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Enhanced anorectic potency of naloxone in rats sham feeding 30% sucrose: Reversal by repeated naloxone administration
Physiology & Behavior, Vol. 47, pp. 419-426. ©Pergamon Press plc, 1990. Printed in the U.S.A.
0031-9384/90 $3.00 + .00
Enhanced Anorectic Potency of Naloxone in Rats Sham Feeding 30% Sucrose: Reversal by Repeated Naloxone Administration T I M C. K I R K H A M 1
School o f Psychology, University o f Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. R e c e i v e d 12 June 1989
KIRKHAM, T. C. Enhanced anorectic potency of naloxone in rats sham feeding 30% sucrose: Reversal by repeated naloxone administration. PHYSIOL BEHAV 47(3) 419-426, 1990.--The time-course of naloxone-anorexia was monitored in gastric-fistulated rats sham feeding sucrose (10%, 20%, 30%) solutions. Naloxone reduced sham intake dose dependently without affecting feeding initiation and in a manner which resembled the effects of progressive sucrose dilution. However, when rats sham fed 30% sucrose there was a 2-fold increase in the anorectic potency of naloxone. This exaggerated response was prevented by prior repeated naloxone treatment (5 mg/kg IP, bi-daily), concurrent with the stabilization of sham intake levels (4 days). A further experiment ruled out the possibility that tolerance develops to naloxone effects on this treatment schedule, since intact rats showed a suppression of wet mash consumption following repeated naloxone treatment which was equivalent to an acute naloxone challenge. It is proposed that 1) repeated sucrose sham feeding enhances opioid release and leads to opioid receptor adaptation (down-regulation); 2) repeated (chronic) naloxone treatments have an opposite effect on opioid receptors (up-regulation); 3) the two manipulations, in combination, counteract each other's effects. These behavioural data demonstrate dynamic changes in sham-feeding performance as a function of sucrose concentration and naloxone treatments, reinforce the importance of palatability in naloxone-anorexia, and support opioid involvement in orosensory reward.
Naloxone Anorectic
Sham feeding Opioids
Sucrose
Palatability
Up-regulation
OPIOID receptor antagonists, such as naloxone and naltrexone, will reduce food and water consumption in a wide range of species. These well-established effects, together with the ability of opioid receptor agonists to elevate consumption, indicate that endogenous opioids are crucially involved in the regulation of ingestive behaviours (6, 7, 18). More specifically, it is believed that opioidergic systems contribute to the mediation of orosensory reward, or palatability, of ingesta. According to this hypothesis, then, the anorectic effects of opioid antagonists may be considered to be due to a reduction in the pleasantness of food. A valuable instrument for investigating the influence of palatability on feeding motivation and, thereby, the effects of drugs on palatability, is the sham-feeding paradigm (22,25). By means of gastric fistulation, ingested food--typically, a highly palatable sucrose solution--is retrieved before it can enter and be absorbed from the intestinal tract. Under these conditions, and in the absence of significant deprivation, satiation processes are minimized and intake is motivated solely by the rewarding properties of the sucrose. Sucrose sham feeding is stereospecifically attenuated by low doses of ualoxone (13,20). Moreover, since sham intake is directly related to sucrose concentration, it has been demonstrated that
Down-regulation
Reward
naloxone's effects on sham-feeding motivation mirror those obtained by simple dilution of the test solution (14); i.e., naloxonetreated rats sham feeding a particular sucrose solution behave as if they were consuming a more dilute, less palatable solution. These effects have been replicated across a range of sucrose concentrations and naloxone doses. However, previous work in this laboratory has revealed a particularly interesting anomaly: that is, rats which have been trained to sham feed a highly palatable 30% sucrose solution develop an exaggerated sensitivity to the suppressive effects of naloxone. The reduction of 30% sucrose intake induced by naloxone in these rats (13) is double that obtained with identical doses in animals sham feeding weaker sucrose solutions (14). The studies reported here represent an attempt to investigate these phenomena in more detail. Thus, the first experiment was designed with two objectives. Firstly, to confirm that naloxone can induce behaviour that is identical to the effect of sucrose dilution; i.e., that sucrose sham-feeding patterns after increasing doses of naloxone can be matched to those obtained under control conditions with successively weaker, less palatable sucrose solutions. Secondly, to demonstrate that the apparent increase in naloxone sensitivity seen with very palatable (30%) sucrose solutions is
~Requests for reprints should be addressed to Tim C. Kirkham, E. W. Bourne Behavioral Research Laboratory, The New York Hospital-Comell Medical Center, White Plains, NY 10605.
