INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

IT IS GENERALLY ASSUMED that some direct stimulus consequences of the accumulation of ingested fluid in the gastrointestinal tract play a major role in terminating a meal. If this were so, then when postingestional stimulation is eliminated, or minimized (15), by the sham-feeding procedure, intake should increase immediately to its maximum in the first sham-feeding test. Contrary to this expectation, all but one of the studies that measured changes in intake as rats gain sham-feeding experience are consistent in showing that it takes three or more consecutive sham-feeding tests before intake in these tests reaches maximum and stabilizes.

Davis and Campbell (3) reported that when rats were given a series of sham-feeding tests after stabilization of intake in real-feeding tests with either sweetened condensed milk or Vivasorb, a chemically defined diet, it took three to four sham-feeding tests before intake reached maximum. After real- and sham-feeding experience with one of these test solutions, the rats were given real-feeding tests with the other. When real intake had stabilized, the rats were given five sham-feeding tests with the second test solution. The rats showed the same pattern of increasing intake with sham-feeding experience when tested with the second solution. Davis and Campbell suggested that the progressive increase in sham intake reflected the extinction of a conditioned control of intake acquired in preceding real-feeding tests rather than behavioral adaptation to the novelty of sham feeding.

Young et al. (18) reported the same phenomenon in rats sham feeding the chemically defined diet EC116, and Mook et al. (11) showed that it occurred when rats were tested with 2 M glucose as well. Weingarten and Kulikovsky (17) and Davis and Smith (4) also reported that rats tested with 0.8 M sucrose in the sham-feeding procedure required three to four sham-feeding tests before intake reached its maximum. The one exception to this rule of progressive increase in intake with sham-feeding experience is the report by Mook and colleagues (11) that showed when 0.25 M glucose was used as the test stimulus, sham intake reached its maximum in the first sham-feeding test. Because 0.25 M glucose is much less concentrated than any of the other solutions used in the sham-feeding studies, their result raises the possibility that the phenomenon underlying the progressive increase in sham intake with experience depends on the concentration of the test solution.

Recently, Davis et al. (7) reported that rats sham feeding three concentrated solutions of sweetened condensed milk (1:2, 1:1, and 2:1 water-to-milk dilutions) required a minimum of three sham-feeding tests before intake reached its maximum. When tested with more dilute concentrations (4:1, 8:1, and 16:1), however, intake reached its maximum in the first sham-feeding test. In the experiments reported here, we sought to expand on that finding and see if it applied to another type of commonly used test solution. To do so, we used sucrose rather than the more complex mixture of carbohydrate, fat, and protein used in the previous experiment. In this experiment, we measured the intake of rats in five consecutive sham-feeding tests using seven concentrations of sucrose varying on a logarithmic scale from 0.025 to 0.8 M.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Subjects. Male albino rats of the Sprague-Dawley strain (200-300 g) obtained from Taconic Farms, NY, were used in the experiment. They were housed individually in plastic cages and maintained on a 12:12-h light-dark cycle (lights off at 1900) at a temperature of 22 ± 1°C. Except for a single water deprivation period before testing, the rats had ad libitum access to pelleted food (Purina Rat Chow 5001) and tap water. All animals were surgically implanted with a gastric cannula according to the method of Schneider et al. (14) and were allowed 2 wk to recover from the surgery before being tested.

Testing procedures. Testing was carried out in 24-cm-wide by 20-cm-high by 29-cm-deep cages. Before surgery, the rats were trained to drink from a metal tube centered on the front of the cage 15 cm above the floor. On the first training session, the rats were offered water in the drinking tubes after 17 h of water deprivation. If a rat did not drink from the drinking tube during the 30-min. test period, it was tested again on the following day under the same conditions. Most rats learned to approach and drink from the drinking tube on the first test, all by the second. From this point on, the rats were tested at 1300 after a 4-h period of food, but not water, deprivation with only the test solution available in the testing cages. After the test, the rats were returned to their home cages where food and water were available ad libitum until 0900 the following day, when food was again removed from the home cages.

