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Naloxone attenuation of sham feeding is modified by manipulation of sucrose concentration
Physiology&Behavior, Vol. 44, pp. 491-494. Copyright©Pergamon Press pie, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
Naloxone Attenuation of Sham Feeding is Modified by Manipulation of Sucrose Concentration I T I M C. K I R K H A M
AND STEVEN
J. C O O P E R
Department o f Psychology, University o f Birmingham, Birmingham BI5 2TT, England
KIRKHAM, T. C. AND S. J. COOPER. Naloxone attenuation of sham feeding is modified by manipulation of sucrose concentration. PHYSIOL BEHAV 44(4/5) 491-494, 1988.--The time course of sucrose (5, I0 and 20%; w/v) sham feeding was monitored in one hour tests. Intake levels increased as a function of concentration. Naloxone (1.25 mg/kg, IP) attenuated the sham feeding of 10% sucrose solution in gastric fistulated rats, without affecting initial intake rates. Furthermore, after naloxone the intake pattern of 10% sucrose was identical to that for 5% sucrose in untreated rats. In a second test, substitution of 10% sucrose by a 20% solution after 15 min of sham feeding reversed the effect of naloxone, restoring intake to 10% baseline levels. Thus naloxone's effect appeared to be behaviourally equivalent to that of sucrose dilution and was counteracted by increasing sucrose concentration. Naloxone was apparently more effective against the lower sucrose concentration, suppressing intake at an earlier stage of testing. The data confirm the importance of oropharyngeal stimulation to the suppressive action of naloxone and support opioid mediation of orosensory reward. Naioxone
Opioids
Sham Feeding
Gastric fistula
E N D O G E N O U S opioid peptides are increasingly implicated in processes regulating ingestive behaviour (2, 3, 15). F o r example, there is some evidence that opioids are released in response to the ingestion of palatable foods (4, 5, 8). Therefore, it is hypothesized that opioids may contribute to the mediation of the rewarding aspects of ingestion, and that underlying the respective hyperphagic or anorectic actions o f opioid agonists and antagonists are changes to the hedonic evaluation of ingesta. Thus, the intake reducing actions of antagonists may be due to a reduction of food or fluid palatability. This is supported by the fact that naloxone and naltrexone markedly attenuate the preference for palatable saccharin or salt solutions in rats (1, 13, 17). F u r t h e r m o r e , the acquisition of saccharin preference can be almost wholly blocked by naloxone treatment given prior to daily two bottle tests (14). In humans, naltrexone has been reported to reduce the perceived pleasantness of sucrose solutions (6), also suggesting that antagonists reduce orosensory reward. That antagonists reduce intake by attenuating the rewarding consequences of gustatory stimuli, rather than by enhancing the postingestional satiating actions of ingesta, is supported by sham feeding experiments. Naloxone will reduce the sham intake o f sucrose solutions in gastric fistulated rats (16), where consumption is driven by the rewarding properties of the sucrose. We have demonstrated (3) that this action involves a dose-dependent and stereospecific interaction with opioid receptors. Such data again suggest that naloxone attenuates intake by reducing the palatability o f
Sucrose
Palatability
Reward
Rats
ingesta. Thus, as sham sucrose intake is directly related to sucrose concentration (19), naloxone may be expected to reduce sham feeding in a manner similar to that produced by dilution of the test solutions. This possibility was examined by monitoring the time course of naloxone suppression of sham sucrose intake, a method previously applied to the analysis of dopamine involvement in food reward (7). Additionally, we have investigated how naioxone's action is modified when sucrose concentration is altered during a sham feeding test. METHOD
Animals Nine male hooded rats (Birmingham General strain), mean weight 200 g on acquisition, were housed singly and maintained under a 12:12 hr light-dark cycle (lights on at 08.30). Except for pretest deprivation and during testing, animals were allowed free access to food and water at all times. All animals acted as their own controls and were tested under each of the conditions described below.
Drugs Naloxone HC1 (Sterling-Winthrop) was dissolved in 0,9% saline and injected IP in a volume of 1 ml/kg body weight, 30 min prior to testing. A single dose of 1.25 mg/kg was used throughout; previous dose-response tests showed this dose
1Proceedings of a conference "Appetite, Thirst and Related Disorders," an official satellite to the 17th Annual Meeting of the Society for Neuroscience, November 12-15, 1987.