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replicable, and to more precisely quantify the effect. Consumption of palatable foods or liquids has been associated with endogenous opioid release (3,8). It has also been reported that elevations of brain opioid levels, in vivo, produce concomitant decreases in specific opiate binding, suggesting down-regulation of opioid receptors (1,4). It is possible, therefore, that heightened opioid release induced by repeated consumption of highly palatable sucrose may bring about similar receptor adaptations. Such changes may account for the enhanced anorectic potency of naloxone, as described above. Fewer receptors could facilitate the ability of a given dose of antagonist to compete with endogenous ligands for binding, thus making antagonism more complete and the consequent behavioural response more pronounced. Conversely, chronic administration of opioid antagonists can rapidly induce opioid receptor up-regulation (15, 24, 28, 29). For example, chronic naltrexone treatment causes a maximal (80%) increase in opiate receptor number in rat brain after about 8 days. Half-maximal increases occur after approximately four days; significant elevations are evident as early as the second day of treatment (29). Thus, any receptor adaptation induced by sucrose ingestion could occur as rapidly, within the period required for the facilitation of naloxone-anorexia in the sham-feeding model (approximately 3 days). Moreover, the ability of opioid antagonists to induce receptor up-regulation permits an indirect test of the putative down-regulating consequences of prolonged sucrose ingestion. Thus, in the second experiment, repeated naloxone administration was paired with daily sham feeding of 30% sucrose to determine whether the increase in sensitivity to acute naloxone could be reversed. In Experiment Three, the effects of daily naloxone treatments on the anorectic response to acute naloxone were tested in intact animals feeding a palatable wet mash diet. This test was included to determine whether any changes in sensitivity to naloxone following repeated administration of the drug in the sham-feeding model could be ascribed to factors, such as tolerance, which might occur independently of the ingestion of sucrose solutions.
returned to home cages and, after 30 min, food was restored. Criteria for successful sham-feeding tests were that ingested fluid should drain freely at all times and that the total volume of liquid recovered should not be less than the volume of sucrose ingested.
Drug Naloxone hydrochloride (Du Pont de Nemours & Co.) was dissolved in isotonic saline and injected IP in a volume of 1 ml/kg body weight. Vehicle and naloxone injections were made 30 min prior to testing. In Experiment One, doses of 0.25, 0.5, 1.0 and 2.0 mg/kg naloxone were tested. In Experiment Two, the effects of acute doses of 1.0 and 5.0 mg/kg naloxone on sham feeding were tested after several naloxone (5.0 mg/kg bi-daily) or saline treatments. A single dose of 5.0 mg/kg was tested in Experiment Three. Previous analyses (7, 13, 14) indicated that a dose of 1.0 mg/kg naloxone will reduce 1-hr sham intake of relatively dilute sucrose solutions (_-<20%), equivalent to the difference in intake produced by halving sucrose concentration. Those experiments also showed that doubling the dose of naloxone has an effect on intake akin to a further halving of concentration. For these reasons, in Experiment One, 1.0 mg/kg naloxone was administered to rats sham feeding 10%, 20% and 30% sucrose. Additionally, 20% sham intake was tested after injection of 2.0 mg/kg naloxone. In the 30% sucrose condition, because of the possibility of increased naloxone sensitivity, the effects of 0.25 and 0.5 mg/kg were also assessed. In Experiment Two, the acute dose of 5 mg/kg naloxone was chosen because in a previous experiment (13) it had produced a maximal suppression of 30% sucrose sham feeding and would, therefore, provide "floor" intake values against which the effects of repeated naloxone treatments on the drug's acute anorectic potency could be easily judged. An acute dose of 1.0 mg/kg was used to provide a standard by which the anorectic potency of naloxone could be compared between different conditions, within the experiment, and also with the data from Experiment One, obtained using lower concentrations of sucrose.
GENERALMETHOD
Animals Sixty-four male hooded rats (Birmingham General Strain), weighing between 200-245 g at time of acquisition, were used. Animals were housed singly and maintained under a 12:12 hr light-dark cycle (lights on at 0830 hr). Except for brief pretest food deprivation (in sham-feeding experiments only) and during test periods, rats had free access to the laboratory diet and drinking water.
