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Lithium chloride, cholecystokinin and meal patterns: Evidence that cholecystokinin suppresses meal size in rats without causing malaise
Apprtitr,
1987, 8. 221-227
Lithium Chloride, Cholecystokinin and Meal Patterns: Evidence that Cholecystokinin Suppresses Meal Size in Rats Without Causing Malaise DAVID B. WEST, M. R. C. GREENWOOD KATHLEEN A. MARSHALL Department
of Biology,
STEPHEN
C. WOODS
Department
of Psychology,
Vassar
College,
University
and
Poughkeepsie,
of Washington,
New
Seattle,
York
Washington
Gut hormoneswhich are proposedas natural satiety factors may act through malaiseto suppressshort-termfeedinginsteadof through a normal physiological process.Evaluation of the ability of candidatesatiety hormonesto serveas the unconditionedstimulusin the developmentof a conditioned tasteaversionis not adequateevidenceof malaisesinceagentswhich do not producemalaisein humans are alsocapableof conditioning aversions.Examination of the patternsof eating behaviorwhendrugsaregiven during a mealin ratsmay serveasa better testof the potential of an agentto produceillness.To evaluatethis approach,LiCl (a known aversiveagent)wasinfusedinto five rats at the start of eachfree-feedingmealat a doseof 1.9mg/meal/ratfor four daysand at 38 mg/meal/ratfor an additional four days. Feedingpatterns during drug infusion werecompare>with patterns for six days when no drugs were infused. LiCl infusion produced a significant dosedependentreduction in feedingfrequencywhile the sizeand duration of mealswere not changed.At the higher doseof lithium, daily mealnumberwasreducedfrom a normal 13.2& l-4 to 9.9+-0.5 meals/day.This pattern of behavior is significantly different from the behavior exhibited when cholecystokinin octapeptide(CCK-8) wasinfusedusingthe sameparadigm.CCK-8 significantly reducedmealsizewhile meal frequency increasedto compensatefor the smaller meals.Thesefindings suggestthat LiCl infusionduring a meal will lead to an aversionto feedingwhile CCK-8 is not effective at producing an aversion.The patterns of behavior when CCK-8 is infused are quite different from the patterns resulting from mealcontingent LiCl infusion.This supportsthe view that patternsof normal ingestive behavior may be useful to distinguishbetweenaversive agentsand those that produce true satiety. INTRODUCTION
A number of gut hormones, with the prototype being cholecystokinin (CCK), are reported to suppress meal size in the rat after peripheral administration (Woods, McKay, Stein, West, Lotter & Porte, 1980). In addition, CCK is known to suppress sham-eating in the rat and to initiate the normal postprandial behaviors (Antin, Gibbs, Holt, Young & Smith, 1975). Because of this and other evidence, it is suggestedthat
This work was supported by NIH grants Nos. AM07332, Correspondence and reprint requests should be addressed Vassar College, Poughkeepsie, NY 12601, U.S.A. 0195-~6663/87/030221+07
$0340/O
AM17844 to David
and CD12637. B. West, Department
!i” 1987 Academic
Press Inc. (London)
of Biology.
