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Ontogeny of glucose inhibition of independent ingestion in preweanling rats
Brain Research Bulletin,
Vol. 17, pp. 673-679, 1986.0 A&ho InternationalInc. printedin the U.S.A.
0361~923tM6 $3.00 + .oo
Ontogeny of Glucose Inhibition of Independent Ingestion in Preweanling Rats CURTIS B. PHIFER, Department
JOSEPH
of Psychology,
A. BROWDE,
JR. AND W. G. HALL
Duke University, Durham, NC 27706
PHIFER, C. B., J. A. BROWDE, JR. AND W. G. HALL. Ontogeny of glucose inhibition of independent ingestion in rats. BRAIN RES BULL 17(S) 673-679, 1!486.-Rat pups that have been maternally and nutritionally deprived will vigorously ingest diet infused directly into the mouth. The development of nutritive controls in this form of
preweanling
iniestion was examined by administering nutritive and non-nutritive gastric preloads to 6- and 15-day-oM pups. In 6 day-old pups, nutritive gastric preloads (0.6 M glucose in distilled Hz0 or saline) and vehicle preloads were followed by similar intakes; only the change in hydrational state caused by distilled HI0 loads appeared to tiect intake. By 15 days of age, intake following nutritive preloads was less than intake following non-nutritive preloads. Also, at 15 days, stomach volume at the termination of intake was less following nutritive preloads. In a separate experiment with 6day-old pups, gastric preloads of an alternative energy source, the ketone b-hydroxybutyrate, also failed to inhibit intake when given at a dose that did not cause excessive gastric distension. These results indicate that a nutritive control of intake termination in rats is not present at 6 days of age but develops by 15 days of age.
Feeding behavior
Satiety
Nutritive control
Intestine
THE ingestive behavior of adult mammals is controlled by a set of central and peripheral mechanisms that interact in complex fashions (e.g, [22,43]). These controls might be more easily understood if they could be studied in a simpler system, particularly if such a system had fewer functional mechanisms. The independent ingestive system of young rat pups may provide such a model system. Ingestion can be studied in infant rats by making oral infusions of diet into their mouths [16,45] or by spreading diet on the floor beneath them. Developmental analysis of this form of ingestion, which occurs away from the mother and suckling, has revealed a system that differs from normal suckling and probably represents the independent function of a separate system that will become the substrate for adult feeding and drinking (8, 11, 191. From birth, this independent ingestion is already adultlike in many ways. Intake volumes are related to length of deprivation 1161; ingestion is suppressed by gastric preloads [ 17,401 and is stimulated by dehydration [6,45]. When young pups terminate their ingestion they show behavioral evidence of a satiety state [ 161 (cf. [2]). Finally, early ingestion appears to have positive consequences because smalI oral infusions of milk in rat pups as young as 1 day of age serve as appetitive reinforcers in both operant and classical conditioning paradigms [26,27]. Despite the early ingestive competence and the anticipatory presence of many components of ingestion (see 1381for a discussion of this developmental phenomenon), the controls of early ingestion are, nonetheless, primitive and immature. In particular, for pups 6 days of age, ingestion
Development
appears to be terminated only by inhibitory signaIs arising from gastric fill. No postgastric or postabsorptive controls seem to contribute to intake termination. The importance of gastric fill was shown indirectly in 6&y-old pups that received nutritive and non-nutritive gastric preloads and then ingested orally-infused diet [ 171. Irrespective of the nutritive content of the load, these pups all stopped ingesting at the same level of gastric fill (though water loads with hydrational consequences did reduce intake and reduce the final level of gastric till). More recently, Phifer, S&es and Hall [40] found that when gastric emptying was prevented by pyloric occlusion, intake in dday-old pups was slightly reduced, but terminated at the same level of gastric fill when either nutritive or non-nutritive solutions (loaded or ingested) were allowed to enter the intestine. Reducing gastric fill by draining stomach contents through an implanted fistula restored ingestion, both in pups with an occluded pylorus and in pups that experienced postgastric stimulation. Postgastric stimulation by a nutritive diet infusion did not cause further inhibition of intake. The absence of postgastric contributions to intake termination by the young rat pup can be contrasted to the potency of postgastric stimulation in inhibiting ingestion by the adult (e.g., [lo, 14,29,42]). Presumably, control mechanisms mature after the first week of life and these render postgastric cues (either pre- or postabsorptive) effective in controlling ingestion. In this study, we compare the effects of nutritive and non-nutritive gastric preioads on ingestion in 6- and 15day-old rat pups. The results suggest the emergence of a nutritive control of ingestion by 15 days of age.
673
PHIFER. BROWDE
674 a. 6-Day-Old Pups
%
6-
x .B
5-
i
4-
c d
3-
3 E
2-
b. I5-Day-Old
AND HALI.
Pups
I-
FIG. 1. Intakes of 6-day-old (a) and U-day-old pups (b) during an ingestion test that began 2 hr following sham gastric loading or loading with 5% b.wt. of either distilled H,O, 0.6 M glucose in distilled H,O, isotonic saline, or 0.6 M glucose in isotonic saline. Values are means-+SEM for 8 .DUDSin each condition in (a) and 7-8 pups in each condition in (b). 1
GENERAL METHOD
Subjects Subjects were the progeny ous Charles River CD strain
of primiparous and multiparrats bred in our laboratory.
Dams were maintained on Purina Formulab Chow No. 5008 and water ad lib. in a colony room kept at 21-23°C and 40-70% relative humidity. Cages were checked daily between 800 and 1700 hours for births and pups found during this time were termed 0 days of age. Litters were culled to 10 pups (5 male and 5 female) at 2 days of age. In all experiments, pups were removed from their mothers on day 5 or 14 and were deprived of food, water and maternal care for 24 hr before testing at 6 or 15 days of age. During deprivation, pups were housed individually in Styrofoam cups (10 cm diameter) in a warm (33-t-05°C) humid (7&90% relative humidity) incubator (Isolette, AirShields, Inc.).
Gastric
Loading
Prior to the administration of gastric loads, pups were stimulated to urinate and defecate by stroking the anogenital region with an artist’s brush. Young pups that are voided do not spontaneously urinate or defecate. Pups were given gavage loads by passing a length of Silastic tubing (0.635 mm ID, 1.19 mm o.d.) down the esophagus and into the stomach [ 171. Then loads equal to 5% of pups’ deprived body weight (b.wt.) were hand-delivered by syringe over approximately 1 min. The solutions used as loads will be described in the methods section for each experiment. Pup weights were taken before loading and at 3-5 min after loading to insure that the intended load volume was delivered and retained. A 2 hr delay between gastric loading and ingestive testing was allowed because a previous study [ 171 revealed that approximately 80% of a 5% b.wt. gastric load of saline leaves the stomach within 2 hr.