Statistical Analysis The effects of acute and chronic naloxone treatments on cumulative sham sucrose intake (at each measurement interval) and wet mash consumption were assessed using analysis of variance or, where appropriate, the Student's t-test. Post hoc comparisons, following significant F values, were made with the Newman-Keuls test. EXPERIMENTONE
Surgery and Sham-Feeding Procedure
Method
Twenty-four rats were selected for sham-feeding tests and, after habituation to housing conditions, handling and injection procedures, acrylic gastric cannulas were surgically implanted according to a previously described method (12). Following recovery from surgery, sham-feeding tests began. To facilitate stomach clearance, rats were deprived of food from 1000 hr on each test day. At 1330 hr cannulas were unsealed and stomachs emptied by repeated flushing with tepid water. Animals were then placed in test cages (identical to home cages). To ensure cannulas were free of obstruction, stomachs were rinsed again immediately before sucrose presentation at 1400 hr. Sucrose solutions (w/v, in distilled water) were contained in calibrated drinking tubes. In all tests intake was measured volumetrically, to the nearest 0.5 ml, at 5-min intervals for 1 hr. On completion of each test, stomachs were rinsed once more and the cannulas closed. Animals were
In all stages of this experiment a single group of 8 gastricfistulated rats was tested, receiving all treatments and acting as their own controls. Using the general procedure described above, the rats were successively trained to sham feed 5%, 10%, 20% and 30% sucrose solutions. For each concentration, dally shamfeeding tests were performed to obtain stable baseline intake rates (typically after 3 days). At least 2 nontest days separated trials with different sucrose concentrations. Having achieved stable intake levels of 10% sucrose, rats were retested on 2 consecutive days following saline (day 1) and 1 mg/kg naloxone (day 2). Similarly, sham intake of 20% sucrose was measured after vehicle and 1 mg/kg naloxone. Following a further test with this concentration (to ensure response stability) the procedure was repeated, over a further 2 days, with saline and 2 mg/kg naloxone. As sham intake after each vehicle treatment did not differ significantly, data
ENHANCED NALOXONE ATTENUATION OF SHAM FEEDING
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FIG. 1. Cumulative intake (ml) of sucrose solutions over 1-hr sham-feeding tests, displaying (a) comparison of baseline intakes for each sucrose concentration tested in Experiment One, and the effects of naloxone pretreatment on sham intake of (b) 30%, (c) 20% and (d) 10% sucrose solutions. All values are means for 8 rats (bars indicate S.E.M.). *p<0.05, **p<0.01, difference from respective vehicle condition.
from these days were averaged to provide control values. Sham intake of 30% sucrose was subsequently measured, over a 2-week period, following successive administration of 1.0, 0.5 and 0.25 mg/kg naloxone. As before, each drug treatment was preceded by a vehicle day and intakes averaged to provide control data. After each drug treatment, rats were retested, without injection, on at least 1 day to detect any response deficit and, if necessary, to restore intake to baseline levels. Results and Discussion As can be seen from the comparison of baseline cumulative sucrose intakes (Fig. la), all concentrations of sucrose generated avid sham feeding. It is also apparent that the rate of intake, and hence the volume ingested, increased as a function of increasing sucrose concentration. Total control intakes for the different solutions (mean___ S.E.M.) were: 76.81 --- 10.13 ml (5%); 104.81 ---6.93 ml (10%); 127.81±4.68 ml (20%) and 130.25---6.83 (30% sucrose). The very small difference between intakes of the 20% and 30% solutions suggests that, at these concentrations, animals were ingesting near to their physical (motor) capacity. It is probable that intake patterns would be more easily distinguishable were the test to be prolonged, with high rates of 30% sucrose ingestion persisting for longer than with the weaker solution. In rats sham feeding 10% sucrose, 1 mg/kg naloxone produced a marked attenuation of intake. As in previous experiments (13,14), initial intake rates were unaffected (Fig. ld); significant suppression did not occur until after 15 min (p<0.05) of continuous ingestion. Thereafter, naloxone-treated rats continued to sham feed at a reduced rate, so that 1-hr intake was reduced by 23% to 80.56±8.38 ml, t(7)=3.93, p<0.01. As expected, this reduction was similar in magnitude to the difference (27%) between baseline totals of 10% and 5% sucrose. Moreover, confirming previous observations (14), the cumulative intake pattern of 10% sucrose after naloxone was equivalent to that for the 5% solution under control conditions (Fig. la, d). One-hour sham intake of 20% sucrose was reduced in a