One hundred and five rats were assigned to seven groups of 15 rats for testing each with a different concentration of sucrose. The test solutions, made from reagent-grade sucrose (Sigma, St. Louis, MO), were 0.025, 0.05, 0.1, 0.2, 0.4, 0.6, and 0.8 M. Some of the rats were lost during the experiment because of problems related to the implantation and use of the gastric cannula either during or after surgery. Others rats were rejected because of failure to recover all the fluid ingested from the stomach during a sham-feeding test. A rat was considered to have successfully completed the experiment if at least 100% of the volume ingested was recovered through the fistula by the end of each of the five sham-feeding tests. Of the original 105 rats assigned to the experiment, 21 were eliminated from data analysis for one of these reasons, leaving 84 that completed the real-feeding and five sham-feeding tests successfully. The final group numbers for the seven groups were: 0.025 M, 13; 0.05 M, 12; 0.1 M, 8; 0.2 M, 15; 0.4 M, 12; 0.6 M, 15; and 0.8, 9.

To adapt the rats to the test solution, they were given access to the assigned solution under real-feeding conditions in daily 30-min tests until intake had stabilized. Intake was judged to be stable when there was no more than a 10% variation in intake over three consecutive 30-min tests. This criterion was typically achieved by the fifth test, and, in no case, were more than seven tests required to achieve it. After real-feeding adaptation to the test solution, the rats were given five consecutive sham-feeding tests with it.

In all tests, real and sham, a collecting tube, occluded in real-feeding and open in sham-feeding tests, was attached to the gastric cannula. This collecting tube directed fluid drained from the stomach during the sham-feeding tests to a pan located below the testing cage. At the end of each sham-feeding test, the volume of fluid in the pan was measured.

Data recording and analysis. The test cages were equipped with a computer-controlled lickometer (Henderson, DiLog Instruments, Tallahassee, FL), which passed a current of <60 nA through the rat each time its tongue made contact with the drinking tube. During the test, the time of each tongue contact, to the nearest millisecond, was stored in a data array in the computer memory. At the end of the test, a file of these contact times was created for each rat for later analysis.

Independent-measures ANOVAs were used to analyze the differences in intake across the six groups, and dependent-measures ANOVAs were used to analyze differences across tests within each group. Statistical analysis was carried out with the Systat 7.0 statistical analysis software program, and graphics were created with Sigma Plot for Windows 2.0. Calculations of the microstructure of licking behavior variables were done with the Quick Lick software program (2).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Significantly more sucrose was ingested in the first sham-feeding tests than in the immediately preceding real-feeding tests with each of the seven concentrations [F(1,76) = 158.1, P < 0.001; Fig. 1]. Even with the weakest concentration (0.025 M), where the increase was the smallest (from 5.3 ± 1.2 to 11.0 ± 3.2 ml), significantly more sucrose was ingested in the first sham-feeding test than in the preceding real one [t(12) = 2.0, P = 0.034]. Overall, there was also a statistically significant increase in intake from the first to third sham-feeding test across the seven concentrations [F(1,76) = 36.87, P < 0.001; Fig. 1]. However, in this case, there was a significant interaction between test and concentration [F(6,76) = 6.8, P < 0.001]. The interaction occurred, because, although intake of the three most concentrated solutions increased significantly from the first to the third sham-feeding test [F(1,33) = 49.3, P < 0.001], it did not in the groups of rats tested with the four least concentrated solutions [F(1,43) = 1.8, P = 0.190; Fig. 1].

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Volume ingested in 30-min tests as a function of logarithm of sucrose concentration for last real- and first and third sham-feeding tests.

The volume ingested in the last real- and in the first and third sham-feeding tests depended on the concentration of the test solution (Fig. 1). In the real-feeding tests, there was a significant quadratic trend in the relationship between intake and concentration [F(1,76) = 16.3, P < 0.001]. The largest intakes were stimulated by the 0.1 and 0.2 M solutions (Fig. 1). In the first sham-feeding tests, a quadratic relationship between intake and concentration was also present [F(1,76) = 7.8, P = 0.007], because, although intake increased with concentration over the three lowest concentrations, there was no statistically significant variation in the sham intake of the five highest concentrations (Fig. 1). By the third sham-feeding test, however, the quadratic trend had disappeared [F(1,76) = 0.04] and was replaced by a linear one [F(1,76) = 47.4, P < 0.001; Fig. 1]. This change in the shape of the sham-intake curve occurred because the intakes of the three highest concentrations in the third sham-feeding test were significantly greater than in the first [F(1,35) = 69.1, P < 0.001]. This was not true for the tests with the four weakest concentrations [F(1,46) = 3.3, P = 0.074].