Oral cannulas, used for delivering milk infusions, were installed in pups at least 1 hr before testing [ 16,391. Briefly, a IO-cm length of PE-10 tubing with a heat-flared end was friction fitted to an end of a curved piece of stainless steel wire (8 cm long, 0.280 mm diameter). The point of the curved end of the wire was then placed into the pup’s mouth and through the soft floor of the oral cavity just posterior to the lower incisors. The tubing was then drawn through the lower jaw until the flared end was seated against the inner surface of the mouth. During the 30 min ingestive tests, oral infusions of milk (commercial Half and Half; 11% fat, 4.6% carbohydrate, 3.2% protein) were delivered by continuous flow from 5 cc syringes mounted in a Harvard infusion pump (model 975). The infusion rate was 0.25% b.wt. per min (total infusion of 7.5% b.wt. over 30 min, based on average pup weight in a litter). When diet is delivered into the anterior oral cavity in this manner, pups must actively lap and move the diet to the back of the mouth for swallowing or it spills to the floor. Ingestive tests were conducted in clear, plastic containers (7x 12x 17.5 cm) inside a 57-lit glass aquarium with a Plexiglas top. The aquarium was kept warm (32-34°C) and humid with a small fan that blew air over an aquarium heater (100 W) and across a tray of water [ 161. Just prior to testing, pups were again voided by anogenital stroking; therefore intake during testing was accurately reflected in weight gain. After being placed in the test containers, pups were allowed a 5 min adaptation period with no infusion. Then, during the 30 min ingestive tests, the behavior of each pup was recorded during 10 set observation periods at 2 min intervals. General activity was rated from O-6 with 6 being the most active; mouthing movements were scored and any spilIing of milk from the mouth was noted [16]. At the termination of testing, pups usually were sacrificed by ether overdose and stomachs were promptly removed. Stomach volume was estimated by subtracting the weight of
ONTOGENY
OF NUTRITIVE
CONTROLS r.6-Day-OldPups
b.
lb-Day-OldPup
FIG. 2. Stomach content volumes at the termination of intake by &day-old (a) and
15day-old pups (b) following the gastric loading conditions listed under Fig. 1. Values are means* SEM for 8 pupsin each condition in (a) and 6-8 pups in each condition in (b).
the empty stomach
from the weight of the full stomach. In the p~~rnin~ portion of Experiments 1 and 2, animals were sacrificed by decapitation.
Statistical Analysis The results were analyzed by using a randomized-block ANOVA with litters as the experimental unit 171. Differences between individual treatment groups were assessed with Fisher’s Least Significant Difference test (LSD). EXPERIMENT I: GLUCOSE LOADS IN 6- AND IS-DAY-OLD PUPS
In most mammals that have been studied, peripheral glucose administration suppresses food intake and glucose anti-metabolites (e.g., 2-deoxyglucose) increase intake (for review see 1371). As an initial test of nutritive effects on intake in developing rats, 6 and 15day-old pups were given gastric loads of glucose or a vehicle prior to tests of ingestion. Method Intake is inhibited in adult rats that have been given gastric preloads of 1.O M glucose at a load volume of 1.25-2.17% b.wt. [S]. However, there was concern that highly concentrated glucose loads (e.g., 1.0 M) might affect the pups’ intake by changing their hydrational status [6,17]. Gastric emptying might also be dramatically slowed by concentrated loads and induce a strong gastric distension signal. To determine an appropriate con~ntmtion for preload solutions in preweanling pups, a preliminary experiment on gastric emptying of different glucose solutions was conducted with 6 and U-day-old pups. The pups received distilled HzO, 0.6 M glucose in distilled H90, 1.2 M glucose in distilled HZO, or a sham load (gavage tube passed down the esophagus and then removed without delivering a load). At 120 min after loading the pups were sacrificed by decapitation and stomach contents were determined. At the time of sacrifice, the fi-day-old pups that had received 1.2 M glucose loads retained volumes in their stomachs equal to 77.226.0% of the initial load volume, compared to 43.2+7.% for the 0.6 M glucose load,
5,2+-O&Z for the distilled HZ0 load and 3.0+0.6% for the sham load (n=8 per condition). The 15&y-old pups that had received 1.2 M glucose loads retained volumes equal to 55.8254% of the load volume, compared to 32.2+5.8% for the 0.6 M glucose load, 9.42 1.0% for the distilled HZ0 load and 3.8-cO.6% for the sham loads (n=8 per condition). These results and other preliminary observations that 1.2 M glucose loads depressed general activity levels in both 6- and U-day-old pups seemed to indicate that this ~ncen~tion was an inappropriate stimulus for testing nutritive effects. Therefore, in the present experiment, pups received loads of distilled HzO, 0.6 M glucose in distilled HsO, isotonic saline (0.7% NaCl), 0.6 M glucose in saline, or sham load. All solutions were prewarmed to body temperature before loading. Results and Discussion In (i-day-old pups, the only gastric loads that suppressed intake below levels of sham-loaded pups were the distilled HZ0 load and the glucose in distilled HZ0 load (Fig. la, p
b. Stomach Content
a. intake
60
8L
1 ,’
76-
+ .I..
5432-
I OI
____I
I
0
I
0.3
0. I
0.6
GlucoseCM)
FIG. 3. Intakes (a) and stomach content volumes (b) of 6-day-old pups following sham gastric loading or loading with 5% b.wt. of either isotonic saline. 0.1 or 0.3 M /3-hydroxybutyrate in isotonic saline. Values are meanstSEM for 8 pups in each condition.
below sham levels and there was no difference in intakes between pups with distilled H,O and saline vehicle loads or between pups with glucose in distilled H,O and glucose in saline. In both 6- and 15-day-old pups, adding glucose (at the concentrations used here) to the vehicle loads had no significant effect on the general activity of pups during the ingestive tests. None of the load conditions significantly affected the stomach volume reached at the termination of intake in 6-day-old pups [Fig. 2a, F(4,28)= 1SO, NS]. But at 1.5days of age, terminal stomach volumes of pups that received glucose loads in either distilled H,O or saline were significantly smaller than the stomach volumes of pups that received vehicle loads [Fig. 2b, F(4,26)=5.53, pCO.01; glucose vs. vehicle comparisons, ~~0.01, LSD]. This result further supports the difference seen in the intakes; that is, the nutritive value of diet begins to affect intake between 6 and 15 days of age. Despite having less in their stomachs, 15-day-old pups with glucose loads still terminated intake earlier than pups without glucose loads. Therefore, some signal in addition to the level of gastric fill, presumably the postgastric nutritive effects of the load, contributed to intake suppression in ISday-old pups. Stomach volumes at the termination of intake were not significantly affected by either the distilled H,O or the saline vehicle load. EXPERIMENT
2: p-HYDROXYBUTYRATE IN &DAY-OLD PUPS
LOADS
Due to limited synthesis of the glycolytic enzymes phosphofructokinase and pyruvate dehydrogenase in infant rats, ketone-body metabolism provides a larger proportion of brain energy requirements in developing rats than in adults [44]. Indeed, acetoacetate and P-hydroxybutyrate may provide up to 7W% of the brain’s energy requirements in preweanling rats [20]. Since glucose preloads did not appear to affect intake in 6-day-old pups, another experiment was conducted to determine if an alternative energy source,
FIG. 4. Inhibition
of intake in IS-day-old pups due to 5% h.wt. gastric loads of either isotonic saline, 0.1, 0.3 or 0.6 M glucose in isotonic
P-hydroxybutyrate, might act as a nutritive stimulus that would decrease intake in these young pups. Peripheral administration of ketones such as P-hydroxybutyrate can inhibit intake in adult rats [28].
In a preliminary experiment, intake in 6-day-old pups was decreased following a gastric preload of 0.6 M p-hydroxybutyrate given at 5% b.wt. However, the pups that received @-hydroxybutyrate loads ended the ingestive tests with a stomach content volume that was greater than their Intake. ‘l‘lus suggested that a large portion of the preload was still in the stomach at the start of the ingestive test and therefore provided a strong gastric-distension signal. To determine the extent of gastric fill present at the start of ingestion tests, both from a 0.6 M load as well as less concentrated P-hydroxybutyrate loads, pups from 6 litters (n=6) were given preloads of either saline, 0.1, 0.3, or 0.6 M @-hydroxybutyrate in saline, or a sham load. Then, following a 2 hr delay, the pups were decapitated and their stomach contents were determined at the time when they would normally have been given feeding tests. At the time of sacrifice, pups that had received 0.6 M P-hydroxybutyrate had volumes in their stomachs equal to 64.0+5.8% of the initial load volume (only 25.2?4.6% for the 0.3 M solution and 6.2-~0.6% for the 0.1 M solution). Such a degree of stomach fill would likely create a confounding suppression of intake by gastric distension mechanisms [32] and probably be an inappropriate test of postgastric (nutritive) factors. Therefore, for our assessment of the possible nutritive effect of /3-hydroxybutyrate, gastric preloads of 0.1 or 0.3 M /3-hydroxybutyrate in saline, saline, or sham loads were given to pups 2 hr before an ingestion test.
