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Am J Physiol Regul Integr Comp Physiol 285: R1170-R1183, 2003. First published June 19, 2003; doi:10.1152/ajpregu.00015.2003
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REGULATION IN GENETICALLY MODIFIED ANIMALS

c-Kit mutant mouse behavioral phenotype: altered meal patterns and CCK sensitivity but normal daily food intake and body weight

Michael M. Chi and Terry L. Powley

Laboratory of Regulatory Psychobiology, Department of Psychological Sciences, Purdue University, West Lafayette, Indiana 47097

Submitted 10 January 2003 ; accepted in final form 12 June 2003


   ABSTRACT
TOP
 ABSTRACT
EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 
The mouse W/Wv mutation of the c-Kit receptor causes extensive loss of gastrointestinal interstitial cells of Cajal and vagal intramuscular arrays (IMAs; one of the two putative mechanoreceptors in gastrointestinal smooth muscle). To characterize the behavioral phenotype of the c-Kit mouse and to evaluate the roles of these mechanoreceptors in controlling food intake, meal patterns and daily intakes of W/Wv mice and controls were examined using solid (20-mg pellets) and liquid (Isocal) maintenance diets. After the meal pattern experiments, CCK (0.5, 1, 2, 4, 8, and 16 µg/kg ip) was administered to examine the role of the interstitial cells and vagal IMA mechanoreceptors in relaying peripheral signals of satiety activated by CCK-A receptors, whereas the specificity of the response was assessed with the antagonist devazepide (300 µg/kg ip). On both diets, the W/Wv mice ate smaller meals for shorter durations, with a compensatory increase in meal number, resulting in daily intakes and body weights similar to the controls. After CCK injections, the mutant mice consistently suppressed intake more (~2x) in 30-min tests, regardless of the test diet (12.5% glucose, chow, pellets, and Isocal). The increased sensitivity of W/Wv mice to CCK reflected an increased potency of the hormone (c-Kit mouse ED50 = 2.4 µg/kg; control ED50 = 6.4 µg/kg) and a shift of the dose-response curve to the left. Devazepide blocked the CCK suppression of ingestion. These results indicate that the selective loss of the interstitial cells and IMAs disrupts short-term feeding of the W/Wv mice by inducing an earlier satiety, possibly by altering gastric accommodation and/or emptying, without affecting the long-term mechanisms controlling overall intake or body weight. The results also suggest that the reduction of interstitial cells and IMAs augments the sensitivity to or increases the efficiency of exogenous CCK.

feeding; devazepide; microstructure; intramuscular arrays; interstitial cells of Cajal

THE W/Wv MOUSE HAS TWO SELECTIVE losses of tissue in its gastrointestinal (GI) tract that suggest the mutant should be a useful model for structure-function analyses in GI physiology. The feeding behavior of the mouse has not yet been analyzed, and such a characterization is the aim of the present experiments.

W/Wv mice have a point mutation at the white spotted locus that results in an abnormal kit gene. Kit is a receptor tyrosine kinase that is expressed in tissues known to be affected by mutations of the W locus in fetal and adult erythropoietic tissues, mast cells, and neural crest-derived melanocytes, and the Wv allele encodes for a product with little kinase activity (11). In the central nervous system (CNS), c-Kit is expressed in postnatal development of the mouse cerebellum (18), although no studies have examined any possible structural effects in the brains of c-Kit-deficient mice. In the periphery, specifically the GI tract, the receptor is essential for the normal development of interstitial cells of Cajal (ICCs) in the wall of the GI tract (17, 35). In particular, ICCs of the intramuscular type (ICCIMs), densely distributed in the forestomach, are selectively ablated in the mutation, whereas other classes of ICCs are spared (2, 36, 37). The ICC-IMs have been implicated in the initiation and coordination of GI motor activity (8, 28). On the basis of such observations, delayed or reduced emptying and nutrient transit in the GI tract would be predicted, and such expectations lead to the prediction that food intake, particularly the size of discrete feeding bouts, or "meals," would be reduced.

The dramatic reduction of ICC-IMs in W/Wv mice has also recently been associated with a loss of one of the two classes of vagal afferents innervating GI smooth muscle. Vagal afferent innervation of muscularis externa consists of two specialized endings, both of which are putative mechanoreceptors (24). One of these two types of endings, the intramuscular array (IMA), is found in close association with the ICC-IMs. The ICC-IMs apparently provide structural and/or trophic support to the vagal IMAs, and it has recently been shown that the loss of ICC-IMs in W/Wvs is accompanied by a depletion of IMAs (10). The loss is selective insofar as the second class of vagal afferents in smooth muscle, the intraganglionic laminar endings (IGLEs) found at smooth muscle-myenteric ganglion interfaces, is normal in number and distribution in the W/Wv mutant (10).

IMAs have provisionally been considered stretch receptors on the basis of their morphology and distribution (24). In their juxtaposition to ICC-IMs and smooth muscle fibers, they appear to be disposed so as to transduce changes in length associated with either contractions or with passive changes in tension. The dense concentration of the IMAs found in the forestomach suggests that they play a role in gastric accommodation reflexes and receptive relaxation initiated by food ingestion. These ideas lead to the expectation that the W/Wv mouse, with its depletion of IMAs in the forestomach, would have impaired accommodation responses during feeding and, consequently, tend to be less capable, compared with controls without the mutation, of taking large meals.

On the basis of the morphological observations on the GI tract structures in the W/Wv mice and on the predictions that these defects might alter food intake either through impaired motility and gastric emptying or through reduced accommodation responses, the present experiments were designed to provide an initial assessment of the feeding patterns of animals with the mutation.


   EXPERIMENT 1
TOP
 ABSTRACT
 EXPERIMENT 1
EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 
Meal pattern analyses, or "microbehavioral" analyses of feeding, have proved particularly powerful in examinations of feeding behavior in rats (3, 6). Measures of meal size, meal number, meal frequency, and other parameters describing separate bouts of feeding often detect significant changes that are missed when the only index of feeding used is cumulative intake. The specific parameters can also often be related to potential physiological processes. The majority of these fine-grained behavioral analyses have been performed using rats; however, the same analytic approaches have also proved informative in mice (12, 15). Such analyses were employed in the present experiment to determine whether the c-Kit mutation might yield disturbed ingestive patterns in the W/Wv mouse.

Materials and Methods

Animals. Seven- to eight-week-old male WBB6F1 strain c-Kit mutant mice (W/Wv) (n = 16) and their controls (+/+) (n = 16) weighing 20-25 g on arrival were obtained from Jackson Laboratories. Mice were housed individually in custom-made plastic cages equipped with computerized food dispensers (Coulbourn Instruments, Allentown, PA) and maintained on a 12:12-h light/dark cycle, with lights on at 0600 and off at 1800. The colony temperature was maintained at 22°C. Mice had ad libitum access to tap water and were weighed on a daily basis.