Although the sham intake of the three most concentrated solutions increased significantly with sham-feeding experience, the number of tests required for sham intake to reach maximum varied with concentration (Fig. 2). When tested with 0.4 M sucrose, intake of the rats increased significantly from the first to the second sham-feeding test [F(1,11) = 26.4, P < 0.001] and reached its maximum on that test. After the second sham-feeding test, the largest difference in intake between successive sham-feeding tests was between the second and third sham-feeding test, but this difference was not statistically significant [F(1,11) = 3.1, P = 0.107]. When tested with 0.6 M sucrose, rats showed a significant increase in intake from the first to second sham-feeding test [F(1,14) = 13.81, P = 0.002] and from the second to third [F(1,14) = 4.6, P = 0.050; Fig. 2]. Sham intake had stabilized by then because there were no significant increases in intake after the third sham-feeding test (largest F ratio = 0.7). Similar results were obtained with the 0.8 M solution, where intake was significantly greater on the second than on the first [F(1,8) = 36.8, P < 0.001] and on the third than on the second [F(1,8) = 6.3, P = 0.037] sham-feeding tests. There were no further significant increases in intake after the third sham-feeding test (Fig. 2).

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Volume ingested in last real (R5)- and 5 sham (S1-S5)-feeding tests for 3 most concentrated solutions used.

The licking behavior of rats is organized into bouts of licking at a high rate separated by pauses of varying duration. These bouts of licking are called clusters and are defined as runs of licking separated by pauses of 0.5 s or more (5). In those cases where there were significant increases in intake with increasing sham-feeding experience, the increase could have occurred either because of an increase in the size or number of clusters. In each case, the increase in intake was due exclusively to an increase in the number of clusters [F(2,22) = 17.7, P < 0.001 for 0.4 M; F(2,28) = 23.2, P < 0.001 for 0.6 M; and F(2,16) = 20.3, P < 0.001 for 0.8 M; Table 1]. Cluster size decreased, but not significantly, over the three sham-feeding tests with 0.6 M [F(2,28) = 0.3] and 0.8 M [F(2,16) = 1.9, P = 0.185; Table 2]. With 0.4 M sucrose, cluster size decreased slightly, but significantly, over the three tests [F(2,22) = 3.6, P = 0.045; Table 2].

It has been reported that the progressive increase in sham intake with sham-feeding experience is due, at least in part, to an increase in the rate of licking early in the test (3, 4, 6, 7). To determine if this occurred in this experiment, we analyzed the changes in the number of licks that occurred in the two halves of the first three sham-feeding tests with the three most concentrated solutions (Fig. 3).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   Number of licks in first half (circles) and second half (diamonds) of last real- and first three sham-feeding tests with the 0.8, 0.6, 0.4, 0.2, 0.1, and 0.05 M sucrose concentrations.

With 0.4 M sucrose, there was a significant increase in the number of licks in the first half of the tests across the three tests [F(2,22) = 4.6, P = 0.022]. The statistical significance, however, was due to a significant increase from the first to the second sham-feeding test. The difference between the number of licks in the first halves of the second and third tests was not statistically significant [F(1,11) = 0.02]. With 0.6 M, there was a statistically significant increase in the number of licks in the first half of the first three sham-feeding tests [F(2,28) = 8.7, P = 0.001]. In this case, the increase from the first to second tests was not statistically significant [F(1,14) = 2.5, P = 0.139], but the increase from the second to the third was [F(1,14) = 5.8, P = 0.031]. With 0.8 M sucrose, the overall difference was statistically significant across the three tests [F(2,16) = 27.4 P < 0.001]. With this solution, the increase in the number of licks in the first half of the tests from the first to the second, and from the second to the third, was statistically significant [F(1,8) = 44.3, P < 0.001; F(1,8) = 10.0, P = 0.013, respectively].

The number of licks in the second half of the tests with the three most concentrated solutions also increased with sham-feeding experience (Fig. 3). With 0.4 M sucrose, the overall significant increase [F(2,22) = 5.3, P = 0.013] was due to a significant increase between the first and second test [F(1,11) = 13.4, P = 0.004]. The difference between the second and third test was not significant [F(1,11) = 0.02]. When tested with 0.6 M sucrose, the rats showed a significant overall increase [F(2,28) = 3.7. P = 0.038]. This was due to a significant increase from the first to the second test [F(1,14) = 5.4, P = 0.034]. There was no statistically significant increase from the second to the third test [F(1,14) = 0.3]. With 0.8 M sucrose, the overall significant increase [F(2,16) = 19.9, P < 0.001] was due to a significant increase from the first to the second [F(1,8) = 18.2, P = 0.003] and from the second to third sham-feeding tests [F(1,8) = 5.7, P = 0.044].