Just
as with
/3-hydroxybutyrate
the
glucose
preloads
failed
loads
in 6-day-old
to suppress
intake
pups, below
ONTOGENY OF NUTRITIVE CONTROLS
671
the levels seen fo~owi~ saline or sham loads [Fig, 3a, F(3,21)=0.55, NS]. Also, none of the load conditions significantly affected stomach volume at the termination of intake [Fig. 3b, F(3,21)=0.97, NS]. Roth the intakes and stomach volumes following P-hydroxybutyrate preloads were in the same range as the intakes and stomach volumes seen following glucose loads in 6&y-old pups (Fig. 1). Therefore, despite evidence that ketones provide a metabolic substrate in young rat pups, this nutrient source does not appear to affect short-term intake, at least as evaluated here. This finding is consistent with the fact that loads of milk have no suppressive effects on intake in young pups other than those related to gastric fill 140’1. EXPERI~E~
3: EFFECT OF GLUCOSE LOAD CONC~~~TION ISDAY-OLD PUPS
IN
The intake-inhibiting effect of the glucose loads in 15day-old pups seen in Experiment 1 could have been due to some non-nutritive effect (e.g., osmotic) of the particular dose (0.6 M) that affected 15day-olds in a way that would not affect younger pups. To examine this possibility another group of 15day-old pups were given varied concentmtions of glucose to evaluate the dose-response relationship between glucose load and intake inhibition. Method
Loads of 5% b.wt, containing 0.1,0.3, or 0.6 M glucose in saline, saline, or a so-loa~ng treatment were each given to 15-day-old rat pups. After a 2 hr delay, the pups were given the same ingestion test used in the previous two experiments. Results and Discussion
The 0.6 M solution was the only glucose load that suppressed intake to a greater degree than the isotonic saline load [Fig. 4, overall load effect, F(4,26)=10.82, p~O.001; saline vs. 0.6 M glucose, pcO.05, LSD]. Also, the 0.6 M glucose load was the only one that resulted in significantly smaller stomach volumes at the termination of ingestion. This result suggests that the threshold dose for glucose loads in 15day-old rat pups appears to lie between 0.3 and 0.6 M. The conclusion that the 0.6 M dose is close to the threshold for the glucose response, together with the observation that this dose did not cause any inhibition of general activity, either in this experiment [F(4,28)= 1.31, NS] or in Experiment 1 [F(4,27)=0.36, NS], suggests that a 0.6 M glucose solution represents a load that is not debilitating or excessively disruptive for 15-day-old pups. Therefore, the intakeinhibiting effect of 0.6 M glucose seen in this experiment and in Experiment I is likely due to a nutritive aspect of the stimulus. A comparison of intake suppression and load volumes was made to determine if intake in the glucose-loaded pups was depressed by an amount equal to the caloric value of the load (i.e., caloric com~nsation). The caloric values of the glucose loads were respectively 0.07,0.215 and 0.43 kcal/ml for the 0.1,0.3 and 0.6 M loads. The intake suppression was calculated by subtracting the volume of milk consumed by pups in each glucose load condition from the amount consumed by the saline-loaded pups and multiplying this result by the caloric value of the milk (1.38 k&/ml). The 0.1 M glucose loads supplied 0.10~0.003 kcal and suppressed intake by 0.11+0.14 kcal; the 0.3 M glucose loads supplied
0.32~0.009 kcal and suppressed intake by 0.15+0.17 kcal; and the 0.6 M glucose loads supplied 0.65-cO.17 kcal and suppressed intake by 0.88ztO.18 kcal. Although intake suppression was greater following loading with solutions of higher caloric value, the poor correlation and large SEM’s suggest that caloric compensation is not exact in pups at this age. GENERAL DISCUSSION The results of these experiments, along with previous findings from our laboratory [ 17,401and others’ (e.g., [30]), demonstrate a major role for gastric fill in the control of independent ingestion by preweanling rat pups. The results also provide further support for the notion that body hydration level and gastric fill are the primary, if not only, controls of intake in very young rat pups (6 days of age). As demonstrated previously by Hall and Bnmo 1171and again in this study, gastric loads of distilled water, which because of their hypotonicity cause cellular rehydration or overhydration, decrease intake in 6-day-old pups that have been deprived for 24 hr. However, adding glucose to the distilled water loads had no additional effect on intake, nor did glucose have an effect when administered in isotonic saline. Thus, the nutritive value of a gastric load does not affect the intake of 6-day-old pups, at least when the nutritive component of the load is glucose or milk [17,40]. Data on stomach volume at the termination of intake indicated that the level of gastric fill totally accounted for intake te~ination (other than hydrational status). Following all treatments, including the sham control, the stomach volume at the termination of intake was approximately 6% of pups’ deprived body weight (b.wt.), with no significant differences between treatments. This result is in close agreement with the earlier finding that approximately 6.5% b.wt, is the level of gastric till that will terminate intake in 6-day-old pups [40]. The slight difference between these two estimates is likely due to a difference in criteria used to indicate te~ination of intake. The sole nutrient source for the suckling mammal is milk. The low carbohydrate to lipid ratio of milk f 1:4), together with the limitations on glycolytic enzymes in neonates force rat pups to depend on lipid-delved metabolites as their primary energy source [20]. However, when the ketone ~-hydroxybutyrate was administered in gastric preloads at a concentration that would not cause excessive gastric distension, this nutrient did not suppress intake. The technical difficulties associated with intravenous infusions in young rat pups precluded a direct circulatory system administration of P-hydroxybutyrate. However, subcutaneous injections of ~-hydroxybuty~te have been shown to increase brain utilization of this nutrient [35]. Further, gastric preloading of the nutrient allowed maximal exposure of both gastric and intestinal epithelium to the nutrient. Thus, we believe this experiment provided an adequate test of the 6-day-old pup’s sensitivity to a second metabolic fuel. By the time pups reach 15 days of age, the nut~tive component of gastric loads begins to make a cont~bution to intake control. Intake was reduced by 50% (relative to sham and vehicle loads) when pups received gastric loads of glucose in either distilled water or isotonic saline vehicles (Fig. 1). Note too, that in contrast to 6-day-olds (in this study and in [ 171) the hypotonicity of gastric loads (distilled water and glucose in distilled water) had no effect on intake; intake following loads of distilled water was equal to intake following loads of saline and intake following glucose in distilled