During the week before testing, the mice were adapted to their cages, exposed to the test pellets (20 mg dustless precision food pellets; Bioserv, French-town, NJ), and accustomed to the feeding stations. Food was made available for 19 h per day, with a 5-h period of no food in the middle of the light period to facilitate monitoring of the equipment, performing food changes and weighing, and providing a defined start time for each day's feeding measures. At the beginning of the adaptation phase, mice were 8-9 wk old; the W/Wvs and the controls weighed, respectively, 27.68 ± 2.5 and 27.02 ± 1.29 g. All procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Purdue University Animal Care and Use Committee.

Meal patterning analyses. Once the animals were accustomed to consuming the test pellets, the feeding stations were used to monitor and dispense the same pellets. Briefly, the state of an infrared beam was monitored by a computer every 100 ms. The beam was interrupted as long as a pellet was in the food magazine. When a mouse removed a pellet (no longer breaking the beam), the computer recorded the time and dispensed another pellet, thus breaking the beam once again. Analyses were performed by exporting the raw data from Graphic State, the Coulbourn data management software, to an Excel (Microsoft, Redmond, WA) macro. The daily 19-h runs were broken down into 1-min intervals. The 19-h runs were started at 1430 and ended at 0930 the following day, so that there was 3.5 h of food availability during light preceding, and a similar interval following, the 12-h dark cycle.

The start of a meal was defined as three pellets consumed in a 7-min period, whereas the end of a meal was specified as the beginning of a 20-min period of no eating (see APPENDIX A) (16, 33).

Data representation and analysis. Meal parameters were determined by averaging, for each mouse, a 5-day block. Then, averages were computed for each 5-day block for all the mice in a group. Feeding data were analyzed using one-way repeated-measures ANOVAs, with group (mutant vs. control) as the independent variable and each of the meal pattern variables examined individually as the dependent variables. The main effect of group of each ANOVA was used to determine significance. Total daily intakes and body weights were analyzed using one-way repeated-measures ANOVAs, using the main effect of group to determine significance.

All graphs were made using Graphpad Prism 3.0 (Graphpad Software; San Diego, CA). Statistical analyses were performed using Statistica 6.0 (StatSoft; Tulsa, OK). Significance was considered to be obtained for P values <0.05.

Results

Cumulative food intake/body weight. Mutant mice had a slightly larger daily food intake, which was significant [F(1,29) = 17.725, P < 0.05; Fig. 1]. However, the W/Wv mice started out with a slightly higher baseline intake, and the overall differences in intake between the two groups remained similar throughout the experiment. The food intakes of both groups significantly declined over the 30-day test period [F(5,145) = 87.218, P < 0.05] but did not result in a significant group-by-time block interaction [F(5,145) = 2.052, P > 0.05]. These differences were perhaps a reflection of a deceleration in the growth rate or an initial overconsumption of the test pellets (compared with the conventional maintenance diet), as evidenced by both groups consuming more food per day early in the 30-day test period and less food per day later. Although the W/Wv mice also maintained a slightly higher body weight throughout the experiment, this was not significant [F(1,29) = 2.304, P > 0.10 (Fig. 1)]; there was a group-by-time block interaction [F(5,145) = 2.479, P < 0.05].



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  		Fig. 1. A: daily food intake for W/Wv mice (n = 16) and controls (n = 16) on the Bio-Serv pelleted diet in 5-day time blocks (means ± SE). W/Wv mice consistently had a larger daily food intake, especially during time blocks 4 and 5, when the controls ate much smaller amounts compared with their baseline intakes. B: daily body weights for W/Wv mice and controls, in 5-day time blocks (means ± SE). W/Wv mice consistently had larger body weights throughout the experiment. As a result of the reduction of intake by control mice during time blocks 4 and 5, there was a larger difference in body weights during and after this time period.

 

Meal analyses. Compared with their controls, mutant mice displayed consistent differences in meal patterns throughout the experiment (Fig. 2). Because the dark cycle is the time period when mice perform the majority of their feeding and because patterns of ingestion vary during the dark and light phases, the data were examined for both the dark cycle and the entire 19-h daily run.



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  		Fig. 2. Meal analysis summary graphs for W/Wv mice (n = 16) and controls (n = 16) maintained on the solid diet during the 12-h dark cycle (means ± SE, averaged over the 30-day experiment). W/Wv mice ate smaller meals (A) for shorter durations (B), yet ate more meals (C) to maintain similar cumulative intakes. Corresponding with their smaller meal sizes, W/Wv mice also had shorter intermeal intervals (D). Ratio of intermeal interval to the size of the previous meal, as indicated by the satiety ratio, was similar for both W/Wv and control mice. For the first 30 min of the dark cycle, both groups of mice ate similar numbers of pellets. *Significant difference (P < 0.05) between W/Wvs and controls.

 

During the dark cycle, W/Wv mice ate significantly smaller meals (22%) [F(1,28) = 29.828, P < 0.05] for shorter durations (24%) [F(1,28) = 4.8992, P < 0.05], but had a larger number of meals (22%) [F(1,28) = 50.544, P < 0.05]. In addition, the W/Wv mice had significantly shorter intermeal intervals (25%) [F(1,28) = 19.1395, P < 0.05] while maintaining nonsignificant differences in their "satiety ratios" [F(1,20) = 1.6963, P > 0.20], or the ratio of meal size to the following intermeal interval, which is often taken to be an indicator of the efficacy of a meal. The amount consumed in the first 30 min of the dark cycle was also not different in the two groups [F(1,28) = 0.7196, P > 0.40]. The rate of feeding, expressed as number of pellets eaten per minute during a meal, was almost identical for the two groups (3.43 controls; 3.27 W/Wv) [F(1,29) = 1.185, P > 0.40].

The results for the total 19-h run were similar, except the values varied to account for the longer time frame. The W/Wv mice ate significantly smaller meals (16%) [F(1,28) = 50.439, P < 0.05] for shorter durations (17%) [F(1,28) = 5.5819, P < 0.05] but had a larger number of meals (21%) [F(1,28) = 61.171, P < 0.05]. As in the dark cycle, the W/Wv mice also had a shorter intermeal interval (21%) [F(1,28) = 25.743, P < 0.05] with nonsignificant differences in their satiety ratios [F(1,28) = 4.055, P > 0.05].

The amount consumed in the first 30 min after food was returned in the light cycle, which reflects the onset of feeding after a 5-h fast, was significant, with the W/Wv mice eating fewer pellets (18%) [F(1,28) = 4.5026, P < 0.05].


   EXPERIMENT 2
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 
Experiment 1 determined that W/Wv animals exhibit distinctly altered patterns of short-term intake when they are maintained on a standard pelleted food. In general, through reciprocal changes in the size and number of meals, the W/Wv mice maintained their body weights at levels similar to their controls, suggesting that the mutants have normal long-term control of body weight and daily food intake.

The smaller and shorter bouts of pellet consumption exhibited by the W/Wv mice are consistent with the proposal that reduced motility, emptying, and accommodation resulting from the loss of ICC-IMs and vagal IMAs might cause food to remain in the stomach longer, leading to earlier satiety or inhibition of meal taking. This hypothesis would be more persuasive if different diets with different properties (e.g., solid and liquid diets) had the same result.