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FIG. 1. Cumulative sucrose intake in sham feeding rats. All values are means from nine rats. (a) Baseline intake of 5, 10 and 20% sucrose, h 5%<20%,p<0.01; 2: 10%<20%,p<0.01; 3: 5%< 10%, p<0.05. (b) The effect of 1.25 mg/kg naloxone (NX) on sham intake of 10% sucrose. 4: p<0.05; 5: p<0.01--difference from vehicle intake. (c) Reversal of naloxone's suppressive action by substitution of 10% sucrose by a 20% solution after 15 min of testing (indicated by arrow) 6: difference from vehicle, p<0.05.
to produce a reduction of 1 hour sham sucrose intake equivalent to the difference in intake produced by halving sucrose concentration [(3); Kirkham and Cooper, unpublished data].
Procedure Prior to surgery animals were repeatedly handled and accustomed to injection procedures. Subsequently, each rat was fistulated using previously described surgical procedures (11). Ultra lightweight gastric cannulas, manufactured from acrylic (Perspex) were implanted in the greater curvature of the stomach and exteriorized through abdominal wall and skin. External aluminum screws prevented leakage of stomach contents. After recovery from surgery (approximately 7 days) the experimental series was commenced, according to the following general procedure. On each day, rats were deprived of food from 10.00 hr. At 13.30 hr, fistulas were opened and stomach contents removed by repeated flushing with tepid water. Injections of vehicle (0.9% saline) or naloxone were given immediately prior to this operation. The rats were then placed in test cages (identical to home cages). Stomachs were rinsed once again, immediately before sucrose presentation at 14.00 hr. Solutions (w/v, in distilled water) were presented in calibrated drinking tubes and intake was measured volumetrically, to the nearest 0.5 ml, at 5 min intervals for 1 hr. Drainage from fistulas was collected in trays placed beneath each cage. On completion of each test, stomachs were again rinsed and fistulas resealed. Rats were then returned to their home cages and, 30 min later, food was restored. The criteria for successful sham feeding were those described by other workers (7,12): i.e., that (1) fluid consumed should drain freely within 15 sec and for the whole duration of ingestion, (2) the total volume of drainage should at least equal the volume consumed during testing.
In the first stage of experimentation daily tests continued until a stable baseline for sham intake of 5% sucrose solution was established. Subsequently, the same animals were retested to obtain reliable baselines, successively, for 10% and 20% sucrose solutions. At least two test-free days separated trials with different concentrations. No injections were given during this stage of testing. Having obtained 20% baseline measures, sham feeding tests were resumed with 10% sucrose (in the same animals, following the above procedure) and continued until intake had restabilized (3 days). On the next 2 days sham intake of 10% sucrose was retested after naloxone or saline injection; treatments were reversed on the second day. After 2 nontest days animals were twice allowed to sham feed the 10% solution to ensure the stability of responding. On the 2 subsequent days, rats were retested using a modified procedure. As before, the animals initially sham fed a 10% solution. However, at 15 min this was removed, and for the remainder of the test rats were given access to 20% sucrose. Prior to the first test animals received an injection of either saline or naloxone; the opposite treatment being administered the following day. The effects of sucrose concentration and/or naloxone on sham feeding were analyzed using A N O V A and the Newman-Keuls test for multiple comparisons, for each measurement interval. RESULTS
The combination of open gastric fistulas and palatable sucrose solutions was clearly effective in generating high levels of intake, with the 20% solution maintaining particularly high intake rates throughout the full 1 hr test. Figure l a displays the effect of sucrose concentration upon the cumulative intake of sham feeding rats. As anticipated, cumulative intake increased as a direct function of concen-
NALOXONE AND SHAM FEEDING tration. Initial intake rates were similar for all concentrations, reliable differences became apparent only after 20 min o f continuous intake. However, by 45 min intake curves show marked separation as rate of intake (especially of the 5% solution) began to decline, with consumption o f each concentration being significantly different from each of the others. Total (60 min) intakes for each solution were: 72.22---7.15 ml (5%); 90.33___7.37 ml (10%); 112.83--.9.87 ml (20%). The effect of naloxone on sham intake of 10% sucrose is illustrated in Fig. lb. It is apparent that the drug did not suppress intake immediately: vehicle and naloxone consumption were identical up to 10 min. It should also be noted that the drug did not appear to affect the latency to ingest; in both conditions rats were observed to begin feeding immediately after sucrose presentation. A significant suppressive (rate reducing) effect of naloxone was not observed until the 20 min measurement (p<0.05). This effect persisted for the remainder of the test, so that total intake was reduced to 67.61___11.69 ml, compared to a control intake of 93.44_+ 10.06 ml (p<0.01). It is important to note that naloxone did not completely suppress sham feeding; rats continued to ingest--albeit at a lower rate--until the end o f the test. Rather, comparison between the data displayed in Fig. l a and b reveals that naloxone induced a pattern of intake (of 10% sucrose) very similar to that of these same rats, in the baseline condition, ingesting a 5% sucrose solution. In the third experiment rats received 10% sucrose from 0-15 min and a 20% solution from 15--60 min. After saline, intakes of the 10 and 20% solutions were equivalent to their respective baselines for the appropriate intervals. Thus, the combined 10/20% intake curve (Fig. lc) was identical to the baseline curve for these rats ingesting 20% sucrose throughout the whole test (Fig. la). Total control intake was 110.56_+ 11.49 ml. As in the previous experiment, naloxone did not significantly attenuate intake of 10% sucrose over the first 15 min (Fig. lc). Moreover, increasing the sucrose concentration at the end of that period considerably delayed the onset of naloxone's suppressive action. Control intake levels were maintained for 20 min after concentration substitution. Intake reduction was not apparent until 35--40 min and became significant only after 45 min of sham feeding. Total intake after naloxone was 88.06___ 11.27 ml. The influence of sucrose concentration on the expression o f naloxone's suppressant action can be seen more clearly in Fig. 2. Interestingly, the net effect of naloxone in the 10/20% condition was a cumulative intake pattern equivalent to the baseline curve for 10% sucrose.