water was equal to intake following glucose in saline. Pups are known to show developmental changes in their ingestive responsiveness to hydrational status; by 20 days they exhibit the adult-typical dehydration anorexia [6]. These 15day-old pups appear to be in a state of transition with respect to hydrational effects on food intake. Rehydration of dehydrated pups neither decreases nor increases intake. In adults, rehydration releases feeding (e.g., [4, 24, 251). Two processes are likely to be involved in the emergent feeding control of U-day-old pups. First, gastric emptying may have become more sensitive to glucose, either to intestinal or gastric stimulation. Thus, at 15 days, glucose-loaded pups may have shown decreased intake because their stomachs contained more of the loaded solution at the start of ingestion (as demonstrated in the preliminary observations described in the Method section of Experiment 1) and they reached the critical level of gastric fill more quickly. Such a process could still rely on simple gastric fill mechanisms for intake termination but incorporates a nutrientbased modulation of gastric emptying rates (e.g.. 1331). However, the decreased intake is not likely to be due only to a gastric fill signal because the stomach volumes at the termination of intake were smaller in glucose-loaded pups than in vehicle-loaded pups (Fig. 2). Thus, the nutritive effect of loads caused pups to terminate their intake at a lower level of gastric fill (i.e., less of a fill signal was needed). Some type of mechanism other than fill-probably either gastric chemoreceptive, intestinal, or metabolic-must also make a contribution to intake termination in these pups. We favor the latter two possibilities because 70% of the glucose load had left the stomach by the beginning of ingestion. This second mechanism also may partly account for the large difference between 6- and 15-day-old pups in gastric fill at the termination of intake, even in control and vehicle conditions. While both 6- and IS-day-old pups were being exposed to nutritive stimulation from the ingested milk, in 15day-olds the additional nutrritive control would add to that of gastric fill causing intake in all conditions to stop at a lower level of fill than in the 6-day-olds. There are, of course, other developmental changes that may contribute to this difference (e.g., growth and size, gustatory changes, digestive system maturation and metabolic changes). Precise caloric compensation following intragastric loading (i.e., intake suppression in calories equal to caloric value of load) has been demonstrated in rhesus monkeys [3 1,321, but see [ 121. Although compensation in rats in less exact than in monkeys (e.g., [5]), large volumes and high concentrations of gastrically loaded solutions do suppress intake more than small volumes and low concentrations. In this study, 15-day-old pups showed a pattern of intake suppression that
is similar to adult rats. More accurate compensation might have been observed if loads had been delivered just prior to or during the ingestive bouts [ 12,411. Conversely, a more physiologically appropriate gastric load (i.e., predigested) might have caused less suppression of intake 1341. The development of nutritive controls of intake has not been studied previously using an independent ingestion paradigm, i.e., away from the dam and suckling [ 161.But. in studies with suckling rats. nutritive gastric loads have the same effect as non-nutritive loads only until 14 to 20 days 01 age [13,18]. The single exception appears to he galactose loads, which inhibit intake as early as 6 days of age, but this effect may be unique to suckling since it disappears by 21 days of age 1131. Also, subcutaneous injections of glucose do not affect suckling until 17 days of age [36]. Thus. there appears to be comparable development of control in suckling and independent ingestive systems. Glucoprivation by 2-deoxyglucose or phloridzin has no stimulatory effect on intake by suckling rats until approximately 28 days of age 19. 13, 15, 231. In numerous unpublished studies, we have failed to see an effect of glucoprivation on independent ingestion by preweanling rats. Thus, the onset of the control of feeding initiation by blood glucoserelated signals does not appear to occur until about 28 days and may represent yet another emergent system. In summary, the results from studies of suckling and the current studies on independent ingestion indicate that the nutritive value of gastric loads begins to affect intake by the start of the third week of life. Interestingly, the emergence of this control coincides with the development of several other ingestion-related events, including the appearance of digestive enzymes that will be present through adulthood (see review [21]) and the control of intake by opioids (31 and endogenous cholecystokinin [ 11. The development of nutritive inhibition also comes just before the onset of the process of weaning in rats and may provide an additional intakelimiting mechanism when pups eat their first solid food. In younger rat pups, gastric fill and hydrational state appear to be the only controls, at least for independent ingestion. The ontogenetic increase in complexity of intake-terminating cues, as revealed in these early behaviors, provides a model system for further analysis of feeding behavior control.
ACKNOWLEDGEMENTS
We thank D. Kucharski and L. Terry for critical reading of the manuscript, and E. Kenny and K. McCall for technical assistance including figure preparation. This work was supported by NfCHD Grant HD-17457 to W. G. Hall.
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Psycho/
95: 1016-1027, 1981. 7. Denenberg, V. H. Some statistical and experimental considerations in the use of the analysis-of-variance procedure. Am .I Ph.vsio/ 246 (Regul Int Comu Physiol 15): R403-R408, 1984 8. Drewett, R. F. The devetopmenr of munvatlonat systems. Prow Brain Res 48: 407-417, 1978. 9. Drewett, R. F. and K. M. Cordall. Control offeeding in suckling rats: Effects of glucose and of osmotic stimuli. Phwriol Bchu~ 16: 711-717, 1976. 10. Ehman, G. K., D. J. Albert and J. L. Jamieson. Injections into the duodenum and induction of satiety in the rat. Can .I Pxvc-ho/ 25: 147-166, 1971.
ONTOGENY OF NUTRITIVE CONTROLS 11. Epstein, A. N. The ontogeny of ingestive behaviors: Control of milk intake by suckling rats and the emergence of feeding and drinkii at weaning. In: Neural and Humoral Controls ofFood Intake, edited by R. Ritter, S. Ritter and C. D. Barnes. New York: Academic, in press. 12. Foltin, R. W. and C. R. Schuster. Response of monkeys to intragastric preloads: Li~~ons on caloric ~orn~ns~on. Physiof Behav 33: 791-798, 1984. 13. Geiselman, P. J., D. A. Vanderweele, S. M. Dray, A. T. Ewing and E. Cryder-Mooney. Ontogeny of chemospeciflc control of ingestive behavior in the rat. Brain Res Bull 5: Suppl4, 37-42, 1980. 14. Gibbs, J., S. P. Maddison and E. T. Rolls. Satiety role of the small intestine examined in shy-fee~g rhesus monkeys. J Comp Physiui Psychol 95: 1803-1015, 1981. 15. Gisel, E. G. and S. J. Henning. Appearance of glucoprivic control of feeding behavior in the developing rat. Physiol Behav 24: 313-318, 1980. 16. Hall, W. G. The ontogeny of feeding in rats: I. Ingestive and behavioral responses to oral infusions. J Comp Physiol Psycho1 93: 977-1000. 1979. 17. Hall, W. G. and J. P. Bnmo. Inhibitor controls of ingestion in bday-old rat pups. Physiol Behav 32: 831-841, 1984. 18. Hall, W. G. and J. S. Rosenblatt. Development of nutritional controls of food intake in suckling- rat __ DUDS. Behav Biol24: 412427, 1978. 19. Hall, W. G. and C. L. Williams. Suckling isn’t feeding, or is it? A search for developmental continuities. In: Advances in the Study of Behavior, vol 13, edited by J. S. Rosenblatt, R. A. Hinde, C. Beer and M-C. Busnel. New York: Academic, 1983, pp. 21s254. 20. Hawkins, R. A., D. H. Williamson and H. A. Krebs. Ketonebody utilization by adult and suckling rat brain in vivo. Biochem J 122: 13-18, 197i. 21. Henning, S. J. Postnatal development: coordination of feeding, dinestion, and metabolism. Am J Physiol241 (Gustrointest Liver Physiol4j: G199-G214, 1981. 22. Houpt, K. A. Gastrointestinal factors in hunger and satiety. Neurosci Biobehav Rev 6: 145164, 1982. 23. Houpt, K. A and A. N. Epstein. Ontogeny of controls of food intake in the rat: GI fill and glucoprivation. Am J Physiol 225: 58-66, 1973, 24. Hsiao, S. and D. J. Langenes. Liquid intake, initiation and amount of eating as determined by osmolality of drinking Iiquids. Physioi Rehav 7: 233-237, 1971. 25. Hsiao, S. and F. Trankina. Thirst-hunger interaction: I. Effects of body fluid restoration on food and water intake in waterdeprived rats. J Comp Physiol Psycho1 69: 448-453, 1969. 26. Johanson. I. B. and W. G. Hall. Annetitive leamina in l-day-old rat pups. Science 295: 419-421, 1979. 27. Johanson, I. B., W. G. Hall and J. M. Polefrone. Appetitive condition~g in neonatal rats: Conditioned ingestive responding to stimuli paired with oral infusions. Dev Psy~hobiol 17: 357381, 1984.