LimIted
222 cholccystokinin, and possibly other gut hormones, contribute to the normal satiety process and are important for the regulation of feeding behavior in rats and other mammals (Smith & Gibbs, 1979). This work has been criticized since the suppression of short-term feeding after i.p. injection of these hormones might be the result of malaise and not due to a normal physiological process. Some cvidencc indicates that i.p. CCK at doses which arc effective at suppressing meal size also are effective at conditioning a taste aversion (CTA) (Deutsch & Hardy, 1977) suggesting that the hormone is producing malaise. This conclusion is supported by a recent report indicating that an antiemetic will attenuate the ability of CCK-8 to suppress feeding in the rat (Moore & Deutsch, 1985). These same doses of CCK can produce atypical duodenal peristalsis indicating an abnormal state which might produce illness (Deutsch, Thiel & Greenberg, 1978). To counter this evidence, others argue that CCK, at doses which suppress food intake in the rat, is not effective at conditioning taste aversions (Holt, Antin, Gibbs. Young & Smith, 1974). Furthermore, it is reported that agents which do not product malaise in humans can be used to produce CTAs in rodents; perhaps not through malaise but by altering the physiological state of the animal (Berger, 1972). This suggests that the ability to produce a CTA is not adequate proof that the agent produces illness. The most powerful counterarguments for a malaise theory of CCK action are several reports that doses of purified CCK, which suppress feeding in humans, do not lead to reports of illness (Kissileff, Pi-Sunyer, Thornton & Smith, 198 1: Stacher, Bauer & Steinringer, 1978). However, the use of an impure preparation of CCK in humans has been associated with illness (Sturdevant & Goetz. 1976). It is clear that this argument has not diminished over the 13 years since a crude intestinal extract containing cholecystokinin was first shown to reduce normal feeding in the rat (Gibbs, Young & Smith, 1973). Other hormones or metabolites which are advocated as endogenous satiety agents also will have to be evaluated for their potential for producing malaise. This testing certainly will include an evaluation of their ability to produce a CTA and this may lead to controversy. Clearly, additional and/or alternative tests to evaluate the potential for producing illness are needed to rule out this action for candidate satiety hormones. VanderWeele, Granja and Deems (1982) suggest that the pattern of feeding behavior in the rat following injection of a compound may be used as an index of illness. They reported that effects on the interval until the next meal and the size of the next meal differ for agents that suppress food intake normally compared with those that produce illness. In that study, behaviors were evaluated for only several hours after a bolus i.p. injection. In this report, we further evaluate the potential for using mealpatterning data to distinguish between malaise and ‘satiety’ by examining the behavioral effects of meal-contingent infusion of LiCl, a known aversive agent (Nachman, 1963) into free-feeding rats.
METHODS
Five adult, male, Long Evans rats were habituated to computer enclosures where food (45 mg Noyes pellets, P. J. Noyes, Inc.) and water were available old lihitum. The animals weighed approximately 300g at the beginning of the experiment and they wcrc kept in a room maintained at a constant temperature with a LD 12: 12 cycle with lights on at 0800 hrs. When a food pellet was removed from a feeding trough by the rat. this
LiCl
AND
MEAL
PATTERNS
“1 -_.
e\ ent was recorded by a microcomputer and another pellet was immediately dispensed (West, Tengan. Smith & Samson, 1983). Following the habituation period, when the growth rate had recovered to normal, feeding behavior was recorded for a 5-day period (L>ays l-5 of the experiment). Throughout the experiment, continuous recordings of feeding behavior were made for approximately 23 hours each day with a one hour interval during the day when the pellet dispensers were inactivated and the program was interrupted. During this interruption the rats were weighed, the cages were cleaned. the volume of water consumed was recorded, and the water and pellet reservoirs were refilled. After baseline behaviors were recorded, the rats were implanted with i.p. silastic catheters which were exteriorized via a head pod and connected to saline-filled syringes mounted on computer-controlled infusion pumps. Following surgery, physiologica! saline (0.29 ml/30 set) was infused at the start of each free-feeding meal after 0.15 g of food had been consumed. After one infusion, another infusion was not allowed for IO minutes. By the fifth day of saline infusion (Day 10 of the experiment), the patterns of feeding behavior and the total daily food intake were not different from baseline patterns and intake. Therefore, the last saline day was combined with the five baseline days to calculate the overall baseline feeding patterns. The animals then received meal-contingent infusions of LiCl (150m~; 19mg LiClimeal/rat) for four days (Days 1 l-14). This was followed by four days (Days 15- 18) of meal-contingent LiCl at a dose of 3.8 mg LiCl/mcal/rat. The criteria for infusions and infusion parameters were the same during 1.9 mgjmeal LiCl infusion as during saline infusion. During the four days of 3.8 mg/meal LiCl infusion, the rats received a volume of 0.58 ml per infusion. For the data analysis, the average daily food intake, water intake, meal size. meal duration and meal number were calculated for each rat over six days of the baseline period, four days of 1.9 mg LiCl infusion and four days of 3.8 mg LiCl infusion. The definition of a meal required a minimum of0.225 g and an interval without eating of five minutes. The means for each animal for every treatment period were used to calculate a group mean during each period. In the text and in the figures the data are reported as means and standard errors. The treatments were compared with a Repeated Measures ANOVA followed by post-hoc t-tests. Growth rates over the different periods were compared with a two-way Repeated Measures ANOVA.