To assess the magnitude of the effect of direct and conditioned negative feedback on intake in real-feeding tests, we calculated the difference in intake between the last real- and first and third sham-feeding tests. In both cases, the magnitude of the difference depended on the concentration of sucrose [first sham  real: F(6,76) = 2.3, P = 0.047; third sham  real: F(1,76) = 10.9, P < 0.001; Fig. 4A]. The differences, in both cases, were a significant increasing linear function of the log of sucrose concentration [first sham  real: F(1,76) = 6.0, P = 0.016; third sham  real: F(1,76) = 55.4, P < 0.001; Fig. 4A]. These trends indicate that oropharyngeal stimulation played a significant role in determining the magnitude of the differences. Therefore, to factor out this influence of oropharyngeal stimulation on the differences, we expressed the real intake of each concentration as a percentage of the first and the third sham intakes for each concentration.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   A: difference in volume ingested between last real- and first sham-feeding tests () and third sham-feeding tests () for each concentration. B: real intake expressed as a percentage of the intakes in first sham ()- and third sham-feeding tests () for each sucrose concentration.

Intake in the real-feeding test expressed as a percentage of sham intake in the following sham-feeding test ranged between ~40 and 60% over the entire concentration range (Fig. 4B), but this variation was not statistically significant [F(6,76) = 1.2, P = 0.322]. When expressed as a percentage of intake in the third sham-feeding test, real intake was ~50% of intake in the sham-feeding test with the five least concentrated solutions (Fig. 4B). In those tests with the 0.6 and 0.8 M solutions, it was considerably less, ~30 and 15%, respectively (Fig. 4B). Across the concentration range, this variation was statistically significant [F(6,76) = 2.9, P = 0.001], but the statistical significance was due solely to the values for the 0.6 and 0.8 M concentrations (Fig. 4B). There was no significant effect of concentration on the percentages among the four lowest concentrations [F(3,43) = 0.7; Fig. 4B].

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Our results show that the type of negative feedback that controls the intake of sucrose depends on its concentration. With the four weakest concentrations (0.025, 0.05, 0.1, and 0.2 M), negative feedback was generated solely by some direct consequence of the accumulation of ingested fluid in the gastrointestinal tract. With the three most concentrated solutions, the direct physiological signal operated in conjunction with a second labile negative feedback that disappeared as the rats acquired sham-feeding experience. It has been reported previously that the sham intake of 0.8 M sucrose increases with sham-feeding experience (4, 6, 17). We show here that this effect is not unique to that concentration but occurs also with 0.6 and 0.4 M sucrose as well.

We previously reported a similar finding when rats were tested with various dilutions of sweetened condensed milk (7). In that experiment, the sham intake of all the dilutions of milk used were significantly greater in the first sham-feeding test than in the immediately preceding real-feeding test. With the weak concentrations (16:1, 8:1, 4:1 water-to-milk dilutions), sham intake reached its maximum in the first sham-feeding test. With the three most concentrated solutions (2:1, 1:1, 1:2), sham intake required up to three sham-feeding tests to reach maximum. Thus the mechanisms of negative feedback control of ingestion have now been shown to depend on the concentration of two different types of test solutions, milk and sucrose. In both cases, a simple direct negative-feedback signal based on gastrointestinal filling operates throughout the concentration range. In both cases, an additional labile negative-feedback signal operates at the upper end of the concentration range.

The transition point between those concentrations of sucrose where intake was controlled by a single negative-feedback signal and those where intake was controlled by two was between 0.2 and 0.4 M. Below 0.2 M, real intake increased with concentration; above 0.4 M, real intake decreased with concentration (Fig. 1). It is interesting to note that this transition point on the concentration range corresponds roughly to the traditional peak of the "preference-aversion" function (16) for sucrose (8, 13). The traditional interpretation of the shape of that function is that oropharyngeal stimulation determines the intake of those sucrose solutions that lie below the peak, and negative feedback plays a major role in controlling the intake of those sucrose concentrations that lie above it.