679 28. Langhans, W., F. Wiesenreiter and E. Scharrer. Different effects of subcutaneous D,L-3-hydroxybutyrate and acetoaeetate injections on food intake in rats. Physiol Behav 31: 483-486, 1983. 29. Liebling, D. S., J. Eisner. 3. Gibbs and G. P. Smith. Intestinal satiety in rats. .I Camp Physiol Psycho1 89~ 955-965, 1975. 30. Lorenz, D. N., S, B. Ellis and A. N. Epstein. Differential effects of upper gastrointestinal fiU on milk ingestion and nipple attachment in the suckling rat. Dev Psychobiof 15: 309-330, 1982. 31. McHugh, P. R. and T. H. Moran. Accuracy of the regulation of caloric intake in the rhesus monkey. Am J Physioi 235 (Regul int Comp Physioi 4): RI&R34, 1978. 32. McHugh, P. R., T. H. Moran and G. N. Barton. Satiety: A graded behavioral phenomenon regulating caloric intake. Science 19th 167-169, 1975. 33. McHugh, P. R., T. H. Moran and J. B. Wirth. Postpyloric regulation of gastric emptying in rhesus monkeys. Am J Physiol243 (Regul Int Comp Physiol 12): R408R415, 1982. 34. Maddison, S. Predigestion and suppression of food intake by gastric loads. Phvsiol Behav 21: 497-501, 1978. 35. Moore, ‘I’. J., A. P. Lione, M. C. Sugden and D. M. Regen. ~-hydroxybuty~te transport in rat brain developmental and dietary modulations. Am .I Physiot 230: 619-630, 1976. 36. Mazes, S., S. Kuchar and J. Koppel. Food intake of suckling rats after administration of glucose and amino acids. Physiol Bohemoslav 32: 460-465, 1983. 37. Novin, D. and D. A. VanderWeele. Visceral involvement in feeding: There is more to regulation than the hydrous. In: Progress in Psy~hobioiogy and Physiological Psyc~fogy, ~017, edited by J. S. Rosenblatt, R. A. Hinde, R. A. Beer and M.-C. Busnel. New York: Academic, 1977, pp. 193-241. 38. Oppenheim, R. W. Ontogenetic adaptations and retrogressive processes in the development of the nervous system and behavior: A neuroembryological perspective. In: Maturation and Development: Biological and Psychological Perspectives, edited by K. J. Connolly and H. R. F. Prechtl. P~~elphia: Lippincott, 1981, pp. 73-109. 39. Phifer, C. B. and W. G. Hall. Development of feeding behavior. In: Methods and Techniques to Study Feeding and Drinking Behavior, edited by F. Toates and N. Rowland. Amsterdam: Elsevier, in press. 40. Phifer, C. B., C. R. Sikes and W. G. Hall. Control of ingestion in dday-old rat pups: Termination of intake by gastric fill alone? Am J Physio~ 258 (Regul fnt Comp Phys~ol 19): R807-R814, 1986. 41. Quartermain, D., H. Kissileff, R. Shapiro and N. E. Miller. Suppression of food intake with intra8astric loading: relation to natural feeding cycle. Science 173: 941-943, 1971. 42. Reidelberger, R. D., T. J. Kalogeris, P. M. B. Lung and V. E. Mendel. Postgastric satiety in the sham-feeding rat. Am J Physiof 244 {Regul int Comp Physioi 13): R872R881, 1983. 43. Rolls, E. T. Central nervous mech~isms related to feeding and appetite. Br Med Bull 37: 131-134, 1981. 44. Stumpf, B. and H. Kraus. Regulatory aspects of glucose and ketone body metabolism in infant rat brain. Pediatr Res 13: 585-590, 1979. 45. Wirth, J. B. and A. N. Epstein. The ontogeny of thirst in infant rats. Am J Physiot 230: 188-198, 1976.
Vol. 17, pp. 673-679, 1986.0 A&ho InternationalInc. printedin the U.S.A.
0361~923tM6 $3.00 + .oo
Ontogeny of Glucose Inhibition of Independent Ingestion in Preweanling Rats CURTIS B. PHIFER, Department
JOSEPH
of Psychology,
A. BROWDE,
JR. AND W. G. HALL
Duke University, Durham, NC 27706
PHIFER, C. B., J. A. BROWDE, JR. AND W. G. HALL. Ontogeny of glucose inhibition of independent ingestion in rats. BRAIN RES BULL 17(S) 673-679, 1!486.-Rat pups that have been maternally and nutritionally deprived will vigorously ingest diet infused directly into the mouth. The development of nutritive controls in this form of
preweanling
iniestion was examined by administering nutritive and non-nutritive gastric preloads to 6- and 15-day-oM pups. In 6 day-old pups, nutritive gastric preloads (0.6 M glucose in distilled Hz0 or saline) and vehicle preloads were followed by similar intakes; only the change in hydrational state caused by distilled HI0 loads appeared to tiect intake. By 15 days of age, intake following nutritive preloads was less than intake following non-nutritive preloads. Also, at 15 days, stomach volume at the termination of intake was less following nutritive preloads. In a separate experiment with 6day-old pups, gastric preloads of an alternative energy source, the ketone b-hydroxybutyrate, also failed to inhibit intake when given at a dose that did not cause excessive gastric distension. These results indicate that a nutritive control of intake termination in rats is not present at 6 days of age but develops by 15 days of age.
Feeding behavior
Satiety
Nutritive control
Intestine
THE ingestive behavior of adult mammals is controlled by a set of central and peripheral mechanisms that interact in complex fashions (e.g, [22,43]). These controls might be more easily understood if they could be studied in a simpler system, particularly if such a system had fewer functional mechanisms. The independent ingestive system of young rat pups may provide such a model system. Ingestion can be studied in infant rats by making oral infusions of diet into their mouths [16,45] or by spreading diet on the floor beneath them. Developmental analysis of this form of ingestion, which occurs away from the mother and suckling, has revealed a system that differs from normal suckling and probably represents the independent function of a separate system that will become the substrate for adult feeding and drinking (8, 11, 191. From birth, this independent ingestion is already adultlike in many ways. Intake volumes are related to length of deprivation 1161; ingestion is suppressed by gastric preloads [ 17,401 and is stimulated by dehydration [6,45]. When young pups terminate their ingestion they show behavioral evidence of a satiety state [ 161 (cf. [2]). Finally, early ingestion appears to have positive consequences because smalI oral infusions of milk in rat pups as young as 1 day of age serve as appetitive reinforcers in both operant and classical conditioning paradigms [26,27]. Despite the early ingestive competence and the anticipatory presence of many components of ingestion (see 1381for a discussion of this developmental phenomenon), the controls of early ingestion are, nonetheless, primitive and immature. In particular, for pups 6 days of age, ingestion
Development
appears to be terminated only by inhibitory signaIs arising from gastric fill. No postgastric or postabsorptive controls seem to contribute to intake termination. The importance of gastric fill was shown indirectly in 6&y-old pups that received nutritive and non-nutritive gastric preloads and then ingested orally-infused diet [ 171. Irrespective of the nutritive content of the load, these pups all stopped ingesting at the same level of gastric fill (though water loads with hydrational consequences did reduce intake and reduce the final level of gastric till). More recently, Phifer, S&es and Hall [40] found that when gastric emptying was prevented by pyloric occlusion, intake in dday-old pups was slightly reduced, but terminated at the same level of gastric fill when either nutritive or non-nutritive solutions (loaded or ingested) were allowed to enter the intestine. Reducing gastric fill by draining stomach contents through an implanted fistula restored ingestion, both in pups with an occluded pylorus and in pups that experienced postgastric stimulation. Postgastric stimulation by a nutritive diet infusion did not cause further inhibition of intake. The absence of postgastric contributions to intake termination by the young rat pup can be contrasted to the potency of postgastric stimulation in inhibiting ingestion by the adult (e.g., [lo, 14,29,42]). Presumably, control mechanisms mature after the first week of life and these render postgastric cues (either pre- or postabsorptive) effective in controlling ingestion. In this study, we compare the effects of nutritive and non-nutritive gastric preioads on ingestion in 6- and 15day-old rat pups. The results suggest the emergence of a nutritive control of ingestion by 15 days of age.