RESULTS
LiCl infusion produced a dose-dependent decrease in the number of meals taken each day. During baseline the rats took an average of 13.2 + 1.4 meals/day. Meal frequency was reduced to 116kO.7 and 9.9kO.5 meals/day by I.9 and 3.8 mg LiCl respectively. The overall reduction of meal frequency by the higher dose of LiCl averaged 23?, and was statistically reliable (p
224
Meal
number
Meal
SIR
FIGURE 1. Meal size and meal number during LiCl infusion expressed as a percentage of baseline value. Asterisks indicate a significant difference from baseline (p < 0.01, ANOVA and p~~,st-hoc’ f-test). I-1.1.9mg/meal; t2, 3.8 mg/meal.
350
340 -
FIGURE2. Body weight during the meal-contingent LiCl infusion. LiCl infusion at 1.9 or 3.8 mg/meal/rat had only a transient effect on body weight. Growth rate was not significantly affected. A, Baseline; l , 1.9 mg LiCl; 0, 3.8 mg LiCI.
Licl
AND MEAL PATTERNS
‘75 ___
Because fewer meals were consumed, the total daily intake was significantly reduced from 28.7 f 0.9 g/day to 22.2 + 2.5 g/day when 3.8 mg LiCl was infused during each meal (p < 005, ANOVA and post-hoc t-test). The daily intake during the infusion of 1.9 mg LiCl was reduced to 25.9 + 0.5 g, but this was not significantly different from baseline intake. Daily water intake was not affected by drug infusion. Water intake averaged 34.1 f 08 ml/day during baseline and 34.5 + 1.1 ml/day and 35.8 f 3.7 ml/day during the periods of drug infusion. The growth of the animals (Figure 2) was only transiently affected by drug infurion and there was no significant effect on body weight for either dose of LiCI. The daily dose of LiCl averaged 23.9* 1.1 mg/rat/day during infusion of I ,9 mg/meal and 38.2 f 2.7 mg/rat/day during the infusion of 3.8 mg/meal of LiCI.
DISCIJSSION
Infusion of LiCl at low doses produced a dose-dependent reduction of food intake due to fewer meals while the meal size of the rats was not affected. This suggests that lithium at this dose (3.8 mg/meal/rat) is aversive to the animal and consequently the rat takes fewer meals to avoid infusions. This conclusion is consistent with the findings 01 aversions developed by rats to vitamin deficient diets (Rozin & Kalat, 1971), however, it is not known if the aversion to vitamin-deficient diet is expressed by a reduction in the frequency of feeding and/or a reduced meal size. The observation that LiCl infusions paired with the taking of a normal meal will lead to an apparent aversion to feeding is also consistent with the observations that LiCl injections into rats can be used to condition aversions to novel tastes (Revusky & Garcia, 1970). It must be stressed that the reduced feeding frequency found with LiCl infusion paired with feeding may be due to other actions of LiCl instead of its known aversive actions. Yet an explanation of this phenomenon based upon the known aversive properties of LiCl is both reasonable and plausible. The effects of meal-contingent lithium infusion on meal patterns in the free-feeding r:it are strikingly different from the effects of meal-contingent infusion of CCK-8. In ;I previously published report (West, Fey & Woods, 1984), using a paradigm identical to that employed in the study described here, CCK-8 (1.1 pg/meal/rat) was infused into five rats at the start of each free-feeding meal over a 6-day period. This manipulation produced a remarkable decrease in meal size (407,, of normal) which was compensated for by an increase in feeding frequency. Total daily food intake was reduced only transiently below normal. One might argue that due to the dramatic decrease in meal size produced by CCK-8 infusion, a compensatory increase in feeding frequency was necessary for the animal to maintain a minimal caloric intake and this overwhelmed the aversive conditioning to the diet produced by meal contingent CCK-8 infusion. However, in a similar study (West, Greenwood, Sullivan, Prescod, Marzullo & Triscari. 1087). CCK-8 was infused towards the end of free-feeding meals in seven rats in large doses ( I.8 /lg;meal;rat) as well as during the intermeal interval. This protocol rcsultcd in a smaller reduction of meal size (29%), but meal number still increased to compensalc for the smaller meals. Therefore, in both studies using meal contingent CCK-8 infusion. feeding frequency was not reduced. The results of VanderWeele et al. (1982) are not entirely consistent with these findings. They showed that a high dose of LiCl(127 mg/kg) given immediately after ;t n~c:~l in r:lts increased the latency until the next spontaneous meal and decrcascd the
226
I). H. WI:S’I
b.7‘ Al..