DISCUSSION
These results confirm the ability of naloxone to reduce the sham intake of sucrose solutions in gastric fistulated rats (16). And, by extending the analysis to include an examination o f the time course o f drug action in this paradigm, the experiments support the involvement of opioidergic processes in the mediation of orosensory reward. Consistent with earlier findings (9,10), the drug did not block the initiation of sham feeding. Under control conditions fistulated rats displayed a high level o f anticipatory arousal and, when the sucrose solution was presented, immediately approached the drinking spout and began avid consumption. Naloxone did not appear to affect the pretest arousal and had no obvious effect on the latency to approach and ingest the sucrose.
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FIG. 2. Comparison of the effects of naloxone on sham intake when rats were sham fed 10% sucrose alone, or 10% followed by 20% (see Fig. lb and c). 1: p<0.05, 2: p<0.01---difference between naloxoneinduced intake in the two experiments.
Moreover, naloxone did not reduce the rate of intake at the earliest stages of testing. Indeed, naloxone's suppressive action was not evident until rats had sham fed for some considerable time and had ingested large quantities of sucrose. The temporal characteristics of naloxone's suppressant action indicate that the drug influences processes involved in the maintenance, rather than initiation, of intake. More specifically, our results suggest that naloxone attenuates the palatability, or reward, of the sweet taste o f sucrose which excites and maintains ingestion in the sham feeding preparation. As noted earlier, and as our baseline data confirm (Fig. la), the orosensory reward o f sucrose solutions (as indicated by the rate and duration of sham intake) is directly related to their concentration. The present data suggest that naloxone has an effect on sham feeding that is functionally equivalent to a dilution of the test solution. The percent reduction o f 1 hr intake induced by 1.25 mg/kg o f naloxone was very similar to the differences between total baseline intake of 5% and 10%, or between 10% and 20% sucrose; i.e., the net effect o f this dose of naloxone was identical to that produced by halving the concentration of the test solutions. When the cumulative intake curves are compared the similarities between naloxone and dilution effects are even more striking. Thus, after naloxone the rats sham feeding 10% sucrose behaved as they had when consuming a 5% solution under baseline conditions; intake being significantly reduced from 15-20 rain onwards. One might expect, therefore, that this effect of naloxone should be opposed by doubling the concentration of the sucrose, restoring intake to control levels. Indeed, substitution of the 10% solution by 20% sucrose after 15 min of sham feeding counteracted naioxone's action to a significant extent; the consequent cumulative intake curve being very similar to the 10% baseline curve for these animals. It is of course possible to account for these latter results as an inevitable outcome of higher control intake levels, as total intake was suppressed to a similar degree in both exper-
494
K I R K H A M AND COOPER
iments. However, upgrading stimulus intensity in the described manner effectively doubled the latency to the appearance of naloxone's suppressive action (from 20 min for 10% sucrose to 45 min for 10/20% combination). Clearly, therefore, the ability of naloxone to attenuate sham intake involves some interaction with the sensory or hedonic properties of the sucrose. Moreover, it seems unlikely that contrast effects underlie the delayed appearance of naloxone's effect following concentration substitution. As the baseline data show, both 10% and 20% solutions generated similar rates of ingestion over the first half of