dose-dependent manner by naloxone, F(2,14) = 20.76, p<0.001. Total intake after 1.0 and 2.0 mg/kg was reduced to 98.44___ 8.77 (p<0.01) and 82.94---7.81 ml (p<0.01), respectively. Again, the drug failed to affect intake rate between 0-5 min, F(2,14) = 1.13, NS, or from 5-10 min, F(2,14)=0.74, NS (Fig. lc). Significant suppression became apparent at 15 min, F(2,14) = 6.32, p<0.025, with both 1.0 and 2.0 mg/kg doses reducing intake (p<0.05). Although, after naloxone treatment, rats continued to sham feed throughout the remainder of the test, intake rate was much reduced by the drug and slowed progressively over time. That this effect was more marked with the higher dose is evident from the fact that not only was total intake after 2.0 mg/kg significantly less than the control value, but also reliably less than the 1.0 mg/kg total
(p<0.05). The 1.0 mg/kg dose of naloxone produced a net (23%) reduction of 20% sucrose intake, close to the reduction obtained with this dose when the rats sham fed the 10% solution. In effect, this dose had reduced total intake of 20% sucrose to the 10% control level. Moreover, as can be seen from Fig. 1, the pattern of 20% sucrose intake after 1.0 mg/kg naloxone closely matched that of 10% sucrose--in the same rats--after vehicle. Thus, the behavioural effect of this dose of naloxone was equivalent to that of halving the sucrose concentration. Additionally, the effect of doubling the dose of naloxone was equivalent to the effect of a further halving of the concentration of the test solution (i.e., to a 5% solution). After 2.0 mg/kg naloxone total intake was reduced by 35%, comparable to the difference (39%) between total baseline intakes of 20% and 5% sucrose. Furthermore, the cumulative intake pattern of 20% sucrose following this dose, although less linear, resembled the 5% control curve.
The effects of naloxone on sham intake of 30% sucrose are illustrated in Fig. lb. Total intake was markedly reduced by the two highest doses, F(3,21)=21.18, p<0.001. The lowest (0.25 mg/kg) dose produced only a slight (6%), nonsignificant reduction to 122.25---6.82 ml. However, the absence of more marked suppression may indicate a failure of detection, rather than a lack
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of effect of this dose. Given the close similarity between baseline intakes of the highest sucrose concentrations over 1 hr, it is possible that any effect of this dose would be discerned only if the test were continued for a longer period. Total intakes after 0.5 and 1.0 mg/kg naloxone were reduced to 87.81_+15.62 ml (p<0.01) and 67.25___13.38 ml (p<0.01), respectively. It is clear, therefore, that 1.0 mg/kg naloxone induced a considerably greater reduction (48%) of 30% sucrose than of the 20% or 10% solutions. Indeed, the net (33%) reduction after 0.5 mg/kg, in this condition, exceeded the suppression obtained with 1.0 mg/kg when rats sham fed the weaker sucrose solutions. Thus, when total intake values are compared, the anorectic potency of naloxone in animals sham feeding the 30% sucrose is approximately double that seen with weaker, less palatable, solutions. As in the previous conditions, naloxone reduced total consumption by inducing a marked decline in the rate of sham feeding. Again, there was no effect of the drug at the beginning (0-5 min) of the test, F(3,21)= 2.39, NS. However, 1.0 mg/kg naloxone suppressed intake at an earlier point (10 min; p<0.05) and induced a far more rapid deceleration of intake rate than in the preceding tests. The effects of 0.5 mg/kg naloxone were less marked, with a more gradual decline of intake rate. Although some depression of cumulative intake was apparent as early as 10-15 min, significant (p<0.05) attenuation of sham feeding did not occur until 20-25 min. After each of these treatments, as in the other tests, naloxone did not produce any obvious behavioural disruption. The animals' behaviour