size of that next meal, while cholecystokinin also increased the latency to the next meal but did not influence the size of that next spontaneous meal. However, these studies examined the effects of a single, large bolus of drug in the animal and did not examine the consequences of repeated pairings of drug and feeding. Therefore. these data may reflect differences in the timing of drug effects on eating, but do not indicate the differential ability of different drugs to produce an aversion to the diet normally supplied to the animal. This interpretation is supported by the variable effects of food intake observed after LiCl injection before access to food by food deprived rats (Ervin &Teeter, 1986). The effects of LiCl in that study depended both on the dose and timing of drug administration. It is important to address the possibility that endogenous agents, such as metabolites and hormones, contribute to the regulation of ingestive behavior by affecting the intermeal interval. Cholecystokinin is not important for determining the interval between meals (West ct Al., 1987), but other hormones may contribute to this process (Mindell, DiPoala, Weiner, Gibbs & Smith, 1985). The paradigm described in this paper may not be useful for distinguishing between a toxic agent like LiCI and an agent which prolongs the intermeal interval since the latter would also be expected to lead to a reduced feeding frequency. Other aspects of feeding behavior may be useful to distinguish between drugs producing malaise and those that suppress feeding through other mechanisms. For instance, VanderWeele, Deems and Gibbs (1984) showed that LiCl and CCK-8 affect the macronutrient proportions self-selected by rodents differently. Components of feeding behavior such as taste sensitivity or the microbehaviors of eating could also serve as independent indicators to distinguish possible satiety from malaise. To conclude, the patterns of ingestive behavior following the meal contingent injection of lithium vs. CCK-8 are significantly different. Indirectly, this supports the hypothesis that CCK-8 reduces meal size through a mechanism other than malaise. These findings indicate that a close examination of the normal feeding behavior of the animal is a useful tool to distinguish between agents that produce illness and those that do not. However, the final analysis will require the testing of putative satiety hormones in humans. Preliminary testing, in rodents and other animal models, can serve only to eliminate from further testing those compounds or hormones which demonstrate obvious toxicity. REFERENCES
Antin, J., Gibbs, J., Halt, J., Young, R. C. & Smith, G. P. Cholecystokinin elicits the complete behavioral sequence of satiety in rats. Journal qfcomparative and Physiological Psychology. 1975,89, 784-790. Berger, B. D. Conditioning of food aversions by injections of psychoactive drugs. journal of’ Comparative und Physiological Psychology, 1972, 81, 21-26. Deutsch, J. A. & Hardy, W. T. Cholecystokinin produces bait shyness in rats. Nuture (London), 1977, 266, 196. Deutsch, J. A., Thiel, T. R. &Greenberg, L. H. Duodenal motility after cholecystokin injection or satiety. Behavioral Biology, 1978, 24, 393-399. Ervin, G. N. & Teeter, M. N. Cholecystokinin octapeptide and lithium produce different effects on feeding and taste aversion learning. Physiology and Behavior, 1986, 36, 507-517. Gibbs, J., Young, R. C. & Smith, G. P. Cholecystokinin decreases food intake in rats. Journal of Comparative and Physiological Psychology, 1973, 84, 488~495. Holt, J., Antin, J., Gibbs, J., Young, R. C. & Smith, G. P. Cholecystokinin does not produce bait shyness in rats. Physiology and Behavior, 1974, 12, 487 498.