673
PHIFER. BROWDE
674 a. 6-Day-Old Pups
%
6-
x .B
5-
i
4-
c d
3-
3 E
2-
b. I5-Day-Old
AND HALI.
Pups
I-
FIG. 1. Intakes of 6-day-old (a) and U-day-old pups (b) during an ingestion test that began 2 hr following sham gastric loading or loading with 5% b.wt. of either distilled H,O, 0.6 M glucose in distilled H,O, isotonic saline, or 0.6 M glucose in isotonic saline. Values are means-+SEM for 8 .DUDSin each condition in (a) and 7-8 pups in each condition in (b). 1
GENERAL METHOD
Subjects Subjects were the progeny ous Charles River CD strain
of primiparous and multiparrats bred in our laboratory.
Dams were maintained on Purina Formulab Chow No. 5008 and water ad lib. in a colony room kept at 21-23°C and 40-70% relative humidity. Cages were checked daily between 800 and 1700 hours for births and pups found during this time were termed 0 days of age. Litters were culled to 10 pups (5 male and 5 female) at 2 days of age. In all experiments, pups were removed from their mothers on day 5 or 14 and were deprived of food, water and maternal care for 24 hr before testing at 6 or 15 days of age. During deprivation, pups were housed individually in Styrofoam cups (10 cm diameter) in a warm (33-t-05°C) humid (7&90% relative humidity) incubator (Isolette, AirShields, Inc.).
Gastric
Loading
Prior to the administration of gastric loads, pups were stimulated to urinate and defecate by stroking the anogenital region with an artist’s brush. Young pups that are voided do not spontaneously urinate or defecate. Pups were given gavage loads by passing a length of Silastic tubing (0.635 mm ID, 1.19 mm o.d.) down the esophagus and into the stomach [ 171. Then loads equal to 5% of pups’ deprived body weight (b.wt.) were hand-delivered by syringe over approximately 1 min. The solutions used as loads will be described in the methods section for each experiment. Pup weights were taken before loading and at 3-5 min after loading to insure that the intended load volume was delivered and retained. A 2 hr delay between gastric loading and ingestive testing was allowed because a previous study [ 171 revealed that approximately 80% of a 5% b.wt. gastric load of saline leaves the stomach within 2 hr.
Oral cannulas, used for delivering milk infusions, were installed in pups at least 1 hr before testing [ 16,391. Briefly, a IO-cm length of PE-10 tubing with a heat-flared end was friction fitted to an end of a curved piece of stainless steel wire (8 cm long, 0.280 mm diameter). The point of the curved end of the wire was then placed into the pup’s mouth and through the soft floor of the oral cavity just posterior to the lower incisors. The tubing was then drawn through the lower jaw until the flared end was seated against the inner surface of the mouth. During the 30 min ingestive tests, oral infusions of milk (commercial Half and Half; 11% fat, 4.6% carbohydrate, 3.2% protein) were delivered by continuous flow from 5 cc syringes mounted in a Harvard infusion pump (model 975). The infusion rate was 0.25% b.wt. per min (total infusion of 7.5% b.wt. over 30 min, based on average pup weight in a litter). When diet is delivered into the anterior oral cavity in this manner, pups must actively lap and move the diet to the back of the mouth for swallowing or it spills to the floor. Ingestive tests were conducted in clear, plastic containers (7x 12x 17.5 cm) inside a 57-lit glass aquarium with a Plexiglas top. The aquarium was kept warm (32-34°C) and humid with a small fan that blew air over an aquarium heater (100 W) and across a tray of water [ 161. Just prior to testing, pups were again voided by anogenital stroking; therefore intake during testing was accurately reflected in weight gain. After being placed in the test containers, pups were allowed a 5 min adaptation period with no infusion. Then, during the 30 min ingestive tests, the behavior of each pup was recorded during 10 set observation periods at 2 min intervals. General activity was rated from O-6 with 6 being the most active; mouthing movements were scored and any spilIing of milk from the mouth was noted [16]. At the termination of testing, pups usually were sacrificed by ether overdose and stomachs were promptly removed. Stomach volume was estimated by subtracting the weight of
ONTOGENY
OF NUTRITIVE
CONTROLS r.6-Day-OldPups
b.
lb-Day-OldPup
FIG. 2. Stomach content volumes at the termination of intake by &day-old (a) and
15day-old pups (b) following the gastric loading conditions listed under Fig. 1. Values are means* SEM for 8 pupsin each condition in (a) and 6-8 pups in each condition in (b).
the empty stomach
from the weight of the full stomach. In the p~~rnin~ portion of Experiments 1 and 2, animals were sacrificed by decapitation.
Statistical Analysis The results were analyzed by using a randomized-block ANOVA with litters as the experimental unit 171. Differences between individual treatment groups were assessed with Fisher’s Least Significant Difference test (LSD). EXPERIMENT I: GLUCOSE LOADS IN 6- AND IS-DAY-OLD PUPS
In most mammals that have been studied, peripheral glucose administration suppresses food intake and glucose anti-metabolites (e.g., 2-deoxyglucose) increase intake (for review see 1371). As an initial test of nutritive effects on intake in developing rats, 6 and 15day-old pups were given gastric loads of glucose or a vehicle prior to tests of ingestion. Method Intake is inhibited in adult rats that have been given gastric preloads of 1.O M glucose at a load volume of 1.25-2.17% b.wt. [S]. However, there was concern that highly concentrated glucose loads (e.g., 1.0 M) might affect the pups’ intake by changing their hydrational status [6,17]. Gastric emptying might also be dramatically slowed by concentrated loads and induce a strong gastric distension signal. To determine an appropriate con~ntmtion for preload solutions in preweanling pups, a preliminary experiment on gastric emptying of different glucose solutions was conducted with 6 and U-day-old pups. The pups received distilled HzO, 0.6 M glucose in distilled H90, 1.2 M glucose in distilled HZO, or a sham load (gavage tube passed down the esophagus and then removed without delivering a load). At 120 min after loading the pups were sacrificed by decapitation and stomach contents were determined. At the time of sacrifice, the fi-day-old pups that had received 1.2 M glucose loads retained volumes in their stomachs equal to 77.226.0% of the initial load volume, compared to 43.2+7.% for the 0.6 M glucose load,
5,2+-O&Z for the distilled HZ0 load and 3.0+0.6% for the sham load (n=8 per condition). The 15&y-old pups that had received 1.2 M glucose loads retained volumes equal to 55.8254% of the load volume, compared to 32.2+5.8% for the 0.6 M glucose load, 9.42 1.0% for the distilled HZ0 load and 3.8-cO.6% for the sham loads (n=8 per condition). These results and other preliminary observations that 1.2 M glucose loads depressed general activity levels in both 6- and U-day-old pups seemed to indicate that this ~ncen~tion was an inappropriate stimulus for testing nutritive effects. Therefore, in the present experiment, pups received loads of distilled HzO, 0.6 M glucose in distilled HsO, isotonic saline (0.7% NaCl), 0.6 M glucose in saline, or sham load. All solutions were prewarmed to body temperature before loading. Results and Discussion In (i-day-old pups, the only gastric loads that suppressed intake below levels of sham-loaded pups were the distilled HZ0 load and the glucose in distilled HZ0 load (Fig. la, p
b. Stomach Content
a. intake
60
8L
1 ,’
76-
+ .I..