Because the digestion and emptying of solid foods depends heavily on antral grinding and mixing, whereas the movement of liquid diets relies more on pressure gradients established between the proximal and distal stomach, W/Wv mice might exhibit different patterns of meal taking if they were maintained on a liquid diet. Furthermore, the use of a second diet would also make it possible to explore the generality of the alterations in feeding patterns when the animals were tested on a diet with different rheological and macro-nutrient characteristics.

Thus, to evaluate the generality of the first experiment and specifically to assess whether a liquid diet would alter the pattern of feeding that the W/Wv mice display, experiment 2 examined the meal-taking patterns of W/Wvs maintained on a nutritionally complete liquid diet. Table 1 summarizes the proportions of macronutrients in the diets used in experiments 1 and 2 as well as the diets employed in experiment 3.


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  		Table 1. Caloric distribution of the diets used throughout the experiments

 

Materials and Methods

Animals. Naive 7- to 8-wk-old male WBB6F1 strain c-Kit mutant mice (W/Wv) (n = 8) and their controls (+/+) (n = 8) weighing 20-25 g on arrival were obtained from Jackson Laboratories. All other conditions were identical to experiment 1, except a liquid diet was used in place of the pelleted diet and liquid dippers were used instead of feeders to dispense the diet.

Meal patterning analyses. Starting when they were 8-9 wk of age (and the W/Wv and control mice weighed, respectively, 26.93 ± 0.69 and 26.14 ± 1.37 g), the mice went through an acclimation procedure similar to that in experiment 1. Once the animals were familiar with the test diet, the liquid dippers (Coulbourn Instruments, Allentown, PA), which consisted of a small metal 0.08-ml cup attached to the end of a metal arm, were used to monitor and dispense the liquid diet (Isocal; Mead Johnson, Evansville, IN). As with the feeders in experiment 1, photocells were used to detect when the animals ate. For the dippers, the beam was uninterrupted during periods of no eating (opposite to the feeder set-up). When a mouse positioned its head in the food magazine (breaking the beam), the computer recorded the time and presented a cup of Isocal for 5 s, after which the cup was retracted. Data exportation and analyses were performed in a manner similar to experiment 1.

Data representation and analysis. Intakes were expressed as number of dipper cups, and not milliliters, because of the variability in the amount of Isocal that was consumed per cup (see APPENDIX B). The start of a meal was defined as five cup deliveries in a 9-min period, whereas the end of a meal was defined as the beginning of a 20-min period of no eating. Feeding data were analyzed using separate one-way repeated-measures ANOVAs, with group (mutant vs. control) as the independent variable and each of the meal pattern variables examined as the dependent variables. The main effect of group of each ANOVA was used to determine significance. Total daily intakes and body weights were analyzed using one-way repeated-measures ANOVAs, using the main effect of group to determine significance.

Graphs were prepared and analyses were performed in the same manner as in experiment 1.

Results

Cumulative food intake/body weight. Mutant mice had a slightly larger daily food intake, but this did not reach significance [F(1,14) = 0.7461, P > 0.40 (Fig. 3)]. As with the mice maintained on the solid diet in experiment 1, the W/Wv mice started out with a slightly larger baseline intake, and the overall differences in intake between the two groups were relatively consistent throughout the experiment, as there was no group-by-time block interaction [F(5,45) = 1.0729, P > 0.30]. The W/Wv mice also started the experiment at a slightly higher body weight than their controls, although they ended the experiment at a slightly lower weight; this crossover in the body weight curves led to a significant group-by-time block interaction [F(5,70) = 17.595, P < 0.01].



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  		Fig. 3. A: daily food intake for W/Wv mice (n = 8) and controls (n = 8) on the liquid diet in 5-day time blocks (means ± SE). W/Wv mice consistently had somewhat larger daily food intakes, although this was not significant. B: daily body weights for W/Wv mice and controls in 5-day time blocks (means ± SE). W/Wv mice started with slightly heavier body weights than their controls, but this pattern was reversed after time block 3.

 

Meal analyses. Control and mutant mice displayed consistent differences in meal patterns that were almost identical to the patterns observed with the solid diet (Fig. 4). Although the parameters examined for the mice on the solid diet in experiment 1 were analyzed with a larger sample size (and a corresponding difference in the statistical power of the analyses), the same parameters for the mice on the liquid diet were also significant or at least almost reached significance. The same time periods were examined (12:12-h light/dark cycle and the entire 19-h daily run). During the dark cycle, W/Wv mice ate smaller meals (29%) [F(1,14) = 5.5391, P < 0.05] for shorter durations (37%) [F(1,14) = 4.1739, P = 0.06], but had a larger number of meals (29%) [F(1,14) = 7.4823, P < 0.05]. In addition, the W/Wv mice had shorter intermeal intervals (14%) [F(1,14) = 4.366, P = 0.055] while maintaining nonsignificant differences in their satiety ratios [F(1,14) = 4.3660, P > 0.30]. The first 30 min of feeding was also nonsignificant [F(1,14) = 0.5361, P > 0.30]. The rate of feeding, expressed as number of cups of Isocal per minute during the meals, was almost identical for the two groups (3.4 controls; 3.6 W/Wv) [F(1,14) = 1.4889, P > 0.20].



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  		Fig. 4. Meal analysis graphs for W/Wv mice (n = 8) and controls (n = 8) on the liquid diet during the 12-h dark cycle averaged over the entire experiment (means ± SE). W/Wv mice ate smaller meals (A) for shorter durations (B), yet ate more meals (C) to maintain similar total intakes. As a result of their smaller meal sizes, the W/Wv mice had shorter intermeal intervals (D). Satiety ratios were similar for both mutant and control mice. For the first 30 min of the dark cycle, both groups of mice drank similar numbers of cups of Isocal. *Significant difference (P < 0.05) between W/Wvs and controls.

 

The results for the total 19-h run were similar, except the values varied once again, reflecting the longer time frame. The W/Wv mice ate smaller meals (24%) [F(1,14) = 4.1756, P = 0.06] for shorter durations (35%) [F(1,14) = 3.92507, P = 0.06], but had a larger number of meals (32%) [F(1,14) = 10.6983, P < 0.05]. As with the dark cycle, the W/Wv mice also had a shorter intermeal interval (20%) [F(1,14) = 13.795, P < 0.05] with nonsignificant differences in their satiety ratios [F(1,14) = 0.0010, P > 0.95]. The first 30 min of feeding in the light cycle, which occurs at the onset of feeding after a 5-h fast, was significant, with the W/Wv mice drinking more cups (97%) [F(1,14) = 10.12387, P < 0.01].


   EXPERIMENT 3
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 
CCK provides one of the important negative feedback loops whereby nutrients arriving in the intestine influence meal size (13, 26) and the hormone affects gastric emptying (19, 29) and vagal mechanoreceptor activity (7). These peripheral effects of CCK are mediated by the CCK-A receptor (22), which is found in the smooth muscle of the proximal GI tract (22). It has also been shown that CCK amplifies the effects of gastric loads by increasing the discharge rates in vagal fibers that are sensitive to gastric distension (29, 30). Vagal gastric stretch or tension receptors could therefore be the pathway for the combined satiety effects of gastric distension and circulating CCK (7).