the test (Fig. la). Therefore, increasing sucrose concentration at 15 min should not be expected to (and did not) cause a sudden upward shift of intake rate. Rather, the substitution had the effect of extending the duration of those early, common and high rates of intake, generating a cumulative intake curve equivalent to that of rats ingesting 20% sucrose throughout the whole test. This relationship between concentration and the persistence of a vigorous sham-feeding response may provide an appropriately parsimonious account of the late onset of naloxone's effect under these conditions. Under control conditions, the latter stages of each shamfeeding test was marked by a gradual decline in the rate of ingestion. The onset of this change was clearly dependent upon sucrose concentration. The more concentrated, and
hence more reinforcing the solution, the longer were high rates maintained before any reduction became apparent. As Fig. 1. indicates, naloxone--in each condition--brought forward the rate attenuation without altering initial levels of ingestion. If a reduction of the reinforcing potency of sucrose underlies this general effect, then we should expect that its onset should also be concentration dependent; i.e., as observed, the latency of the suppressive effect will increase as sucrose concentration is increased. It is possible to predict, therefore, that the onset ofnaloxone's suppressive effect will be advanced as sucrose concentration is diluted (for a given dose), or as dosage is increased (for a given sucrose concentration). More comprehensive analyses of the influence of a wider range of antagonist doses on different sucrose concentrations are required to examine this issue further. In conclusion, these data indicate that the suppressant effect of naloxone in rats sham feeding sucrose solutions is behaviourally equivalent to the effect of concentration dilution. Furthermore, the onset of naloxone's action is dependent upon sucrose concentration, occurring earlier with weaker solutions. This confirms the importance of preabsorptive factors to antagonist anorexia. Overall, the data suggest that naloxone reduces the reward associated with sweet taste and support the hypothesis that opioids contribute to the mediation of the sensory reward of eating and drinking.
REFERENCES 1. Cooper, S. J.; Gilbert, D. B. Naloxone suppresses fluid consumption in tests of choice between sodium chloride solutions and water in male and female rats. Psychopharmacology (Berlin) 84:362-367; 1984. 2. Cooper, S. J.; Jackson, A.; Kirkham, T. C. Endorphins and food intake: kappa opioid receptor agonists and hyperphagia. Pharmacol. Biochem. Behav. 23:889-901; 1985. 3. Cooper, S. J.; Jackson, A.; Kirkham, T. C.; Turkish, S. Endorphins, opiates and food intake. In: Rodgers, R. J.; Cooper, S. J., eds. Endorphins, opiates and behavioural processes. Chichester: John Wiley; 1987:143-186, 4. Dum, J.; Gramsch, C.; Herz, A. Activation of hypothalamic fl-endorphin pools by reward induced by highly palatable food. Pharmacol. Biochem. Behav. 18:443-447; 1983. 5. Dum, J.; Herz, A. Endorphinergic modulation of natural reward systems indicated by behavioural changes. Pharmacol Biochem. Behav. 21:259-266; 1984. 6. Fantino, M.; Hosotte, J.; Apfelbaum, M. An opioid antagonist, naltrexone, reduces preference for sucrose in humans. Am. J. Physiol. 251:R91-R96; 1986. 7. Geary, N.; Smith, G. P. Pimozide decreases the positive reinforcing effect of sham fed sucrose in the rat. Pharmacol. Biochem. Behav. 22:787-790; 1985. 8. Getto, C. J.; Fullerton, D. T.; Carlson, I. H. Plasma immunoreactive beta endorphin response to glucose ingestion in human obesity. Appetite 5:329-335; 1984. 9. Kirkham, T. C.; Blundell, J. E. Dual action of naloxone on feeding revealed by behavioural analysis: separate effects on initiation and termination of eating. Appetite 5:45-52; 1984.