The pattern of increasing intake with increasing concentration in the left limb of the function was interpreted by McCleary (10) and Jacobs (9) as a response to increasing orosensory stimulation provided by the increasing concentration of the solution. Negative feedback was assumed to play little or no role in controlling the intake of those solutions. The decrease in intake with increasing concentration above the peak was interpreted as a response to increasing negative feedback provided by the increasing osmolality of the solution. The magnitude of negative feedback was assumed to grow faster than the stimulating effect of orosensory stimulation beyond the peak, resulting in decreasing intake with increasing concentration. Our results suggest a different interpretation.

With all concentrations of sucrose, intake in the first sham-feeding test was greater than in the preceding real-feeding test. This means that negative feedback stimulated by the accumulation of ingested fluid in the gastrointestinal tract played a significant role in limiting intake in real-feeding tests with all concentrations. In fact, there was no significant variation in real intake from the least to the most concentrated solutions when the influence of oropharyngeal stimulation on real and sham intake was removed by expressing real intake as a percentage of intake in the following sham-feeding test (Fig. 4B). The presence of this direct negative-feedback signal derived from the accumulation of fluid in the gastrointestinal tract suppressed real intake by ~50% regardless of the concentration of the test solution (Fig. 4B).

Above the transition point (concentrations >0.2 M), the decline in intake with concentration is not due directly to the increasing osmolality or nutritive value of the solution, both of which are correlated with concentration. Rather, it is due to the presence of a conditioned negative-feedback signal. Sham intake of all three solutions lying above the transition point in the real intake function (0.4, 0.6, and 0.8 M) increased with sham-feeding experience. It has been demonstrated in a number of studies (3, 4, 17) that this increase in sham intake reflects the extinction of an acquired inhibitory control on ingestion. The higher concentrations required more tests for sham intake to reach maximum. This suggests that the magnitude of the labile negative-feedback signal is proportional to the concentration of the solution. From this point of view, the declining right hand limb of the "preference-aversion" function is seen as a response to increasingly greater conditioned negative feedback, not the increasingly greater magnitude of an unconditioned negative-feedback signal.

It should be noted that the sham intake of the weakest concentration of sucrose used (0.025 M) was greater than in the immediately preceding real-feeding test. This is at odds with results reported by Nissenbaum and Sclafani (12). They reported that the sham intake of 1% sucrose (0.025 M sucrose) was no greater than the real intake of that solution, implying that negative feedback is not involved in the control of the intake of very weak sucrose solutions. Why we found an increase in the sham intake of 0.025 M sucrose and they did not with virtually the same concentration of sucrose is unclear. A difference in the sex of the animals (females were used in their experiment, males in ours), the strain of rats, or some unspecified difference in the testing procedure may be responsible for the discrepancy. Another distinct possibility is that this concentration may be very close to a threshold below which negative feedback does not operate. If this were true, then slight variations in the strain of animals and/or testing conditions could push the results one way or the other.

Previous studies have reported that the increase in intake with sham-feeding experience occurs, at least in part, because of an increase in the rate of licking early in the meal (3, 4, 6). Recently we reported that the same was true when concentrated solutions of milk were used as test solutions. It was also true for the three concentrated solutions used in this study where sham-feeding experience was required for sham intake to reach its maximum. Over the first three sham-feeding tests with the three most concentrated solutions, there was a significant increase in the number of licks in the first half of the tests. There was also an increase in the number of licks in the second halves of those tests. This indicates that the conditioned control of ingestion of these solutions operates not just at the beginning of the meal but throughout it as well.

In summary, the results of this experiment show that the mechanisms controlling the ingestion of sucrose solutions in the rat depend on an interaction between at least three types of stimulus control. These are orosensory stimuli acting to stimulate intake and two different types of negative feedback acting to inhibit it. The control exerted by oropharyngeal stimulation can be seen in the function relating the concentration of sucrose to intake in the third sham-feeding test. In those tests, neither direct physiological nor the labile negative-feedback signals could have played any significant role in limiting intake. Therefore, intake had to have been under the sole influence of the orosensory stimulation. Direct physiological negative feedback inhibited the intake in real-feeding tests of all sucrose concentrations, because, in the first sham-feeding test, when it was no longer present, intake increased significantly with each concentration. With the three most concentrated solutions, a labile negative-feedback signal, primarily based on an association between oropharyngeal stimulation and stimulation from gastrointestinal filling, acted jointly to inhibit intake.

Perspectives