Thus, because CCK operates primarily in the short-term control of intake and does so largely through its feedback on vagal afferents, the present experiment examined the sensitivity of W/Wv mutant mice to the hormone. To evaluate whether the effects were expressed through the CCK-A receptor, the ability of the selective antagonist devazepide (25) to block the CCK effects was also examined. Given that diets did influence some observations in experiments 1 and 2 (e.g., compared with controls, W/Wv mice ate smaller solid meals during the first 30 min of the dark phase, but larger liquid meals during the same period), the effect of diet was also examined. Four test diets were employed in different trials.

Materials and Methods

CCK suppression tests. On completion of their respective feeding tests, all mice previously used in experiments 1 and 2 (n = 24 controls, n = 24 W/Wv) went through consecutive series of CCK tests. For at least 1 wk, or until a consistent intake was achieved, mice were presented daily with glucose solutions [12.5% anhydrous D(+)-dextrose in distilled water] for 30 min. The mice were then tested with injections of the hormone or vehicle every 2-3 days. On test days, after an overnight fast, 2 µg/kg of CCK-8 sulfated (catalog no. H-2080; Bachem) or an equivalent volume of the saline vehicle (0.9% sodium chloride; Abbott Laboratories, North Chicago, IL) was administered intraperitoneally. All injections were performed between 0900 and 1000. Interspersed saline trials were used so that CCK was not injected on consecutive test trials. Five minutes after the injections, the mice were given access to the test solution for 30 min. At the completion of each test, mice were again provided access to their maintenance diet. All mice were given at least 24 h between tests. On nontest days, the mice also had access to the glucose test solution (with no injections) for 30 min.

Four different types of diets were used to measure intake during the CCK tests (see Table 1). All mice initially received the glucose test diet while being maintained on their respective diets from experiments 1 or 2 (precision pellets or Isocal). The mice were then tested for CCK suppression with chow (5001 Rodent Diet; PMI Nutrition International, Brentwood, MO), Bio-Serv precision food pellets, and Isocal as test diets in randomized orders. The additional test diets, just as with the glucose solution, were presented to the animals for a 30-min period that began between 0900 and 1000. Except for the glucose trials, all mice were also maintained on the same diet that was being used as the test diet at that time (e.g., Isocal maintenance diet when Isocal was used as the test diet). For each test diet there were four sets of CCK and saline injections, with the first set excluded from the final analyses to account for adjustment to each new diet.

Devazepide tests. Devazepide tests were performed in a manner similar to the CCK tests. At the end of the four sets of CCK/diet trials, a subset of the animals was used (n = 10 controls, n = 6 W/Wv). Animals were adapted to a maintenance diet of chow and accustomed to drinking glucose for 60 min between 0900 and 1000. For each test, after an overnight fast, the mice were given one of three combinations of intraperitoneal injections: saline followed by saline (sal/sal), saline followed by CCK (sal/CCK), and devazepide followed by CCK (dev/CCK). The first injection, given between 0900 and 0930, was followed by the second injection 15 min later. Then the test solution of 12.5% glucose was provided 5 min after the second injection. The dose of CCK used was the same as in the previous CCK tests (2 µg/kg) and the dose of devazepide (a gift from ML Laboratories, Leicestershire, UK) was 300 µg/kg, which was previously shown to produce complete attenuation of suppression by peripheral injections of CCK (4). The devazepide was prepared by adding 1% DMSO and diluting it with saline to reach the desired concentration. Intakes were recorded at 30 and 60 min after presentation of the glucose.

Data representation and analysis. On the assumption that the animals' responses to CCK might be influenced by their dietary histories from experiments 1 and 2, respectively, the data from the animals from the two experiments were analyzed separately. For each mouse, the mean of its intakes after saline injections and the mean after CCK injections were each computed, and a resulting percent suppression for each mouse was calculated. CCK and devazepide results were analyzed using one-way ANOVAs, with group as the independent variable and percent suppression as the dependent variable.

Graphs and analyses were prepared as in experiments 1 and 2.

Results

CCK suppression tests. All mice reached a consistent level of glucose intake (1-1.5 ml) after a few days, but were still given at least 1 wk of acclimation before beginning the injections. In the case of the mice maintained on the solid diet in experiment 1, the W/Wv mice consistently and significantly had a larger suppression of intake after injections of CCK than their controls, regardless of the test diet (Fig. 5). Both controls and mutants consumed identical baseline amounts of glucose (1.55 ml) after injections of saline, but after injections of CCK, the control mice consumed an average of 1.10 ml (29% suppression), whereas the W/Wv mice consumed an average of 0.66 ml (57% suppression) [F(1,34) = 67.2746, P < 0.01]. Similarly for the pelleted test diet, controls ate 0.63 g after saline and 0.49 g after CCK (22% suppression); whereas W/Wv mice ate 0.68 g after saline but 0.32 g after CCK (53% suppression) [F(1,31) = 38.0830, P < 0.01]. For the chow test diet, controls ate 0.66 g after saline and 0.47 g after CCK (29% suppression), whereas the W/Wv mice ate 0.81 g after saline and 0.44 g after CCK (46% suppression) [F(1,12) = 5.61824, P < 0.05]. Finally, on the Isocal test diet, controls drank 1.66 ml after saline and 1.12 ml after CCK (33% suppression), whereas the W/Wv mice drank 1.33 ml after saline and 0.59 ml after CCK (56% suppression) [F(1,9) = 6.09245, P < 0.05]. In all cases, the suppression values of the mutants were significantly greater than those of the controls (P < 0.05).



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  		Fig. 5. Percent suppression of intake (mean difference in intake after CCK and saline trials as a percentage of mean intake after saline trials) for W/Wv mice (n = 24) and controls (n = 24) on the solid diet in experiment 1 for each of the 4 test diets. *Significant difference (P < 0.05) between W/Wvs and controls.

 

Similar results were obtained for the mice that had been maintained on the liquid diet in experiment 2 (Fig. 6). Both controls and mutants consumed similar amounts of glucose after injections of saline (1.26 ml for controls; 1.24 ml for W/Wv mice), but after injections of CCK, the control mice once again consumed more (0.82 ml; 35% suppression) than the W/Wv mice (0.34 ml; 73% suppression) [F(1,14) = 19.0921, P < 0.01]. For the pelleted test diet, controls ate 1.04 g after saline and 0.65 g after CCK (38% suppression), whereas the W/Wv mice ate 1.11 g after saline and 0.35 g after CCK (68% suppression) [F(1,10) = 25.7330, P < 0.01]. The chow test diet showed the smallest difference in intakes, with the controls eating 0.71 g after saline and 0.46 g after CCK (35% suppression), whereas the W/Wv mice ate 0.60 g after saline and 0.33 g after CCK (45% suppression) [F(1,13) = 1.1430, P > 0.30]. Finally, on the Isocal test diet, the controls drank 1.70 ml after saline and 1.03 ml after CCK (39% suppression), whereas the W/Wv mice drank 1.36 ml after saline and 0.59 ml after CCK (57% suppression) [F(1,13) = 7.7271, P < 0.05].