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AND
MEAL
PA'ITEKYS
“7 --
Kissileff, H. R., Pi-Sunyer, F. X., Thornton. J. CG Smith, G. P. C-terminal octapeptide c)f cholecystokinin decreases food intake in man. /lmericurt .Iorrrnrrl ofC/iniccrl Nufririorl. 1% I. 34. I 54m160. hlindell, S., DiPoala, J. A.. Weiner, S., Gibbs. J. &Smith, G. P. Bombesin increases postprandt.tl Intermeal interval. Socier_r ,fiw Ncuroscicnce Ah,strcKfs. 1985. I I (1 ), 3X. hloorc. B. 0. & Deutsch, J. A. An antiemetic is antidotal to the satiety effects ofcholccystokllltn Nature (London), 1985, 315, 321-322. Nachman, M. Learned aversion to the taste of lithium chloride and generalization to the other salts. Jourml of Comparatirv und Physiologicill P,s~~cho/oq~~. 1963. 56. 343349. Revusky, S. H. & Garcia, J. Learned associations over long delays. In G. H. Bower (Ed.). I‘ll(, ps~choloq_r of learning and motivation, Vol. 4. New York: Academic Press, 1970. Rozin, P. & Kalat, J. W. Specific hungers and poison avoidance as adaptive specializations of learning. Psycho/ogy Reuiews, 197 I, 78, 459486. Smith, G. P. &Gibbs. J. Postprandial Satiety. In J. M. Sprague & A. N. Epstein (Eds.), Pro~/rc.s.s irk ps~&ohioiogr and physioloyicui psJ&~logv. Vol. 8. Pp. 179 243. New York: Academic Press, 1979. Stacher, G.. Bauer, H., Steinringer, H. Cholecystokinin decreases appetite and activation cvokcd by stimuli arising from the preparation of a meal in man. Physiology cznd Beh~wior. 1979. 2.1. 3255331. Sturdevant, R. A. L. & Goetz, H. Cholecystokinin both stimulates and inhibits human food intake. Nature (London), 1976, 261, 717 719. VanderWeele, D. A.? Granja, J. A. & Deems, D. A. Discomfort or satiety: The spontaneous meal pattern may serve as a predictor. In B. G. Hoebel and D. Novin (Eds.), The nrurcd Arc.w 01 J&ding clnd reward. Pp. 167--173. Haer Institute: Brunswick, Maine, 1982. VanderWeele, D. A., Deems, D. A. &Gibbs, J. Cholecystokinin. lithium, and diet self-selection in the rat: lithium chloride decreases protein, while cholccystokinin lowers fat and carhohy drate ingestion. .Nutrition and Brhwior. 1984. ,?. I27 I.75 West. D. B., Fey. D. & Woods, S. C. Cholecystokinin persistently suppresses meal size but IIOI food intake in free-feeding rats. Arnericrm Journal of Physiology, 1984, 246. R776 R787. West. D. B.. Greenwood, M. R. C., Sullivan, A. c‘., Prcscod, L., Marzullo. L. R. & Triscart. J Infusion of cholecystokinin between meals into free-feeding rats fails to prolong the intermeal interval. Physioloyy and Beharior. 1987. 39, I 1 1 I 1.5. West, D. B., Tcngan, C., Smith W. S. & Samson, H. H. A microcomputer-based data acquisttton system for continuous recording of feeding and drinking by rats. Phv.sio/oq~~ md H~hurI‘~r. 1983, JI, 125-132. Woods, S. c’.. McKay, L. D., Stein, L. J., West, D. B.. Lotter. E. C. & Porte. Jr.. D. Neuroendocrine regulation of food and body weight. Brain Rrseurch Bullrtirl. 1980. 5 (Suppl. 4). I 5.
1987, 8. 221-227
Lithium Chloride, Cholecystokinin and Meal Patterns: Evidence that Cholecystokinin Suppresses Meal Size in Rats Without Causing Malaise DAVID B. WEST, M. R. C. GREENWOOD KATHLEEN A. MARSHALL Department
of Biology,
STEPHEN
C. WOODS
Department
of Psychology,
Vassar
College,
University
and
Poughkeepsie,
of Washington,
New
Seattle,
York
Washington
Gut hormoneswhich are proposedas natural satiety factors may act through malaiseto suppressshort-termfeedinginsteadof through a normal physiological process.Evaluation of the ability of candidatesatiety hormonesto serveas the unconditionedstimulusin the developmentof a conditioned tasteaversionis not adequateevidenceof malaisesinceagentswhich do not producemalaisein humans are alsocapableof conditioning aversions.Examination of the patternsof eating behaviorwhendrugsaregiven during a mealin ratsmay serveasa better testof the potential of an agentto produceillness.To evaluatethis approach,LiCl (a known aversiveagent)wasinfusedinto five rats at the start of eachfree-feedingmealat a doseof 1.9mg/meal/ratfor four daysand at 38 mg/meal/ratfor an additional four days. Feedingpatterns during drug infusion werecompare>with patterns for six days when no drugs were infused. LiCl infusion produced a significant dosedependentreduction in feedingfrequencywhile the sizeand duration of mealswere not changed.At the higher doseof lithium, daily mealnumberwasreducedfrom a normal 13.2& l-4 to 9.9+-0.5 meals/day.This pattern of behavior is significantly different from the behavior exhibited when cholecystokinin octapeptide(CCK-8) wasinfusedusingthe sameparadigm.CCK-8 significantly reducedmealsizewhile meal frequency increasedto compensatefor the smaller meals.Thesefindings suggestthat LiCl infusionduring a meal will lead to an aversionto feedingwhile CCK-8 is not effective at producing an aversion.The patterns of behavior when CCK-8 is infused are quite different from the patterns resulting from mealcontingent LiCl infusion.This supportsthe view that patternsof normal ingestive behavior may be useful to distinguishbetweenaversive agentsand those that produce true satiety. INTRODUCTION
A number of gut hormones, with the prototype being cholecystokinin (CCK), are reported to suppress meal size in the rat after peripheral administration (Woods, McKay, Stein, West, Lotter & Porte, 1980). In addition, CCK is known to suppress sham-eating in the rat and to initiate the normal postprandial behaviors (Antin, Gibbs, Holt, Young & Smith, 1975). Because of this and other evidence, it is suggestedthat
This work was supported by NIH grants Nos. AM07332, Correspondence and reprint requests should be addressed Vassar College, Poughkeepsie, NY 12601, U.S.A. 0195-~6663/87/030221+07
$0340/O
AM17844 to David
and CD12637. B. West, Department
!i” 1987 Academic
Press Inc. (London)
of Biology.
