decrease in baseline intake of the 1.2% solution. The cause of this decrease is unknown. In an independent pilot study, we found that NPY produced a small, but again nonsignificant, increase in intake of a 0.8% concentration of saline. Thus, it appears that any stimulatory effect of NPY on saline ingestion in nondeprived animals is weak at best, limited to a narrow range of concentrations, and probably of limited physiological significance. Water intake was not significantly stimulated by NPY in any of the three tests in Experiment 1. The reason for this failure to stimulate water drinking is uncertain. Morley et al. (7) reported increases in water drinking following ICV NPY, but their animals had access to tbod at the time of testing. However, Stanley and Leibowitz (12) reported that NPY injected directly into the paraventricular hypothalamic nucleus stimulates water intake independently of food availability. Based on the present results, it appears that NPY injected into the lateral ventricles does not significantly stimulate water intake in the absence of food intake. The difference between the present results and those of Stanley and Leibowitz may be due to the greater effectiveness or more selective nature of site-specific hypothalamic injections. Results from Experiment 2 show that associating NPY with one of two equally preferred novel-flavored sucrose solutions results in a shift in preference toward the NPY-associated flavor. This suggests that NPY may promote ingestion of an otherwise palatable solution by strengthening a gustatory preference. Whether this shift is due to a positively reinforcing effect of NPY or is secondary to ingestion stimulated by NPY remains to be determined. We noted that preference for the NPY-associated flavor appeared to increase over the 3 days following training. Over this period, intake of the flavor associated with NPY increased relative to intake of the nonassociated flavor. As a result, relative preference for the NPY-associated flavor increased with additional drinking time. One possible explanation for this apparent increase in preference from day I to day 3 is that the choice of the preferred flavor developed gradually. Consistent with this idea, we found that overall preference for the trained flavor increased from 56.4% at the end of the first hour to 65. 1% by 24 h on test day 1. Thus, animals seemed to sample both flavors during the early hours of test day 1 before making a firm choice in favor of the trained flavor.
The results of Experiment 2, showing a preference tbr the NPY-associated flavor, are clearly at variance with a recent report of NPY-conditioned taste aversion by Sipols el al. (10). Two differences between these studies seem potentially important. In the Sipols study the tastants used were unsweetened Kool-Aid~,R., solutions. Since unsweetened Kool-Aid~) is sour to humans, it may have been aversive to rats. In addition, food was apparently available during conditioning trials in the Sipols study. The failure to restrict food intake during conditioning is potentially important because NPY may have stimulated excessive amounts of food intake, resulting in subsequent gastric distress or nausea. Thus, the aversion to flavored solutions reported by Sipols et al. may not have been due directly to NPY-flavor association, but instead either to the aversive quality of the tastants or the aversive consequences of excess ingestion. Taken together, the results of the present experiments extend previous findings in at least two ways. First, the results of Experiment I suggest that, in addition to its ability to stimulate carbohydrate intake (7), NPY has the ability to stimulate ingestion via an orosensory mechanism involving sweet taste. The stimulation of saccharin intake in nondeprived animals suggests either that NPY facilitates a previously learned association between sweet taste and the carbohydrate quality or that it stimulates an orosensory appetite for sweet. Whether NPY's stimulant effect on saccharin intake might extinguish with repeated testing, as the former hypothesis suggests, is unknown. Second, the results of Experiment 2 suggest that NPY is capable of enhancing intake by strengthening an association between a novel flavor and the carbohydrate or caloric quality of an ingested solution. How important this mechanism might be to the effect of NPY on the ingestion of food under ad lib feeding conditions remains to be determined. In general, the results of these studies suggest that centrally injected NPY is capable of stimulating intake of sweet solutions on the basis of the oral sensor3.' qualities of the ingested substances and independently of caloric content. ACKNOWLEDGEMENTS This work was supported by the Department of Veterans Affairs Medical Center and NIDA ROI-DAO3999. The authors wish to acknowledge the technical assistance of Ms. Wendy Welch.
REFERENCES 1. Billington, C. J.; Briggs, J. E.; Grace, M.; Levine, A. S. Effects of intracerebroventricular injection of neuropeptide Y on energy metabolism. Am. J. Physiol. 260:R321-R327; 1991. 2. Brady, L. S.; Smith, M. A.; Gold, P. W,; Herkenham, M. Altered expression of hypothalamic neuropeptide mRNAs in food-restricted and food-deprived rats. Neuroendocrinol 52:441-447; 1990. 3. Clark, J. T.; Kalra, P. S.; Crowley, W. R.; Kalra, S. P. Neuropeptide Y and human pancreatic polypeptide stimulate feeding behavior in rats. Endocrinology 115:427-429; 1984. 4. Grace, M.; Welch, W.; Billington, C. J.; Levine, A. S. The effect of neuropeptide Y on intake of flavored solutions. Soc. Neurosci. Abstr. 15:894; 1989. 5. Kalra, S. P.; Dube, M. G.; Sahu, A.; Phelps, C. P.; Kalra, P. S. Neuropeptide Y secretion increases in the paraventricular nucleus in association with increased appetite for food. Proc. Natl. Acad. Sci. USA 88:10931-10935; 1991. 6. Levine, A. S.; Morley, J. E. Neuropeptide Y: A potent inducer of consummatory behavior in rats. Peptides 5:1025-1029; 1984. 7. Morley, J. E.; Levine, A. S.; Gosnell, B. A.; Kneip, J.; Grace, M. Effect of neuropeptide Y on ingestive behaviors in the rat. Am. J. Physiol. 252:R599-R609; 1987.
8. Paez, X.; Nyce, J. W.; Myers, R. D. Differential feeding responses evoked in the rat by NPY and NPY~_27 injected intracerebroventricularly. Pharmacol. Biochem. Behav. 38:379-384; 1991. 9. Pfaffmann, C.; Norgren, R.; Grill, H. J. Sensory affect and motivation. In: Wenzel, B. M.; Zeigler, H. P., eds. Tonic functions in sensory systems. New York: New York Academy of Sciences; 1977:18-34. 10. Sipols, A. J.; Brief, D. J.; Ginter, K. L.; Saghafi, S.; Woods, S. C. Neuropeptide Y paradoxically increases food intake yet causes conditioned flavor aversions. Physiol. Behav. 51(6): 1257-1260; 1992. 11. Stanley, B.; Daniel, D.; Chin, A.; Leibowitz, S. F. Paraventricular nucleus injections ofpeptide YY and neuropeptide Y preferentially enhance carbohydrate ingestion. Peptides 6:1205-1211 ; 1985. 12. Stanley, B. G.; Leibowitz, S. F. Neuropeptide Y: Stimulation of feeding and drinking by injection into the paraventricular nucleus. Life Sci. 35:2635-2642; 1984. 13. Tatemoto, K.; Carlquist, M.; Mutt, V. Neuropeptide Y-a novel brain peptide with structural similarities to peptide YY and pancreatic polypeptide. Nature 296:659-660; 1982. 14. Tempel, D. L.; Leibowitz, S. F. Neuropeptide Y effect on feeding behavior: Relationship to norepinephrine and the circadian system. Ann. NY Acad. Sci. 611:529-530; 1990.