was, therefore, entirely compatible with the response of fistulated rats ingesting a more dilute solution in the absence of naloxone, supporting the hypothesis that the drug effectively reduces the palatability of the test solution. Moreover, this experiment has clearly demonstrated that ingestion of a highly palatable, very concentrated sucrose solution can alter the sensitivity of sham-feeding rats to naloxone's anorectic action. The mechanism(s) underlying this alteration are unknown, but it is possible that, as discussed earlier, elevated central opioid activity may induce opioid receptor down-regulation. Several researchers have demonstrated that both naloxone and naltrexone will induce opioid receptor up-regulation, when administered chronically over several days [e.g., (23, 24, 29)]. In Experiment Two, daily antagonist treatment was paired with the acquisition of stable rates of 30% sucrose sham feeding. It was expected that, by assessing the extent to which such treatment affected the response of sham-feeding rats to acute naloxone challenge, some inference concerning the role of receptor changes in increased sensitivity to the antagonist seen in Experiment One could be made. EXPERIMENT TWO
Method
Sixteen naive rats (2 groups of N = 8) were trained to sham feed 30% sucrose, using the same general procedure described above. Daily 1-hr intake tests were performed on each of 5 successive days. Stable baselines were obtained by day 3 for both groups. On day 4 all rats received saline injection before the test to obtain control data. Typically, in studies where up-regulation has been obtained after chronic antagonist administration, the drugs were administered via IV infusion or implantation of osmotic minipumps, usually at a rate of 10 mg/kg/day (15,24). The same daily dosage of naloxone was adopted in this study. However, to avoid interference with the development of high baseline sham intake, the dally dosage was equally split and administered in bi-daily injections.
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FIG. 2. Enhanced anorectic potency of 5 (a) or 1 mg/kg (b) naloxone in rats sham feeding 30% sucrose solution under control conditions and the attenuated effects of these doses following repeated naloxone administration. Circles represent data from rats given repeated saline treatment; squares are values for repeated naloxone treatment. Solid symbols indicate saline pretreatment; open symbols indicate acute naloxone pretreatment. 1: ©
Thus, on days 1-4, rats in group 1 received 5 mg/kg (IP) naloxone at both 0900 and 1700 hr. Over the same period, and at the same times, control animals (group 2) were injected with saline. On day 5, the 0900 injection of naloxone or saline was omitted. Instead, sham intake of 30% sucrose was retested (at 1400) 30 min after pretreatment with 5 mg/kg naloxone. After 2 test-free days, the above procedure was repeated, with naloxone (group 1) or saline (group 2) administration on days 8-11. On day 12, the test of acute effects of naloxone on sham feeding were repeated (as for day 5), but on this occasion each rat was pretreated with 1.0 mg/kg naloxone. Results and Discussion
It is clear from Fig. 2a that naioxone treatment, over days 1-4, did not impair the acquisition of a vigorous sham-feeding response to the 30% sucrose. Total control intakes after daily naioxone (group 1) and saline (group 2) treatments were 108.5_+4.69 and t 11.5 _+8.61 ml, respectively. Naloxone (5 mg/kg) pretreatment on day 5 produced a marked attenuation of 1-hr sham intake in both groups, F(1,14) = 67.27, p<0.001. Moreover, there was a significant interaction between chronic treatment and the magnitude of intake suppression after acute naloxone, F(1,14)= 4.903, p<0.05, with control animals being more sensitive. Total sham intake of the control group was reduced by 70% to 32.56_+ 11.77 ml (p<0.01). This reduction was due to a very marked slowing of intake rate, with a significant reduction already apparent at 10 min (p<0.05). Only 2 out of 8 subjects continued to sham feed for longer than 40 min. Termination of consumption was associated with bouts of grooming and exploratory ambulation, which continued sporadically through the remainder of the test. Although these behaviours are components of the behavioural satiety sequence (22), in this situation there was no occurrence of the resting or sleeping typical of normally satiated rats. It is notable that a similar degree of suppression and pattern of 30% sucrose intake was induced by this dose of naloxone in a previous study (13). In contrast to those extreme effects, total intake of rats in group