5432-
I OI
____I
I
0
I
0.3
0. I
0.6
GlucoseCM)
FIG. 3. Intakes (a) and stomach content volumes (b) of 6-day-old pups following sham gastric loading or loading with 5% b.wt. of either isotonic saline. 0.1 or 0.3 M /3-hydroxybutyrate in isotonic saline. Values are meanstSEM for 8 pups in each condition.
below sham levels and there was no difference in intakes between pups with distilled H,O and saline vehicle loads or between pups with glucose in distilled H,O and glucose in saline. In both 6- and 15-day-old pups, adding glucose (at the concentrations used here) to the vehicle loads had no significant effect on the general activity of pups during the ingestive tests. None of the load conditions significantly affected the stomach volume reached at the termination of intake in 6-day-old pups [Fig. 2a, F(4,28)= 1SO, NS]. But at 1.5days of age, terminal stomach volumes of pups that received glucose loads in either distilled H,O or saline were significantly smaller than the stomach volumes of pups that received vehicle loads [Fig. 2b, F(4,26)=5.53, pCO.01; glucose vs. vehicle comparisons, ~~0.01, LSD]. This result further supports the difference seen in the intakes; that is, the nutritive value of diet begins to affect intake between 6 and 15 days of age. Despite having less in their stomachs, 15-day-old pups with glucose loads still terminated intake earlier than pups without glucose loads. Therefore, some signal in addition to the level of gastric fill, presumably the postgastric nutritive effects of the load, contributed to intake suppression in ISday-old pups. Stomach volumes at the termination of intake were not significantly affected by either the distilled H,O or the saline vehicle load. EXPERIMENT
2: p-HYDROXYBUTYRATE IN &DAY-OLD PUPS
LOADS
Due to limited synthesis of the glycolytic enzymes phosphofructokinase and pyruvate dehydrogenase in infant rats, ketone-body metabolism provides a larger proportion of brain energy requirements in developing rats than in adults [44]. Indeed, acetoacetate and P-hydroxybutyrate may provide up to 7W% of the brain’s energy requirements in preweanling rats [20]. Since glucose preloads did not appear to affect intake in 6-day-old pups, another experiment was conducted to determine if an alternative energy source,
FIG. 4. Inhibition
of intake in IS-day-old pups due to 5% h.wt. gastric loads of either isotonic saline, 0.1, 0.3 or 0.6 M glucose in isotonic
P-hydroxybutyrate, might act as a nutritive stimulus that would decrease intake in these young pups. Peripheral administration of ketones such as P-hydroxybutyrate can inhibit intake in adult rats [28].
In a preliminary experiment, intake in 6-day-old pups was decreased following a gastric preload of 0.6 M p-hydroxybutyrate given at 5% b.wt. However, the pups that received @-hydroxybutyrate loads ended the ingestive tests with a stomach content volume that was greater than their Intake. ‘l‘lus suggested that a large portion of the preload was still in the stomach at the start of the ingestive test and therefore provided a strong gastric-distension signal. To determine the extent of gastric fill present at the start of ingestion tests, both from a 0.6 M load as well as less concentrated P-hydroxybutyrate loads, pups from 6 litters (n=6) were given preloads of either saline, 0.1, 0.3, or 0.6 M @-hydroxybutyrate in saline, or a sham load. Then, following a 2 hr delay, the pups were decapitated and their stomach contents were determined at the time when they would normally have been given feeding tests. At the time of sacrifice, pups that had received 0.6 M P-hydroxybutyrate had volumes in their stomachs equal to 64.0+5.8% of the initial load volume (only 25.2?4.6% for the 0.3 M solution and 6.2-~0.6% for the 0.1 M solution). Such a degree of stomach fill would likely create a confounding suppression of intake by gastric distension mechanisms [32] and probably be an inappropriate test of postgastric (nutritive) factors. Therefore, for our assessment of the possible nutritive effect of /3-hydroxybutyrate, gastric preloads of 0.1 or 0.3 M /3-hydroxybutyrate in saline, saline, or sham loads were given to pups 2 hr before an ingestion test.
Just
as with
/3-hydroxybutyrate
the
glucose
preloads
failed
loads
in 6-day-old
to suppress
intake
pups, below
ONTOGENY OF NUTRITIVE CONTROLS
671
the levels seen fo~owi~ saline or sham loads [Fig, 3a, F(3,21)=0.55, NS]. Also, none of the load conditions significantly affected stomach volume at the termination of intake [Fig. 3b, F(3,21)=0.97, NS]. Roth the intakes and stomach volumes following P-hydroxybutyrate preloads were in the same range as the intakes and stomach volumes seen following glucose loads in 6&y-old pups (Fig. 1). Therefore, despite evidence that ketones provide a metabolic substrate in young rat pups, this nutrient source does not appear to affect short-term intake, at least as evaluated here. This finding is consistent with the fact that loads of milk have no suppressive effects on intake in young pups other than those related to gastric fill 140’1. EXPERI~E~
3: EFFECT OF GLUCOSE LOAD CONC~~~TION ISDAY-OLD PUPS
IN
The intake-inhibiting effect of the glucose loads in 15day-old pups seen in Experiment 1 could have been due to some non-nutritive effect (e.g., osmotic) of the particular dose (0.6 M) that affected 15day-olds in a way that would not affect younger pups. To examine this possibility another group of 15day-old pups were given varied concentmtions of glucose to evaluate the dose-response relationship between glucose load and intake inhibition. Method
Loads of 5% b.wt, containing 0.1,0.3, or 0.6 M glucose in saline, saline, or a so-loa~ng treatment were each given to 15-day-old rat pups. After a 2 hr delay, the pups were given the same ingestion test used in the previous two experiments. Results and Discussion
The 0.6 M solution was the only glucose load that suppressed intake to a greater degree than the isotonic saline load [Fig. 4, overall load effect, F(4,26)=10.82, p~O.001; saline vs. 0.6 M glucose, pcO.05, LSD]. Also, the 0.6 M glucose load was the only one that resulted in significantly smaller stomach volumes at the termination of ingestion. This result suggests that the threshold dose for glucose loads in 15day-old rat pups appears to lie between 0.3 and 0.6 M. The conclusion that the 0.6 M dose is close to the threshold for the glucose response, together with the observation that this dose did not cause any inhibition of general activity, either in this experiment [F(4,28)= 1.31, NS] or in Experiment 1 [F(4,27)=0.36, NS], suggests that a 0.6 M glucose solution represents a load that is not debilitating or excessively disruptive for 15-day-old pups. Therefore, the intakeinhibiting effect of 0.6 M glucose seen in this experiment and in Experiment I is likely due to a nutritive aspect of the stimulus. A comparison of intake suppression and load volumes was made to determine if intake in the glucose-loaded pups was depressed by an amount equal to the caloric value of the load (i.e., caloric com~nsation). The caloric values of the glucose loads were respectively 0.07,0.215 and 0.43 kcal/ml for the 0.1,0.3 and 0.6 M loads. The intake suppression was calculated by subtracting the volume of milk consumed by pups in each glucose load condition from the amount consumed by the saline-loaded pups and multiplying this result by the caloric value of the milk (1.38 k&/ml). The 0.1 M glucose loads supplied 0.10~0.003 kcal and suppressed intake by 0.11+0.14 kcal; the 0.3 M glucose loads supplied