LimIted
222 cholccystokinin, and possibly other gut hormones, contribute to the normal satiety process and are important for the regulation of feeding behavior in rats and other mammals (Smith & Gibbs, 1979). This work has been criticized since the suppression of short-term feeding after i.p. injection of these hormones might be the result of malaise and not due to a normal physiological process. Some cvidencc indicates that i.p. CCK at doses which arc effective at suppressing meal size also are effective at conditioning a taste aversion (CTA) (Deutsch & Hardy, 1977) suggesting that the hormone is producing malaise. This conclusion is supported by a recent report indicating that an antiemetic will attenuate the ability of CCK-8 to suppress feeding in the rat (Moore & Deutsch, 1985). These same doses of CCK can produce atypical duodenal peristalsis indicating an abnormal state which might produce illness (Deutsch, Thiel & Greenberg, 1978). To counter this evidence, others argue that CCK, at doses which suppress food intake in the rat, is not effective at conditioning taste aversions (Holt, Antin, Gibbs. Young & Smith, 1974). Furthermore, it is reported that agents which do not product malaise in humans can be used to produce CTAs in rodents; perhaps not through malaise but by altering the physiological state of the animal (Berger, 1972). This suggests that the ability to produce a CTA is not adequate proof that the agent produces illness. The most powerful counterarguments for a malaise theory of CCK action are several reports that doses of purified CCK, which suppress feeding in humans, do not lead to reports of illness (Kissileff, Pi-Sunyer, Thornton & Smith, 198 1: Stacher, Bauer & Steinringer, 1978). However, the use of an impure preparation of CCK in humans has been associated with illness (Sturdevant & Goetz. 1976). It is clear that this argument has not diminished over the 13 years since a crude intestinal extract containing cholecystokinin was first shown to reduce normal feeding in the rat (Gibbs, Young & Smith, 1973). Other hormones or metabolites which are advocated as endogenous satiety agents also will have to be evaluated for their potential for producing malaise. This testing certainly will include an evaluation of their ability to produce a CTA and this may lead to controversy. Clearly, additional and/or alternative tests to evaluate the potential for producing illness are needed to rule out this action for candidate satiety hormones. VanderWeele, Granja and Deems (1982) suggest that the pattern of feeding behavior in the rat following injection of a compound may be used as an index of illness. They reported that effects on the interval until the next meal and the size of the next meal differ for agents that suppress food intake normally compared with those that produce illness. In that study, behaviors were evaluated for only several hours after a bolus i.p. injection. In this report, we further evaluate the potential for using mealpatterning data to distinguish between malaise and ‘satiety’ by examining the behavioral effects of meal-contingent infusion of LiCl, a known aversive agent (Nachman, 1963) into free-feeding rats.