ENHANCED NALOXONE ATTENUATION OF SHAM FEEDING
1 was reduced by only 42%, to 63.13+-11.31 ml (p<0.01). Control rates of intake continued for 15 min before any suppression was evident, and cumulative intake was not reliably affected until 30 min (p<0.05). In accordance with the more gradual decline of intake rate in this test, group 1 animals were observed to ingest for longer; 7 out of the 8 rats were still sham feeding after 40 min. The pattern of cumulative intake induced by 5.0 mg/kg naloxone, in these chronically treated rats, was thus more similar to that of 30% sucrose generated in the previous experiment after 1.0 mg/kg naloxone (Fig. lb). Similar, although less-pronounced, results were obtained during the second week of testing (Fig. 2b). Both groups maintained high levels of sham feeding, with control intakes on day 11 being of the same order as on day 4; again there were no differences between total control intake for group 1 (107.63---4.19 ml) and group 2 (115.69+-9.4 ml). On day 12, 1.0 mg/kg naloxone significantly attenuated 1-hr intake of each group, F(1,14)= 25.80, p<0.001. However, the animals which had received daily saline treatment (group 2) were again more sensitive to the actions of the drug. Generally, in group 2, 1.0 mg/kg naloxone produced behavioural effects which resembled those induced by the same dose when rats sham fed 30% sucrose in Experiment One (Fig. la). Total intake was reduced by 45% to 64.0__. 13.75 ml (p<0.01); again, a result of reduced intake rates. Control intake rate was maintained over 0-10 min, with a significant (p<0.05) reduction obtained after 25 min of continuous ingestion. The majority of rats (6 out of 8) were still ingesting at 50 min and half continued to sham feed for the full hour. As before, repeated naloxone administration successfully attenuated the anorectic effect of the 1.0 mg/kg dose. Total 30% sucrose intake of group 1 rats on day 12 was reduced by only 25% to 81.25+- 11.17 ml (p<0.01). Importantly, this degree of suppression was equivalent to that obtained with 1.0 mg/kg in animals sham feeding 20% and 10% sucrose solutions (Fig. lc, d) in Experiment One. Once more, intake suppression involved a gradual decline of intake rate following a period (0-25 min) of normal ingestion: significant (p<0.05) suppression was not evident until after 45 min. This experiment has demonstrated once again that relatively brief periods of sham feeding with 30% sucrose (1 hour on each of 4 successive days) are sufficient to induce extreme sensitivity to the acute anorectic effect of the drug. The potency of naloxone under these present conditions far exceeds that observed in the same paradigm, with weaker sucrose solutions, or in other reports of antagonist effects on food intake. Indeed, in intact, mildly food-deprived rats doses of 60-80 mg/kg of naloxone (which are unlikely to be opioid receptor specific) are required to produce comparable reductions of 1-hr food consumption [unpublished data (9)]. Perhaps more important is the observation that the development of enhanced naloxone sensitivity, as animals stabilize their sham intake of 30% sucrose, can be prevented by concurrent, bi-dally naloxone administration. The present data indicate that this manipulation acts to normalize the suppressive effect of naloxone, so that the acute effects of naloxone are of a similar magnitude to those obtained with animals sham feeding more dilute sucrose solutions. Repeated naloxone administration appears, therefore, to oppose whatever processes induce the increased sensitivity of control rats to acute naloxone. Before these data can be properly considered in terms of the current receptor regulation hypothesis, it is necessary to obtain some further indication that repeated antagonist administration does actually counteract adaptations specifically provoked by ingestion of the 30% sucrose. It is possible that prolonged exposure to naloxone would render animals less sensitive to its
423
TABLE 1 LACKOF EFFECTOF REPEATEDNALOXONEADMINISTRATIONON WETMASHINTAKEAFTERSALINEOR NALOXONEPRETREATMENT Repeated Treatment (Day 1-4)
Acute Treatment(Day 5) Saline
Naloxone
Saline
17.98 (0.93)
9.72 (1.47)
<0.01
Naloxone
16.56 (0.61)
10.45 (1.16)
<0.01
p
N.S.
N.S.