10. Kirkham, T. C.; Blundell, J. E. Effects of naloxone and naltrexone on the development of satiation measured in the runway: Comparisons with d-amphetamine and d-fenfiuramine. Pharacol. Biochem. Behav. 25:123-128; 1986. 11. Kirkham, T. C.; Cooper, S. J. The pyrazoloquinoline, CGS 8216, reduces sham feeding in the rat. Pharmacol. Biochem. Behav. 26:497-501; 1987. 12. Kraly, F. S.; Carty, W. J.; Smith, G. P. Effect ofpregastric food stimuli on meal size and intermeal interval in the rat. Physiol. Behav. 20:779-784; 1978. 13. Lynch, W. C. ; Libby, L. Naloxone suppresses intake of highly preferred saccharin in food deprived and sated rats. Life Sci. 33:1909-1914; 1983. 14. Lynch, W. C. Opiate blockade inhibits saccharin intake and blocks normal preference acquisition. Pharmacol. Biochem. Behav. 24:833-836; 1986. 15. Reid, L. D. Endogenous opioid peptides and regulation of drinking and feeding. Am. J. Clin. Nutr. 42:1099-1132; 1985. 16. Rockwood, G. A.; Reid, L. D. Naloxone modifies sugar-water intake in rats drinking with open gastric fistulas. Physiol. Behav. 29:1175-1178; 1982. 17. Siviy, S. M.; Reid, L. D. Endorphinergic modulation of acceptability of putative reinforcers. Appetite 4:249-257; 1983. 18. Smith, G. P.; Gibbs, J. Postprandial satiety. In: Sprague, J.; Epstein, A., eds. Progress in psychobiology and physiological psychology, vol. 8. New York: Academic Press; 1979:179-242. 19. Weingarten, H. P.; Watson, S. D. Sham feeding as a procedure for assessing the influence of diet palatability on food intake. Physiol. Behav. 28:401--407; 1982.
0031-9384/88 $3.00 + .00
Naloxone Attenuation of Sham Feeding is Modified by Manipulation of Sucrose Concentration I T I M C. K I R K H A M
AND STEVEN
J. C O O P E R
Department o f Psychology, University o f Birmingham, Birmingham BI5 2TT, England
KIRKHAM, T. C. AND S. J. COOPER. Naloxone attenuation of sham feeding is modified by manipulation of sucrose concentration. PHYSIOL BEHAV 44(4/5) 491-494, 1988.--The time course of sucrose (5, I0 and 20%; w/v) sham feeding was monitored in one hour tests. Intake levels increased as a function of concentration. Naloxone (1.25 mg/kg, IP) attenuated the sham feeding of 10% sucrose solution in gastric fistulated rats, without affecting initial intake rates. Furthermore, after naloxone the intake pattern of 10% sucrose was identical to that for 5% sucrose in untreated rats. In a second test, substitution of 10% sucrose by a 20% solution after 15 min of sham feeding reversed the effect of naloxone, restoring intake to 10% baseline levels. Thus naloxone's effect appeared to be behaviourally equivalent to that of sucrose dilution and was counteracted by increasing sucrose concentration. Naloxone was apparently more effective against the lower sucrose concentration, suppressing intake at an earlier stage of testing. The data confirm the importance of oropharyngeal stimulation to the suppressive action of naloxone and support opioid mediation of orosensory reward. Naioxone
Opioids
Sham Feeding
Gastric fistula
E N D O G E N O U S opioid peptides are increasingly implicated in processes regulating ingestive behaviour (2, 3, 15). F o r example, there is some evidence that opioids are released in response to the ingestion of palatable foods (4, 5, 8). Therefore, it is hypothesized that opioids may contribute to the mediation of the rewarding aspects of ingestion, and that underlying the respective hyperphagic or anorectic actions o f opioid agonists and antagonists are changes to the hedonic evaluation of ingesta. Thus, the intake reducing actions of antagonists may be due to a reduction of food or fluid palatability. This is supported by the fact that naloxone and naltrexone markedly attenuate the preference for palatable saccharin or salt solutions in rats (1, 13, 17). F u r t h e r m o r e , the acquisition of saccharin preference can be almost wholly blocked by naloxone treatment given prior to daily two bottle tests (14). In humans, naltrexone has been reported to reduce the perceived pleasantness of sucrose solutions (6), also suggesting that antagonists reduce orosensory reward. That antagonists reduce intake by attenuating the rewarding consequences of gustatory stimuli, rather than by enhancing the postingestional satiating actions of ingesta, is supported by sham feeding experiments. Naloxone will reduce the sham intake o f sucrose solutions in gastric fistulated rats (16), where consumption is driven by the rewarding properties of the sucrose. We have demonstrated (3) that this action involves a dose-dependent and stereospecific interaction with opioid receptors. Such data again suggest that naloxone attenuates intake by reducing the palatability o f
Sucrose
Palatability
Reward
Rats
ingesta. Thus, as sham sucrose intake is directly related to sucrose concentration (19), naloxone may be expected to reduce sham feeding in a manner similar to that produced by dilution of the test solutions. This possibility was examined by monitoring the time course of naloxone suppression of sham sucrose intake, a method previously applied to the analysis of dopamine involvement in food reward (7). Additionally, we have investigated how naioxone's action is modified when sucrose concentration is altered during a sham feeding test. METHOD
Animals Nine male hooded rats (Birmingham General strain), mean weight 200 g on acquisition, were housed singly and maintained under a 12:12 hr light-dark cycle (lights on at 08.30). Except for pretest deprivation and during testing, animals were allowed free access to food and water at all times. All animals acted as their own controls and were tested under each of the conditions described below.