0.32~0.009 kcal and suppressed intake by 0.15+0.17 kcal; and the 0.6 M glucose loads supplied 0.65-cO.17 kcal and suppressed intake by 0.88ztO.18 kcal. Although intake suppression was greater following loading with solutions of higher caloric value, the poor correlation and large SEM’s suggest that caloric compensation is not exact in pups at this age. GENERAL DISCUSSION The results of these experiments, along with previous findings from our laboratory [ 17,401and others’ (e.g., [30]), demonstrate a major role for gastric fill in the control of independent ingestion by preweanling rat pups. The results also provide further support for the notion that body hydration level and gastric fill are the primary, if not only, controls of intake in very young rat pups (6 days of age). As demonstrated previously by Hall and Bnmo 1171and again in this study, gastric loads of distilled water, which because of their hypotonicity cause cellular rehydration or overhydration, decrease intake in 6-day-old pups that have been deprived for 24 hr. However, adding glucose to the distilled water loads had no additional effect on intake, nor did glucose have an effect when administered in isotonic saline. Thus, the nutritive value of a gastric load does not affect the intake of 6-day-old pups, at least when the nutritive component of the load is glucose or milk [17,40]. Data on stomach volume at the termination of intake indicated that the level of gastric fill totally accounted for intake te~ination (other than hydrational status). Following all treatments, including the sham control, the stomach volume at the termination of intake was approximately 6% of pups’ deprived body weight (b.wt.), with no significant differences between treatments. This result is in close agreement with the earlier finding that approximately 6.5% b.wt, is the level of gastric till that will terminate intake in 6-day-old pups [40]. The slight difference between these two estimates is likely due to a difference in criteria used to indicate te~ination of intake. The sole nutrient source for the suckling mammal is milk. The low carbohydrate to lipid ratio of milk f 1:4), together with the limitations on glycolytic enzymes in neonates force rat pups to depend on lipid-delved metabolites as their primary energy source [20]. However, when the ketone ~-hydroxybutyrate was administered in gastric preloads at a concentration that would not cause excessive gastric distension, this nutrient did not suppress intake. The technical difficulties associated with intravenous infusions in young rat pups precluded a direct circulatory system administration of P-hydroxybutyrate. However, subcutaneous injections of ~-hydroxybuty~te have been shown to increase brain utilization of this nutrient [35]. Further, gastric preloading of the nutrient allowed maximal exposure of both gastric and intestinal epithelium to the nutrient. Thus, we believe this experiment provided an adequate test of the 6-day-old pup’s sensitivity to a second metabolic fuel. By the time pups reach 15 days of age, the nut~tive component of gastric loads begins to make a cont~bution to intake control. Intake was reduced by 50% (relative to sham and vehicle loads) when pups received gastric loads of glucose in either distilled water or isotonic saline vehicles (Fig. 1). Note too, that in contrast to 6-day-olds (in this study and in [ 171) the hypotonicity of gastric loads (distilled water and glucose in distilled water) had no effect on intake; intake following loads of distilled water was equal to intake following loads of saline and intake following glucose in distilled
water was equal to intake following glucose in saline. Pups are known to show developmental changes in their ingestive responsiveness to hydrational status; by 20 days they exhibit the adult-typical dehydration anorexia [6]. These 15day-old pups appear to be in a state of transition with respect to hydrational effects on food intake. Rehydration of dehydrated pups neither decreases nor increases intake. In adults, rehydration releases feeding (e.g., [4, 24, 251). Two processes are likely to be involved in the emergent feeding control of U-day-old pups. First, gastric emptying may have become more sensitive to glucose, either to intestinal or gastric stimulation. Thus, at 15 days, glucose-loaded pups may have shown decreased intake because their stomachs contained more of the loaded solution at the start of ingestion (as demonstrated in the preliminary observations described in the Method section of Experiment 1) and they reached the critical level of gastric fill more quickly. Such a process could still rely on simple gastric fill mechanisms for intake termination but incorporates a nutrientbased modulation of gastric emptying rates (e.g.. 1331). However, the decreased intake is not likely to be due only to a gastric fill signal because the stomach volumes at the termination of intake were smaller in glucose-loaded pups than in vehicle-loaded pups (Fig. 2). Thus, the nutritive effect of loads caused pups to terminate their intake at a lower level of gastric fill (i.e., less of a fill signal was needed). Some type of mechanism other than fill-probably either gastric chemoreceptive, intestinal, or metabolic-must also make a contribution to intake termination in these pups. We favor the latter two possibilities because 70% of the glucose load had left the stomach by the beginning of ingestion. This second mechanism also may partly account for the large difference between 6- and 15-day-old pups in gastric fill at the termination of intake, even in control and vehicle conditions. While both 6- and IS-day-old pups were being exposed to nutritive stimulation from the ingested milk, in 15day-olds the additional nutrritive control would add to that of gastric fill causing intake in all conditions to stop at a lower level of fill than in the 6-day-olds. There are, of course, other developmental changes that may contribute to this difference (e.g., growth and size, gustatory changes, digestive system maturation and metabolic changes). Precise caloric compensation following intragastric loading (i.e., intake suppression in calories equal to caloric value of load) has been demonstrated in rhesus monkeys [3 1,321, but see [ 121. Although compensation in rats in less exact than in monkeys (e.g., [5]), large volumes and high concentrations of gastrically loaded solutions do suppress intake more than small volumes and low concentrations. In this study, 15-day-old pups showed a pattern of intake suppression that
is similar to adult rats. More accurate compensation might have been observed if loads had been delivered just prior to or during the ingestive bouts [ 12,411. Conversely, a more physiologically appropriate gastric load (i.e., predigested) might have caused less suppression of intake 1341. The development of nutritive controls of intake has not been studied previously using an independent ingestion paradigm, i.e., away from the dam and suckling [ 161.But. in studies with suckling rats. nutritive gastric loads have the same effect as non-nutritive loads only until 14 to 20 days 01 age [13,18]. The single exception appears to he galactose loads, which inhibit intake as early as 6 days of age, but this effect may be unique to suckling since it disappears by 21 days of age 1131. Also, subcutaneous injections of glucose do not affect suckling until 17 days of age [36]. Thus. there appears to be comparable development of control in suckling and independent ingestive systems. Glucoprivation by 2-deoxyglucose or phloridzin has no stimulatory effect on intake by suckling rats until approximately 28 days of age 19. 13, 15, 231. In numerous unpublished studies, we have failed to see an effect of glucoprivation on independent ingestion by preweanling rats. Thus, the onset of the control of feeding initiation by blood glucoserelated signals does not appear to occur until about 28 days and may represent yet another emergent system. In summary, the results from studies of suckling and the current studies on independent ingestion indicate that the nutritive value of gastric loads begins to affect intake by the start of the third week of life. Interestingly, the emergence of this control coincides with the development of several other ingestion-related events, including the appearance of digestive enzymes that will be present through adulthood (see review [21]) and the control of intake by opioids (31 and endogenous cholecystokinin [ 11. The development of nutritive inhibition also comes just before the onset of the process of weaning in rats and may provide an additional intakelimiting mechanism when pups eat their first solid food. In younger rat pups, gastric fill and hydrational state appear to be the only controls, at least for independent ingestion. The ontogenetic increase in complexity of intake-terminating cues, as revealed in these early behaviors, provides a model system for further analysis of feeding behavior control.
ACKNOWLEDGEMENTS
We thank D. Kucharski and L. Terry for critical reading of the manuscript, and E. Kenny and K. McCall for technical assistance including figure preparation. This work was supported by NfCHD Grant HD-17457 to W. G. Hall.
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