METHODS
Five adult, male, Long Evans rats were habituated to computer enclosures where food (45 mg Noyes pellets, P. J. Noyes, Inc.) and water were available old lihitum. The animals weighed approximately 300g at the beginning of the experiment and they wcrc kept in a room maintained at a constant temperature with a LD 12: 12 cycle with lights on at 0800 hrs. When a food pellet was removed from a feeding trough by the rat. this
LiCl
AND
MEAL
PATTERNS
“1 -_.
e\ ent was recorded by a microcomputer and another pellet was immediately dispensed (West, Tengan. Smith & Samson, 1983). Following the habituation period, when the growth rate had recovered to normal, feeding behavior was recorded for a 5-day period (L>ays l-5 of the experiment). Throughout the experiment, continuous recordings of feeding behavior were made for approximately 23 hours each day with a one hour interval during the day when the pellet dispensers were inactivated and the program was interrupted. During this interruption the rats were weighed, the cages were cleaned. the volume of water consumed was recorded, and the water and pellet reservoirs were refilled. After baseline behaviors were recorded, the rats were implanted with i.p. silastic catheters which were exteriorized via a head pod and connected to saline-filled syringes mounted on computer-controlled infusion pumps. Following surgery, physiologica! saline (0.29 ml/30 set) was infused at the start of each free-feeding meal after 0.15 g of food had been consumed. After one infusion, another infusion was not allowed for IO minutes. By the fifth day of saline infusion (Day 10 of the experiment), the patterns of feeding behavior and the total daily food intake were not different from baseline patterns and intake. Therefore, the last saline day was combined with the five baseline days to calculate the overall baseline feeding patterns. The animals then received meal-contingent infusions of LiCl (150m~; 19mg LiClimeal/rat) for four days (Days 1 l-14). This was followed by four days (Days 15- 18) of meal-contingent LiCl at a dose of 3.8 mg LiCl/mcal/rat. The criteria for infusions and infusion parameters were the same during 1.9 mgjmeal LiCl infusion as during saline infusion. During the four days of 3.8 mg/meal LiCl infusion, the rats received a volume of 0.58 ml per infusion. For the data analysis, the average daily food intake, water intake, meal size. meal duration and meal number were calculated for each rat over six days of the baseline period, four days of 1.9 mg LiCl infusion and four days of 3.8 mg LiCl infusion. The definition of a meal required a minimum of0.225 g and an interval without eating of five minutes. The means for each animal for every treatment period were used to calculate a group mean during each period. In the text and in the figures the data are reported as means and standard errors. The treatments were compared with a Repeated Measures ANOVA followed by post-hoc t-tests. Growth rates over the different periods were compared with a two-way Repeated Measures ANOVA.
RESULTS
LiCl infusion produced a dose-dependent decrease in the number of meals taken each day. During baseline the rats took an average of 13.2 + 1.4 meals/day. Meal frequency was reduced to 116kO.7 and 9.9kO.5 meals/day by I.9 and 3.8 mg LiCl respectively. The overall reduction of meal frequency by the higher dose of LiCl averaged 23?, and was statistically reliable (p
224
Meal
number
Meal
SIR
FIGURE 1. Meal size and meal number during LiCl infusion expressed as a percentage of baseline value. Asterisks indicate a significant difference from baseline (p < 0.01, ANOVA and p~~,st-hoc’ f-test). I-1.1.9mg/meal; t2, 3.8 mg/meal.
350
340 -
FIGURE2. Body weight during the meal-contingent LiCl infusion. LiCl infusion at 1.9 or 3.8 mg/meal/rat had only a transient effect on body weight. Growth rate was not significantly affected. A, Baseline; l , 1.9 mg LiCl; 0, 3.8 mg LiCI.
Licl
AND MEAL PATTERNS
‘75 ___
Because fewer meals were consumed, the total daily intake was significantly reduced from 28.7 f 0.9 g/day to 22.2 + 2.5 g/day when 3.8 mg LiCl was infused during each meal (p < 005, ANOVA and post-hoc t-test). The daily intake during the infusion of 1.9 mg LiCl was reduced to 25.9 + 0.5 g, but this was not significantly different from baseline intake. Daily water intake was not affected by drug infusion. Water intake averaged 34.1 f 08 ml/day during baseline and 34.5 + 1.1 ml/day and 35.8 f 3.7 ml/day during the periods of drug infusion. The growth of the animals (Figure 2) was only transiently affected by drug infurion and there was no significant effect on body weight for either dose of LiCI. The daily dose of LiCl averaged 23.9* 1.1 mg/rat/day during infusion of I ,9 mg/meal and 38.2 f 2.7 mg/rat/day during the infusion of 3.8 mg/meal of LiCI.