All values are mean (S.E.M.) 30-rain intake (g) for 10 rats (separate groups for each treatment). Significance levels were determined by Newman-Keulstest.
anorectic effect independent of sucrose access, perhaps through the development of tolerance. This possibility was tested in Experiment Three, by subjecting intact rats to the same dally naloxone regime as before and subsequently testing acute effects of the drug on consumption of a highly palatable test diet. EXPERIMENTTHREE Me~od
Forty unoperated rats (as described in the General Method section) were randomly assigned to 4 groups of N = 10. Over 3 successive days all animals were habituated to a palatable wet mash diet. This diet consisted of 150 ml powdered food (rat and mouse expanded ground diet No. 1, Special Diet Services Ltd., Essex, U.K.) and 200 ml water. At 1430 on each day animals received approximately 40 g of freshly prepared mash, in their home cages. After 30 min, any remaining food (plus spillage) was recovered and reweighed. Animals were not deprived prior to these, or subsequent tests. After 3 test-free days the daily dose regime was commenced. Animals in groups 1 and 2 received 5 mg/kg naloxone IP at 0900 and at 1700 (i.e., a total of 10 mg/kg/day) on each of 4 successive days. Groups 3 and 4 were given saline injections at those times, over the same period. On day 5, no morning injection was made. At 1400 groups 1 and 3 were treated with 5 mg/kg IP naloxone. The remaining rats were injected with saline at the same time. At 1430, all rats were presented with a weighed portion of the wet mash. After 30 min the remaining food and spillage were collected and reweighed. Results and Discussion
Wet mash intake had stabilized by day 3 of the habituation stage, with all groups consuming similar quantities. Respective 30-min intakes for groups 1--4 were: 15.03- 1.08, 15.11 -+0.83, 14.6--- 1.07 and 16.38---0.59 g, F(3,36)=0.71, NS. The effects of the separate treatments on day 5 intake are summarized in Table 1. Statistical analysis revealed that naloxone pretreatment reliably reduced wet mash intake of both group 1 and group 3, F(1,36)= 43.66, p<0.001. However, there was no evidence of any interaction between treatment on days 1-4 and naloxone's suppressive effect on day 5, F(1,36)=0.98, NS: repeated naloxone treatment had no effect on either control intakes or acute effects of the drug, F(1,36) =0.10, NS. These data, therefore, provide no evidence that rats become tolerant to the anorectic effects of naloxone with repeated administration (at the dose and time-course used here). Moreover,
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comparison of day 5 intake of the two vehicle groups (groups 2 and 4) failed to reveal any effect of chronic naloxone on normal feeding responses. It is worth noting that although control intakes of the palatable mash were high, these values do not represent the maximum limit to consumption. Thus, this feeding model (unlike the 1-hour sham-feeding tests) would be capable of detecting both positive and negative changes in acceptability (or palatability) of the diet, which might have been induced by multiple naloxone treatments. The absence of any reduction in the anorectic potency of naloxone implies that repeated administrationof the drug, alone, is not sufficient to account for the changes observed in Experiment Two (it will, of course, be necessary to similarly assess the long-term consequences of naloxone on the drug's acute suppressive effect in the sham-feeding model, using more dilute sucrose solutions). Overall, these results tend to consolidate the "normalization" hypothesis discussed above. In the absence of conditions (e.g., sham feeding 30% sucrose) which induce increased sensitivity to naloxone, bi-daily antagonist administration does not, by itself, bring about any alteration in an animal's anorectic response. The implications of this will be discussed more fully in the following section. GENERALDISCUSSION The results of the present experiments add to a growing literature which relates opioid processes to the control of ingestive behaviours and, more particularly, to the mediation of food palatability or orosensory reward (18). As discussed previously, it is believed that a reduction in the palatability of ingesta underlies the anorectic effects of opioid antagonists. The changes in shamfeeding behaviour (which is predominantly motivated by the rewarding properties of sucrose) following naloxone administration are wholly consistent with this hypothesis. Confirming the results of earlier studies (13,14), in Experiment One the effects of naloxone were behaviourally equivalent to those obtained by a dilution of the test solution. Thus, the cumulative sham intake curves in naloxone-treated rats were, qualitatively and quantitatively, very similar to those obtained with weaker sucrose solutions under control conditions. Moreover, in each case, naloxone failed to exert any influence on the initiation of sham feeding. This is an important observation as, in agreement with earlier reports (10,11), it indicates that naloxone interferes with neural processes activated by ingestion, rather than anticipation, of the sucrose. This, in turn, suggests that opioids are not directly involved in initiating ingestive behaviour, but may have a positive role in maintaining consumption once begun. As food palatability is a major determinant of the maintenance of consumption, the fact that motivational effects of naloxone occur only