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  		Fig. 6. Percent suppression of intake (mean difference in intake after CCK and saline trials as a percentage of mean intake after saline trials) for W/Wv mice (n = 24) and controls (n = 24) on the liquid diet in experiment 2 for each of the 4 test diets. *Significant difference (P < 0.05) between W/Wvs and controls.

 

Devazepide tests. The initial portion of the devazepide tests replicated the previous CCK tests (Fig. 7). Although the animals were handled and injected twice for each trial, the mice still showed almost identical trends in suppression compared with the previous single injections of CCK. In this case, the controls suppressed 25.5% compared with 63.6% for the W/Wv mice. When devazepide was injected before CCK, the controls actually drank more in the first 30 min (19.4% increase), whereas the W/Wv mice attenuated their suppression to 14.9%.



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  		Fig. 7. Percent suppression of intake during the first 30 min for W/Wvs (n = 6) and controls (n = 10) for the devazepide tests of experiment 3. Suppressions after injections of saline/CCK (sal/CCK) were similar to their suppressions after single injections of CCK in the initial portion of experiment 3. Administration of devazepide before CCK (Dev/CCK) was able to block the effects of CCK and attenuate suppression to baseline levels or greater, as in the case of the control mice. *Significant difference (P < 0.05) between W/Wvs and controls.

 

For the second 30-min period, both groups of mice drank relatively small amounts after injections of sal/sal (0.39 ml controls, 0.25 ml W/Wv). After sal/CCK, both groups drank larger amounts of glucose, presumably to make up for the suppression of intake in the first 30 min (1.33 ml controls, 0.98 ml W/Wv).

Although both groups drank large amounts in the first 30 min after injections of dev/CCK, both groups continued to drink larger amounts in the second 30 min (1.10 ml controls, 1.14 ml W/Wv).


   EXPERIMENT 4
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 
Experiment 3 held the dose of CCK constant at a moderate level and varied maintenance and test diet conditions to explore the sensitivity of W/Wv mice to the hormone under different dietary conditions. The results illustrated that W/Wv mice are more sensitive than controls to 2 µg/kg of CCK and that the degree of increased sensitivity was similar across the diets tested.

The present experiment held the maintenance and test diet conditions constant and varied the CCK dose to examine systematically the dose-response function for CCK in W/Wv mice. To avoid any interactions with dietary histories or previous testing, new groups of naive W/Wv and control animals were used.

In addition, the limited devazepide trials in experiment 3 suggested that a second, and more comprehensive, series of trials in which devazepide and a higher dose of CCK were administered factorially would be instructive. A separate set of naive animals was employed for these trials.

Materials and Methods

Animals. Additional 7- to 8-wk-old male WBB6F1 strain W/Wv mice (n = 15; Jackson Laboratories) and their controls (+/+) (n = 16) weighing 20-25 g on arrival were obtained. General housing and maintenance conditions were the same as in the previous experiments.

CCK dose-response tests. For an adaptation period of 1 wk, W/Wv mice (n = 7; weighing 26.2 ± 0.87 g) and controls (n = 8; weighing 25.0 ± 0.70 g) were fasted overnight, then presented daily with a glucose solution [12.5% anhydrous D(+)-dextrose in distilled water] for 30 min. After the glucose test, mice were provided with access to their maintenance diet (5001 Rodent Diet; PMI Nutrition International, Brentwood, MO) for 31 h (1 complete day plus part of the next day until the beginning of a fast).

After the animals were adapted to the test situation, they were administered injections of CCK or vehicle every 2-3 days, 5 min before receiving access to the glucose test solution for 30 min. On different test days, animals were injected intraperitoneally with 1, 2, 4, or 8 µg/kg of CCK-8 sulfated in randomized order, with at least one saline vehicle injection test intervening between doses of CCK-8. The randomized dose-response test was then repeated for three replications. On intervening noninjection days, animals also were given 30 min of access to the glucose solution; on all days, the mice had access to the maintenance diet at the conclusion of the glucose presentation.

After the randomized trials of the dose-response series were finished, two additional doses were tested. The exceptionally high dose of 16 µg/kg of CCK-8 had not been included in the original series because of concern that it might produce a conditioned aversion and depress intakes in subsequent tests. An exceptionally low dose of 0.5 µg/kg also had not been used, in this case because such a low concentration has commonly been found to be below threshold. On the basis of the results from the randomized trials, however, it appeared that the extreme doses might be instructive. In additional trials, the animals were first given a series of three tests with 0.5 µg/kg CCK-8 on every second to third day, alternating with saline, and then, finally a series of two tests with 16 µg/kg CCK on every second or third day, alternating with saline. After two tests with the high dose of CCK, trials were discontinued because the injections were producing a general behavioral depression.

Devazepide/CCK tests. Additional groups of naive W/Wvs (n = 8) and controls (n = 8) were adapted to the same feeding/testing schedule used for the CCK dose-response series, and weighed 24.4 ± 0.99 and 25.3 ± 1.26 g, respectively, at the start of this period. In this case, however, the period of access to glucose was lengthened to 60 min between 0830 and 0930 to detect any longer-lived drug effects. For each test day, animals received two intraperitoneal injections. The first injection consisted of devazepide (300 µg/kg, diluted in normal saline containing 1% DMSO; gift from ML Laboratories) or normal saline 20 min before glucose solution access. The second injection was CCK (4 µg/kg) or saline vehicle 5 min before access to glucose. Intakes were recorded at 30 and 60 min after glucose had been made available.

Data analysis. For each mouse, the mean of its intakes after saline injections and the mean after CCK injections were each computed, and a resulting percent suppression for each mouse was calculated. Results were analyzed using one-way ANOVAs, with group as the independent variable and percent suppression as the dependent variable. To determine if the intakes after injections of 0.5 µg/kg were significantly different from saline injections, independent t-tests were performed for each group. ED50 values were determined using Graphpad. To examine any differences between the combinations of injections vs. the sal/sal control, intake values for controls and W/Wv mice were analyzed using one-way ANOVAs. Graphs and analyses were prepared as in experiments 1, 2, and 3.

Results

CCK dose-response tests. The baseline glucose consumptions of W/Wvs and their controls were stable after the week of adaptation, and the two groups did not differ in the amount of glucose solution they consumed during the 30-min access period. Figure 8, which expresses the two groups' suppressions to each of the doses of CCK-8 as a function of the percent of glucose intake on saline injection days, summarizes the dose-response functions for W/Wvs and their controls. The functions show that the mutants exhibit an increased sensitivity to CCK-8 intake that is characterized by a shift of their response curve to the left. As expected for such a shift, calculations of the ED50s for the two groups revealed that CCK-8 was more potent in the mutants than in the controls (ED50s, respectively, 2.4 and 6.4 µg/kg).