Drugs Naloxone HC1 (Sterling-Winthrop) was dissolved in 0,9% saline and injected IP in a volume of 1 ml/kg body weight, 30 min prior to testing. A single dose of 1.25 mg/kg was used throughout; previous dose-response tests showed this dose
1Proceedings of a conference "Appetite, Thirst and Related Disorders," an official satellite to the 17th Annual Meeting of the Society for Neuroscience, November 12-15, 1987.
491
492
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FIG. 1. Cumulative sucrose intake in sham feeding rats. All values are means from nine rats. (a) Baseline intake of 5, 10 and 20% sucrose, h 5%<20%,p<0.01; 2: 10%<20%,p<0.01; 3: 5%< 10%, p<0.05. (b) The effect of 1.25 mg/kg naloxone (NX) on sham intake of 10% sucrose. 4: p<0.05; 5: p<0.01--difference from vehicle intake. (c) Reversal of naloxone's suppressive action by substitution of 10% sucrose by a 20% solution after 15 min of testing (indicated by arrow) 6: difference from vehicle, p<0.05.
to produce a reduction of 1 hour sham sucrose intake equivalent to the difference in intake produced by halving sucrose concentration [(3); Kirkham and Cooper, unpublished data].
Procedure Prior to surgery animals were repeatedly handled and accustomed to injection procedures. Subsequently, each rat was fistulated using previously described surgical procedures (11). Ultra lightweight gastric cannulas, manufactured from acrylic (Perspex) were implanted in the greater curvature of the stomach and exteriorized through abdominal wall and skin. External aluminum screws prevented leakage of stomach contents. After recovery from surgery (approximately 7 days) the experimental series was commenced, according to the following general procedure. On each day, rats were deprived of food from 10.00 hr. At 13.30 hr, fistulas were opened and stomach contents removed by repeated flushing with tepid water. Injections of vehicle (0.9% saline) or naloxone were given immediately prior to this operation. The rats were then placed in test cages (identical to home cages). Stomachs were rinsed once again, immediately before sucrose presentation at 14.00 hr. Solutions (w/v, in distilled water) were presented in calibrated drinking tubes and intake was measured volumetrically, to the nearest 0.5 ml, at 5 min intervals for 1 hr. Drainage from fistulas was collected in trays placed beneath each cage. On completion of each test, stomachs were again rinsed and fistulas resealed. Rats were then returned to their home cages and, 30 min later, food was restored. The criteria for successful sham feeding were those described by other workers (7,12): i.e., that (1) fluid consumed should drain freely within 15 sec and for the whole duration of ingestion, (2) the total volume of drainage should at least equal the volume consumed during testing.
In the first stage of experimentation daily tests continued until a stable baseline for sham intake of 5% sucrose solution was established. Subsequently, the same animals were retested to obtain reliable baselines, successively, for 10% and 20% sucrose solutions. At least two test-free days separated trials with different concentrations. No injections were given during this stage of testing. Having obtained 20% baseline measures, sham feeding tests were resumed with 10% sucrose (in the same animals, following the above procedure) and continued until intake had restabilized (3 days). On the next 2 days sham intake of 10% sucrose was retested after naloxone or saline injection; treatments were reversed on the second day. After 2 nontest days animals were twice allowed to sham feed the 10% solution to ensure the stability of responding. On the 2 subsequent days, rats were retested using a modified procedure. As before, the animals initially sham fed a 10% solution. However, at 15 min this was removed, and for the remainder of the test rats were given access to 20% sucrose. Prior to the first test animals received an injection of either saline or naloxone; the opposite treatment being administered the following day. The effects of sucrose concentration and/or naloxone on sham feeding were analyzed using A N O V A and the Newman-Keuls test for multiple comparisons, for each measurement interval. RESULTS
The combination of open gastric fistulas and palatable sucrose solutions was clearly effective in generating high levels of intake, with the 20% solution maintaining particularly high intake rates throughout the full 1 hr test. Figure l a displays the effect of sucrose concentration upon the cumulative intake of sham feeding rats. As anticipated, cumulative intake increased as a direct function of concen-