DISCIJSSION
Infusion of LiCl at low doses produced a dose-dependent reduction of food intake due to fewer meals while the meal size of the rats was not affected. This suggests that lithium at this dose (3.8 mg/meal/rat) is aversive to the animal and consequently the rat takes fewer meals to avoid infusions. This conclusion is consistent with the findings 01 aversions developed by rats to vitamin deficient diets (Rozin & Kalat, 1971), however, it is not known if the aversion to vitamin-deficient diet is expressed by a reduction in the frequency of feeding and/or a reduced meal size. The observation that LiCl infusions paired with the taking of a normal meal will lead to an apparent aversion to feeding is also consistent with the observations that LiCl injections into rats can be used to condition aversions to novel tastes (Revusky & Garcia, 1970). It must be stressed that the reduced feeding frequency found with LiCl infusion paired with feeding may be due to other actions of LiCl instead of its known aversive actions. Yet an explanation of this phenomenon based upon the known aversive properties of LiCl is both reasonable and plausible. The effects of meal-contingent lithium infusion on meal patterns in the free-feeding r:it are strikingly different from the effects of meal-contingent infusion of CCK-8. In ;I previously published report (West, Fey & Woods, 1984), using a paradigm identical to that employed in the study described here, CCK-8 (1.1 pg/meal/rat) was infused into five rats at the start of each free-feeding meal over a 6-day period. This manipulation produced a remarkable decrease in meal size (407,, of normal) which was compensated for by an increase in feeding frequency. Total daily food intake was reduced only transiently below normal. One might argue that due to the dramatic decrease in meal size produced by CCK-8 infusion, a compensatory increase in feeding frequency was necessary for the animal to maintain a minimal caloric intake and this overwhelmed the aversive conditioning to the diet produced by meal contingent CCK-8 infusion. However, in a similar study (West, Greenwood, Sullivan, Prescod, Marzullo & Triscari. 1087). CCK-8 was infused towards the end of free-feeding meals in seven rats in large doses ( I.8 /lg;meal;rat) as well as during the intermeal interval. This protocol rcsultcd in a smaller reduction of meal size (29%), but meal number still increased to compensalc for the smaller meals. Therefore, in both studies using meal contingent CCK-8 infusion. feeding frequency was not reduced. The results of VanderWeele et al. (1982) are not entirely consistent with these findings. They showed that a high dose of LiCl(127 mg/kg) given immediately after ;t n~c:~l in r:lts increased the latency until the next spontaneous meal and decrcascd the
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size of that next meal, while cholecystokinin also increased the latency to the next meal but did not influence the size of that next spontaneous meal. However, these studies examined the effects of a single, large bolus of drug in the animal and did not examine the consequences of repeated pairings of drug and feeding. Therefore. these data may reflect differences in the timing of drug effects on eating, but do not indicate the differential ability of different drugs to produce an aversion to the diet normally supplied to the animal. This interpretation is supported by the variable effects of food intake observed after LiCl injection before access to food by food deprived rats (Ervin &Teeter, 1986). The effects of LiCl in that study depended both on the dose and timing of drug administration. It is important to address the possibility that endogenous agents, such as metabolites and hormones, contribute to the regulation of ingestive behavior by affecting the intermeal interval. Cholecystokinin is not important for determining the interval between meals (West ct Al., 1987), but other hormones may contribute to this process (Mindell, DiPoala, Weiner, Gibbs & Smith, 1985). The paradigm described in this paper may not be useful for distinguishing between a toxic agent like LiCI and an agent which prolongs the intermeal interval since the latter would also be expected to lead to a reduced feeding frequency. Other aspects of feeding behavior may be useful to distinguish between drugs producing malaise and those that suppress feeding through other mechanisms. For instance, VanderWeele, Deems and Gibbs (1984) showed that LiCl and CCK-8 affect the macronutrient proportions self-selected by rodents differently. Components of feeding behavior such as taste sensitivity or the microbehaviors of eating could also serve as independent indicators to distinguish possible satiety from malaise. To conclude, the patterns of ingestive behavior following the meal contingent injection of lithium vs. CCK-8 are significantly different. Indirectly, this supports the hypothesis that CCK-8 reduces meal size through a mechanism other than malaise. These findings indicate that a close examination of the normal feeding behavior of the animal is a useful tool to distinguish between agents that produce illness and those that do not. However, the final analysis will require the testing of putative satiety hormones in humans. Preliminary testing, in rodents and other animal models, can serve only to eliminate from further testing those compounds or hormones which demonstrate obvious toxicity. REFERENCES
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