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  		Fig. 8. Percent suppression of intake (mean difference in intake after CCK and saline trials as a percentage of mean intake after saline trials) for W/Wv mice (n = 8) and controls (n = 8) for each of the 6 doses used in the CCK dose-response tests of experiment 4. Both controls and W/Wvs showed a dose-dependent suppression of intake, with the W/Wvs showing a much larger suppression at all doses, including the dose of 0.5 µg/kg, previously considered sub-threshold. These differences were significant at the 0.5, 1, 2, and 4 µg/kg doses, and reached asymptotic levels for both groups at the higher doses. *Significant difference (P < 0.05) between W/Wvs and controls.

 

Univariate comparisons were used to evaluate for which doses of CCK-8 the mutants displayed significantly greater suppression of glucose intake. Both groups drank similar amounts of glucose after injections of saline (2.49 ml controls; 2.39 ml W/Wvs). At the lowest dose of 0.5 µg/kg, W/Wvs consumed 2.02 ml (15% suppression), whereas the controls drank 2.65 ml (-7% suppression) [F(1,14) = 43.3269, P < 0.001]. The mutants also displayed significantly more suppression of glucose intake than did controls at 1, 2, and 4 µg/kg of CCK-8. At 1 µg/kg, controls drank 2.11 ml (15% suppression), whereas the W/Wvs drank 1.47 ml (38% suppression) [F(1,14) = 17.5031, P < 0.001]. At 2 µg/kg, controls drank 1.82 ml (27% suppression), whereas the W/Wvs drank 1.28 ml (46% suppression) [F(1,14) = 15.3799, P < 0.01]. At 4 µg/kg, controls drank 1.5 ml (40% suppression), whereas the W/Wvs drank 0.84 ml (65% suppression) [F(1,14) = 24.1489, P < 0.001]. At the two highest doses, the two groups showed evidence of reaching a similar asymptote. At 8 µg/kg of CCK-8, the suppression of the intake in mutants compared with their controls neared, but failed to reach, significance, with controls drinking 1.01 ml (59% suppression), whereas the W/Wvs drank 0.62 ml (74% suppression) [F(1,14) = 3.4683, P = 0.08]. At 16 µg/kg, there was no difference between the controls who drank 0.86 ml (66% suppression) and the W/Wvs who drank 0.59 ml (75% suppression) [F(1,14) = 1.2253, P = 0.29]. This asymptote in suppression scores at the two highest doses of CCK-8 suggests that mutants do not differ from their controls in their maximal response or drug efficacy. The data at the end of the 60 min of glucose access (not shown) were similar to those at 30 min.

Dev/CCK trials. Figure 9 illustrates the results of the dev/CCK trials. Glucose consumption baselines did not differ between mutants and controls on the days animals received saline in both injections (2.51 ml, controls; 2.19 ml, W/Wvs) [F(1,13) = 4.0868, P > 0.05], so on the different tests using CCK-8 and/or its antagonist, intake suppression scores are expressed as percent of the sal/sal trials. As expected from the dose-response function described above, when the two groups received 4 µg/kg CCK-8 without a prior injection of antagonist (sal/cck), both groups suppressed, and W/Wvs suppressed their intake significantly more than controls. The controls drank 1.3 ml (48% suppression), whereas the W/Wvs drank 0.7 ml (68% suppression) [F(1,13)=8.0731, P < 0.05]. When the two groups received devazepide before the CCK injection, the antagonist completely blocked the suppression of their respective food intakes, and the two groups did not differ in their suppression scores. The controls drank 2.43 ml (3% suppression), whereas the W/Wvs drank 2.03 ml (6% suppression) [F(1,13)=0.3488, P > 0.5]. When devazepide was injected alone (dev/sal trials), the percent suppression scores of both groups were negative, suggesting a trend for the devazepide to facilitate ingestion by blocking endogenous CCK, but the two groups' percentages were not significantly different from each other, as the controls drank 2.67 ml (-6% suppression), whereas the W/Wvs drank 2.43 ml (-11% suppression) [F(1,13) = 1.1816, P > 0.25]. They also did not differ from their respective sal/sal baselines {[F(1,14) = 1.120, P > 0.25] controls; [F(1,12) = 1.3036, P > 0.25] W/Wvs}.



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  		Fig. 9. Percent suppression of intake during the first 30 min for W/Wvs (n = 7) and controls (n = 8) for the Dev/CCK trials of experiment 4. As with the CCK tests of experiments 3 and 4, W/Wvs suppressed intake significantly more after sal/CCK injections. Devazepide was able to completely block the effects of the peripheral CCK injections (Dev/CCK), although neither group drank significantly different amounts of glucose after Dev/CCK or Dev/sal. Dev/sal, however, did cause both groups to drink more glucose than the sal/sal condition. *Significant difference (P < 0.05) between W/Wvs and controls.

 


   DISCUSSION
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 
The present experiments indicate that the W/Wv mouse, which has losses of GI ICC-IMs and IMAs, exhibits altered control of feeding bouts and an exaggerated sensitivity to CCK. Similar alterations in feeding patterns and exaggerations in sensitivity to CCK occurred on diets that varied in both form (solid/liquid) and proportions of macronutrients (see Table 1). The increased sensitivity was reflected in a shift of the W/Wv CCK dose-response curve to the left and a greater CCK potency defined in terms of a lower ED50, without a detectable change in the maximal effectiveness or efficacy of the peptide. In contrast to the alterations in the short-term control of food intake, the c-Kit mutant appears to have essentially normal long-term controls of energy balance as reflected in its maintenance of normal, or near-normal, body weights and daily measures of food consumption. These observations lead to several inferences about the roles of ICCIMs and/or the putative vagal mechanoreceptors in short-term feeding. Before these suggestions are examined, however, an issue of specificity should be raised, and the long-term energy regulation in the W/Wv mouse should be considered.

Mutations of the kit gene influence a variety of tissues throughout the body. Included in the effects of loss or partial loss of kit gene function are deficits in the survival and development of stem cells involved in hematopoiesis, melanogenesis, and gametogenesis (11, 17, 27). Any or all of these effects of the kit mutation might potentially be the cause(s) of the altered feeding responses we described, although hypothetical mechanisms that might translate most of these defects into the particular alterations in meal patterns are not obvious. In contrast, whereas the effects of kit mutations on other organ systems seem to have only tenuous relationships to the immediate physiological controls of feeding, the effects of the kit mutation on the GI tract, specifically on its ICC-IMs and IMAs, provide candidate mechanisms that might account for the behavioral changes we have observed. Because the ICC-IM and IMA losses in the GI tract are the two GI defects associated with the kit mutation, these losses and their postulated effects on GI motility, gastric accommodation, and receptive relaxation would seem to provide a useful method that may help tease apart the functions of the mechanoreceptors in a manner previously unavailable with surgical methods.