NALOXONE AND SHAM FEEDING tration. Initial intake rates were similar for all concentrations, reliable differences became apparent only after 20 min o f continuous intake. However, by 45 min intake curves show marked separation as rate of intake (especially of the 5% solution) began to decline, with consumption o f each concentration being significantly different from each of the others. Total (60 min) intakes for each solution were: 72.22---7.15 ml (5%); 90.33___7.37 ml (10%); 112.83--.9.87 ml (20%). The effect of naloxone on sham intake of 10% sucrose is illustrated in Fig. lb. It is apparent that the drug did not suppress intake immediately: vehicle and naloxone consumption were identical up to 10 min. It should also be noted that the drug did not appear to affect the latency to ingest; in both conditions rats were observed to begin feeding immediately after sucrose presentation. A significant suppressive (rate reducing) effect of naloxone was not observed until the 20 min measurement (p<0.05). This effect persisted for the remainder of the test, so that total intake was reduced to 67.61___11.69 ml, compared to a control intake of 93.44_+ 10.06 ml (p<0.01). It is important to note that naloxone did not completely suppress sham feeding; rats continued to ingest--albeit at a lower rate--until the end o f the test. Rather, comparison between the data displayed in Fig. l a and b reveals that naloxone induced a pattern of intake (of 10% sucrose) very similar to that of these same rats, in the baseline condition, ingesting a 5% sucrose solution. In the third experiment rats received 10% sucrose from 0-15 min and a 20% solution from 15--60 min. After saline, intakes of the 10 and 20% solutions were equivalent to their respective baselines for the appropriate intervals. Thus, the combined 10/20% intake curve (Fig. lc) was identical to the baseline curve for these rats ingesting 20% sucrose throughout the whole test (Fig. la). Total control intake was 110.56_+ 11.49 ml. As in the previous experiment, naloxone did not significantly attenuate intake of 10% sucrose over the first 15 min (Fig. lc). Moreover, increasing the sucrose concentration at the end of that period considerably delayed the onset of naloxone's suppressive action. Control intake levels were maintained for 20 min after concentration substitution. Intake reduction was not apparent until 35--40 min and became significant only after 45 min of sham feeding. Total intake after naloxone was 88.06___ 11.27 ml. The influence of sucrose concentration on the expression o f naloxone's suppressant action can be seen more clearly in Fig. 2. Interestingly, the net effect of naloxone in the 10/20% condition was a cumulative intake pattern equivalent to the baseline curve for 10% sucrose.
DISCUSSION
These results confirm the ability of naloxone to reduce the sham intake of sucrose solutions in gastric fistulated rats (16). And, by extending the analysis to include an examination o f the time course o f drug action in this paradigm, the experiments support the involvement of opioidergic processes in the mediation of orosensory reward. Consistent with earlier findings (9,10), the drug did not block the initiation of sham feeding. Under control conditions fistulated rats displayed a high level o f anticipatory arousal and, when the sucrose solution was presented, immediately approached the drinking spout and began avid consumption. Naloxone did not appear to affect the pretest arousal and had no obvious effect on the latency to approach and ingest the sucrose.
493 9O 8O 7O
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FIG. 2. Comparison of the effects of naloxone on sham intake when rats were sham fed 10% sucrose alone, or 10% followed by 20% (see Fig. lb and c). 1: p<0.05, 2: p<0.01---difference between naloxoneinduced intake in the two experiments.
Moreover, naloxone did not reduce the rate of intake at the earliest stages of testing. Indeed, naloxone's suppressive action was not evident until rats had sham fed for some considerable time and had ingested large quantities of sucrose. The temporal characteristics of naloxone's suppressant action indicate that the drug influences processes involved in the maintenance, rather than initiation, of intake. More specifically, our results suggest that naloxone attenuates the palatability, or reward, of the sweet taste o f sucrose which excites and maintains ingestion in the sham feeding preparation. As noted earlier, and as our baseline data confirm (Fig. la), the orosensory reward o f sucrose solutions (as indicated by the rate and duration of sham intake) is directly related to their concentration. The present data suggest that naloxone has an effect on sham feeding that is functionally equivalent to a dilution of the test solution. The percent reduction o f 1 hr intake induced by 1.25 mg/kg o f naloxone was very similar to the differences between total baseline intake of 5% and 10%, or between 10% and 20% sucrose; i.e., the net effect o f this dose of naloxone was identical to that produced by halving the concentration of the test solutions. When the cumulative intake curves are compared the similarities between naloxone and dilution effects are even more striking. Thus, after naloxone the rats sham feeding 10% sucrose behaved as they had when consuming a 5% solution under baseline conditions; intake being significantly reduced from 15-20 rain onwards. One might expect, therefore, that this effect of naloxone should be opposed by doubling the concentration of the sucrose, restoring intake to control levels. Indeed, substitution of the 10% solution by 20% sucrose after 15 min of sham feeding counteracted naioxone's action to a significant extent; the consequent cumulative intake curve being very similar to the 10% baseline curve for these animals. It is of course possible to account for these latter results as an inevitable outcome of higher control intake levels, as total intake was suppressed to a similar degree in both exper-
494
K I R K H A M AND COOPER
iments. However, upgrading stimulus intensity in the described manner effectively doubled the latency to the appearance of naloxone's suppressive action (from 20 min for 10% sucrose to 45 min for 10/20% combination). Clearly, therefore, the ability of naloxone to attenuate sham intake involves some interaction with the sensory or hedonic properties of the sucrose. Moreover, it seems unlikely that contrast effects underlie the delayed appearance of naloxone's effect following concentration substitution. As the baseline data show, both 10% and 20% solutions generated similar rates of ingestion over the first half of the test (Fig. la). Therefore, increasing sucrose concentration at 15 min should not be expected to (and did not) cause a sudden upward shift of intake rate. Rather, the substitution had the effect of extending the duration of those early, common and high rates of intake, generating a cumulative intake curve equivalent to that of rats ingesting 20% sucrose throughout the whole test. This relationship between concentration and the persistence of a vigorous sham-feeding response may provide an appropriately parsimonious account of the late onset of naloxone's effect under these conditions. Under control conditions, the latter stages of each shamfeeding test was marked by a gradual decline in the rate of ingestion. The onset of this change was clearly dependent upon sucrose concentration. The more concentrated, and
hence more reinforcing the solution, the longer were high rates maintained before any reduction became apparent. As Fig. 1. indicates, naloxone--in each condition--brought forward the rate attenuation without altering initial levels of ingestion. If a reduction of the reinforcing potency of sucrose underlies this general effect, then we should expect that its onset should also be concentration dependent; i.e., as observed, the latency of the suppressive effect will increase as sucrose concentration is increased. It is possible to predict, therefore, that the onset ofnaloxone's suppressive effect will be advanced as sucrose concentration is diluted (for a given dose), or as dosage is increased (for a given sucrose concentration). More comprehensive analyses of the influence of a wider range of antagonist doses on different sucrose concentrations are required to examine this issue further. In conclusion, these data indicate that the suppressant effect of naloxone in rats sham feeding sucrose solutions is behaviourally equivalent to the effect of concentration dilution. Furthermore, the onset of naloxone's action is dependent upon sucrose concentration, occurring earlier with weaker solutions. This confirms the importance of preabsorptive factors to antagonist anorexia. Overall, the data suggest that naloxone reduces the reward associated with sweet taste and support the hypothesis that opioids contribute to the mediation of the sensory reward of eating and drinking.
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10. Kirkham, T. C.; Blundell, J. E. Effects of naloxone and naltrexone on the development of satiation measured in the runway: Comparisons with d-amphetamine and d-fenfiuramine. Pharacol. Biochem. Behav. 25:123-128; 1986. 11. Kirkham, T. C.; Cooper, S. J. The pyrazoloquinoline, CGS 8216, reduces sham feeding in the rat. Pharmacol. Biochem. Behav. 26:497-501; 1987. 12. Kraly, F. S.; Carty, W. J.; Smith, G. P. Effect ofpregastric food stimuli on meal size and intermeal interval in the rat. Physiol. Behav. 20:779-784; 1978. 13. Lynch, W. C. ; Libby, L. Naloxone suppresses intake of highly preferred saccharin in food deprived and sated rats. Life Sci. 33:1909-1914; 1983. 14. Lynch, W. C. Opiate blockade inhibits saccharin intake and blocks normal preference acquisition. Pharmacol. Biochem. Behav. 24:833-836; 1986. 15. Reid, L. D. Endogenous opioid peptides and regulation of drinking and feeding. Am. J. Clin. Nutr. 42:1099-1132; 1985. 16. Rockwood, G. A.; Reid, L. D. Naloxone modifies sugar-water intake in rats drinking with open gastric fistulas. Physiol. Behav. 29:1175-1178; 1982. 17. Siviy, S. M.; Reid, L. D. Endorphinergic modulation of acceptability of putative reinforcers. Appetite 4:249-257; 1983. 18. Smith, G. P.; Gibbs, J. Postprandial satiety. In: Sprague, J.; Epstein, A., eds. Progress in psychobiology and physiological psychology, vol. 8. New York: Academic Press; 1979:179-242. 19. Weingarten, H. P.; Watson, S. D. Sham feeding as a procedure for assessing the influence of diet palatability on food intake. Physiol. Behav. 28:401--407; 1982.