Long-term controls of food intake and body weight appear to be essentially uncompromised in W/Wv mice, even with the extensive losses of ICC-IMs and IMAs from the smooth muscle of their GI tracts and with the multiplicity of defects in their nongastrointestinal tissues. The differences in long-term ingestion and body weight patterns between the W/Wv mice and their controls were, at most, subtle (i.e., not consistently significant from experiment to experiment) and may have reflected, at least in some instances, problems in matching groups that result when small shipments of animals are obtained. In experiment 1, which employed Bio-Serv precision pellets as a diet, W/Wv mice compared with their controls had slightly but significantly larger daily food intakes as well as slightly larger baseline body weights compared with the controls. The differences between groups existed at the beginning of the testing and appeared to be maintained throughout the experiment. The largest differences on the solid diet occurred during time blocks 4 and 5 (days 20-25), in which a few (n = 3) control mice reduced their intakes, which lowered the overall group average. Although these controls ate far less than their baseline values during this time period, their data were incorporated into the calculations because no criteria were developed for excluding undereating before the start of the experiment. In experiment 2, which employed the Isocal liquid diet, the W/Wv mice had slightly larger daily intakes, consistent with somewhat larger starting body weights, although in this experiment the differences in their intakes failed to reach significance. The subtle food intake differences were maintained although there was a modest reversal in the average weights of the two groups. In any event, it is noteworthy that any tendency for W/Wvs to be somewhat heavier than their controls or to have slightly greater 24-h cumulative intakes than their controls would not account for the changes observed in their meal patterns. Specifically, any such differences in long-term control of energy balance between the W/Wvs and their controls would predict that the mutants might eat larger meals to generate the greater daily total, not the smaller individual meals actually observed.

The short-term controls of intake, as reflected in meal pattern data and in responses to CCK, were clearly affected by the mutation of the kit gene. On either a pelleted diet (experiment 1) or a liquid diet (experiment 2), W/Wv mice consistently ate smaller meals of shorter duration and ate more of them by eating with shorter intermeal intervals. The same meal patterns held when only the meals taken during the 12-h dark period were analyzed as well as when the lights-off data were pooled with the data on the feeding that occurred during the lights-on phase. Most likely, an attenuation of gastric accommodation caused by the reduction of IMAs and disturbances in motility and emptying caused by concomitant losses of ICC-IMs led to the W/Wvs becoming satiated more quickly. Interestingly, although the mutants and controls consumed their meals of different sizes, the satiety ratio, an indicator of how filling a meal is, was almost identical for the W/Wv mice and controls, both for the 12 h of the dark phase and for the entire 19 h in which food was available each day. This ratio suggests that although the two groups reach satiety in different ways, that is by varying the size and duration of meals and the length of the intermeal interval, they seem to adjust their meals so as to generate comparable satiety ratios as well as the long-term patterns discussed above. In a similar vein, it is instructive that the mutants and controls ate at the same speed because the feeding rates were essentially identical for both groups of mice on both types of diets.

Analyses of the initial bouts of eating (i.e., the first 30 min after food was returned during the light phase and the first 30 min of the dark phase, when a burst of eating often occurs) appear instructive. In particular, the diet had a substantial influence on intake during the 30-min period after food was returned in the light phase. After the 5-h fast, W/Wv animals ate significantly more liquid food than did their controls, whereas the mutants ate significantly less solid food than their controls. Potentially, these contrasting patterns might reflect the amount of residual food in the stomach at the time of testing. The 5-h maintenance interval without food is equivalent to seven or eight times the intermeal interval of the mutants on Isocal, and this diet would be expected to empty more readily than the solid diet. As a consequence, the significant (and otherwise uncharacteristic) overeating by the W/Wvs after the fast might reflect some loss of mechanoreceptor feedback from the stomach, until the feeding bout reestablished prepotent feedback cues resulting from impaired accommodation and motility. In contrast, however, the mutants maintained on solid food ate significantly less than controls during the same 30-min window, although the 5-h interval without food would have been roughly five times their typical intermeal interval. Apparently, the accommodation to and emptying of solids is impaired enough in W/Wvs that even the 5-h interval was insufficient to prepare the stomach for a large meal. Inasmuch as animals generally consume little during the light phase, similar arguments indicating that the animals' stomachs were relatively empty at the beginning of the dark phase might at least partially explain how W/Wvs were able to eat as much of both the liquid and solid diets as their controls during the initial 30 min of the dark phase.

In the third experiment, the W/Wv mice consistently and significantly suppressed their intakes more than the controls after intraperitoneal injections of CCK, regardless of the test diet (glucose, Bio-Serv pellets, Isocal, or chow) that was provided. In the fourth experiment, the increased sensitivity of the W/Wv mice was determined to reflect a shift of the dose-response function to the left, with a corresponding increase in CCK's potency as measured by the ED50 values. The devazepide trials in experiments 3 and 4 indicated that the CCK effects were the result of binding to the CCK-A receptor, not a nonspecific pharmacological effect of the hormone treatment. Several experiments have established that low to moderate doses of CCK given peripherally operate on CCK-A receptors in the periphery to reduce feeding (1, 5, 14, 20) and that this negative feedback is relayed to the CNS by vagal afferents (21, 32). Which vagal afferents mediate the increased CCK sensitivity of the c-Kit mutant mouse is, however, unclear. CCK-A receptors are found in myenteric neurons as well as in fibers in both smooth muscle and mucosa of the stomach and proximal small intestine (34). Recordings from vagal load-sensitive mechanoreceptors innervating the stomach indicate that their activity is modified by CCK (7, 9), suggesting that some of the vagal afferents responsible for relaying the CCK suppression to the CNS are these gastric mechanoreceptors. On the other hand, there is evidence that the suppressive effects of CCK on feeding can also be elicited from the proximal intestinal region perfused by the superior pancreaticoduodenal artery (5). As summarized earlier, the c-Kit mutation produces a loss of ICC-IMs and vagal IMAs in the stomach, with a sparing of vagal IGLEs in the stomach and small intestine (10). The status of vagal afferents to the mucosa of the small intestines (or stomach) of the W/Wv mouse has not been examined, but the losses of ICC-IMs are confined to smooth muscle, so the organization of afferents in the mucosa may be spared direct disruptions.

The earlier observations on the W/Wv mutant as well as previous experiments on the CCK effects on vagal afferents suggest a working hypothesis involving vagal mechanoreceptors. If the W/Wvs had slower gastric emptying, as suggested by analyses of ICC-IM physiology (8, 28), and reduced accommodation reflexes, as suggested by other analyses of vagal IMAs (24), then the mechanoreceptors remaining in the GI tract of the W/Wvs would be more strongly stimulated. Furthermore, because the activity of load-sensitive vagal afferents to CCK is the product of both the mechanical stimulus and the hormonal modulation (30), the increase in responsiveness to CCK may simply reflect delayed nutrient handling and impaired accommodation in the W/Wv mouse.

Previously reported observations on meal patterns and CCK are consistent with this hypothesis that alterations in gastric fill and nutrient emptying might exaggerate GI feedback and CCK sensitivity. West and colleagues (38) demonstrated that augmenting CCK feedback by chronically yoking exogenous CCK infusions to ad libitum feeding results in a decrease in meal size with a compensatory increase in meal number, without altering long-term mechanisms controlling total intake or body weight. Thus an altered sensitivity to CCK such as that observed in experiment 3 might produce the decreased meal size and increased meal number, with long-term regulation of near normal levels of body weight and daily food intake observed in experiments 1 and 2.

Which of the classes of vagal mechanoreceptors might be involved in mediating the effects of CCK has been unclear. Inasmuch as CCK binding sites have not been found on vagal afferents in the gut (23, 34), the effects of the hormone on mechanoreceptor sensitivity may be mediated indirectly through smooth muscle or accessory cells associated with the afferents. The present experiments establish, however, that the c-Kit mutant with its extensive loss of intramuscular arrays exhibits not only normal, but even greater than normal, sensitivity to CCK. On the basis of the morphological survey by Fox and colleagues (10), it is clear that the W/Wv mouse may still have some (~35%) IMAs, but it seems unlikely that a reduced population, even if it expressed a compensatory increase in sensitivity, would generate an exaggerated behavioral response to CCK. It seems more probably that an alternate type, or types, of mechanoreceptor must be able to signal the effects of peripheral CCK to the brain. This receptor might be the other class of vagal mechanoreceptors found in GI smooth muscle, namely the IGLE, or the fast-adapting receptor that appears to be located in the mucosa (24). Although a hypothesis involving accommodation and emptying appears to point to the IGLEs or mucosal endings in the stomach, the indirect changes in nutrient handling of the proximal small intestine could also potentially involve intestinal IGLEs or mucosal endings in the increased sensitivity to CCK. Conceivably, mechanoreceptors mediating the effect could also be the higher threshold endings associated with the sympathetic innervation (31), although experiments employing behavioral protocols similar to the present one have consistently implicated the vagus, not the sympathetics, in the mediation of the effects of peripherally administered CCK (7, 21, 32).

The arguments just discussed in the context of CCK are again relevant in a more general assessment of the relative contributions of the two types of vagal mechanoreceptors in smooth muscle, IMAs and IGLEs, to short-term meal-related satiety. By a number of measures, W/Wv or c-Kit mutants with dramatic losses of IMAs (and ICC-IMs) from their forestomachs respond to early meal-associated signals, many of them presumably mechanical stimuli, at least as effectively as their controls. These measures illustrating that W/Wv mice are able to maintain their overall long-term goals of body weight and food intake regulation include meal size, rate of eating, and the satiety ratio index, in addition to the response to CCK discussed above. It is difficult to assign such relatively normal sensitivities to meal-related feedback signals, and particularly to gastric fill, to vagal IMAs, because only a fraction (~35%) of them remain in the W/Wv and a significant number of this residual population appears to be malformed (10). By such arguments it would seem more probable that the IGLEs, the other class of vagal mechanoreceptors in smooth muscle, which are normal in number and distribution in the mutant, are able to relay the mechanical aspects of the meal-related feedback to the CNS. Whether the same signals are also carried by the IGLE fibers in normal animals without mutations of the c-Kit gene, or whether the situation in the W/Wvs reflects functional redundancies and overlaps between the different mechanoreceptors or a plasticity resulting from the absence of IMAs throughout development, is an empirical question.


   APPENDIX A
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
APPENDIX B
 DISCLOSURES
 REFERENCES
 
Identification of Meal Criteria for the Pelleted Diet

Inasmuch as relatively few meal patterning analyses have been conducted with mice (even fewer using the Coulbourn feeding stations), the criteria for meal initiations and terminations were evaluated using pilot mice (n = 4).

For each mouse of this pilot series, the number of putative meals was calculated using a wide range of criterion values for defining the start of a meal (from 1 to 10 pellets in a time range of 7-20 min). Specifically, a meal was considered to have begun in the first minute of a criterion block of time (between 7 and 20 min) in which a criterion number of pellets (from 1 to 10 pellets) was removed from the dispenser. The greatest stability in such estimates of overall numbers of meals occurred with a criteria between two and five pellets for a time period of 7-20 min. Calculated numbers of meals were less stable between one and two pellets, corresponding to the part of the function with a high rate of change (steep slope), became relatively stable between three and five pellets, and once again became relatively unstable when five to ten pellets were used as the criteria for the start of a meal per unit time. Over the range examined, the time parameter had minimal to no effect on putative meal number, with only negligible changes occurring between 7 and 20 min. On the basis of these pilot observations and similar patterns previously obtained for the rat (16, 33), the criterion of three pellets in 7 min defining a meal was deemed appropriate.

Examination of the pilot data indicated that a widely used criterion for the end of a meal, namely the beginning of the first 20-min time block during a meal in which no additional pellets were consumed (15), provided a stable and reliable definition of the termination of a meal.


   APPENDIX B
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
DISCLOSURES
 REFERENCES
 
The results of solid diet trials were expressed as grams of food eaten because each pellet was entirely or mostly eaten and very little spillage was observed on inspection of the cage bottoms. Therefore, the number of pellets was multiplied by the weight of each pellet (20 mg), and the total weight consumed was calculated.

The same type of calculation could not be performed for the liquid diet because the mice did not invariably drink the entire cup, nor did they always drink the identical amount with each cup. For this reason, we supplemented the measure of number of dipper cups dispensed by manually measuring the total amount of Isocal consumed each test day for each mouse. This was performed by calculating the difference in weight of the diet reservoirs before and after each test session. To account for evaporation from the reservoirs, a separate control tray of Isocal was also weighed before and after each test session and the difference was assumed to be the amount evaporated.

As a sample calculation, the mice on the solid diet ate ~3.5 g of food over a given test session. Multiplying this amount by 3.623 kcal/g, the mice ate ~12.7 kcal. The mice on the liquid diet drank ~16 ml of Isocal over a given test session. Multiplying this volume by 1 kcal/ml, the mice drank ~16 kcal.

Although the numbers are not exact, these are just approximations and estimates based on the means and are shown merely to indicate that the amount of kilocalories consumed by the mice for both solid and liquid diets were similar.


   DISCLOSURES
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-27627.


   ACKNOWLEDGMENTS
 
We thank Drs. R. Meisel and S. Swithers, as well as Dr. R. Phillips and K. Fugo, for comments on earlier drafts of this manuscript. We also thank ML Laboratories for generously providing the devazepide. Finally, we thank Dr. M. Covasa for advice on the use of the devazepide.

A preliminary report of a portion of this work appeared in abstract form at the 32nd annual meeting of the Society for Neuroscience (MM Chi and TL Powley, 2002).


   FOOTNOTES
 

Address for reprint requests and other correspondence: T. L. Powley, Dept. of Psychological Sciences, Purdue Univ., 703 Third St., West Lafayette, IN 47907-2004 (E-mail: powleytl{at}psych.purdue.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 EXPERIMENT 1
 EXPERIMENT 2
 EXPERIMENT 3
 EXPERIMENT 4
 DISCUSSION
 APPENDIX A
 APPENDIX B
 DISCLOSURES
 REFERENCES
 

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