at later stages of testing again supports a role of opioids in the mediation of orosensory reward. The importance of palatability factors to the anorectic effects of naloxone is further indicated by the exaggerated response to the drug in animals sham feeding 30% sucrose. In Experiment One, baseline intake of 30% sucrose was only marginally greater than that of the 20% solution which suggests that concentration (stimulus intensity), rather than volume consumed, is the crucial factor. So far, this enhanced anorectic potency has only been obtained with 30% sucrose, although the absolute concentration threshold remains to be determined. The onset of this change occurs very rapidly; increased naloxone sensitivity can be obtained after only 2 days (1 hr/day) of sham feeding with the solution. It remains to be determined whether similar effects may be obtained with more dilute solutions over a longer period of training. In rats, ingestion of palatable, sweet solutions has been reported to stimulate opioid release within the brain, correlated
behaviourally with the induction of naltrexone-reversible analgesia (3,8). As the data in Fig. la demonstrate, the rate, duration and volume of sham sucrose intake is a direct function of the palatability of the test solutions. If, as is proposed, activation of opioid systems underlies the rewarding effects of ingestion, it is possible that opioid activity will also be directly related to sucrose concentration. In this light, the magnified response to naioxone in rats sham feeding the most palatable solution may be due to some neural adaptation induced by chronically elevated levels of endogenous opioids. It has been found that long-term exposure to opioid peptides, in vitro, induces a reduction in the number of opiate receptor-binding sites (2,16). There is also evidence to indicate that such downregulation may occur in vivo, following manipulations known to stimulate the release of endogenous ligands for these receptors. For example, 72-hr food deprivation, which has been associated with increased hypothalamic beta-endorphin levels (19), produced a significant reduction of 3H-naloxone-binding sites in rat hypothalamus and medulla oblongata (1). Also in rats, increased levels of neurohypophyseal dynorphin following chronic dehydration were correlated with marked reductions in specific 3H-bremazocine binding in the neurohypophysis, suggesting a down-regulation of kappa receptors (4). These data suggest that down-regulation of receptors at opioidergic synapses may be a homeostatic, regulatory response to overstimulation by increased levels of endogenous ligand (4). It is possible, therefore, that the opioid release induced during repeated sessions of sham feeding with 30% sucrose may also be of sufficient magnitude to provoke opioid receptor down-regulation. This, in turn, may explain the enhanced anorectic potency of naloxone under these same conditions. A given dose of naloxone could more completely antagonize opioid actions in a downregulated system than might the same dose in an unadapted system, competing more effectively with an endogenous ligand for occupation of a reduced number of opioid receptor-binding sites; the net behavioural effect being equivalent to that of a higher dose of naloxone under other conditions. Such an account would be compatible with the established ability of chronic naloxone or naltrexone administration to induce opioid receptor up-regulation, and with the results of Experiment Two. Chronic antagonist treatment has been shown to increase the number of brain mu, delta and kappa opiate receptors [e.g., (17,29)]. These changes appear to be specific to opioid systems. Up-regulation is associated with the development of functional supersensitivity to the effects of opioid receptor agonists, both in vivo and in vitro (21, 23, 24, 27, 28), but without any apparent change in sensitivity to serotonin, epinephrine (21), benzodiazepines (28), or to muscarinic ligands (29). It is important to note that the doses of naloxone used in this experiment have also been shown to interact stereospecifically with opioid receptors (13). As reported above, in this study repeated naloxone treatment appeared to normalize the anorectic response of animals sham feeding 30% sucrose. When chronic antagonist administration was paired with daily 30% sucrose sham feeding, the magnified response to acute naloxone seen under control conditions was abolished. The degree of intake suppression was thus no greater than would be expected had these same doses been administered to animals sham feeding less palatable solutions. If the observed increase in naloxone sensitivity can be ascribed to a regulatory response to increased opioid levels, the restoration (or maintenance) of more typical levels of anorexia with repeated naloxone may represent the consequences of a counter-regulatory effect of the drug. According to this hypothesis, then, the up-regulation generated by prolonged naloxone blockade of opioid receptors could oppose the putative down-regulation produced by sucroseinduced opioid release. In line with the data from Experiment
ENHANCED NALOXONE ATTENUATION OF SHAM FEEDING
Two, these opposing influences could result in an effective stabilization of basal receptor number and, hence, a "normal" level of naloxone sensitivity. An effect of repeated naloxone administration to stabilize opioid receptor number, rather than to induce a net increase, may help to account for the lack of effect of this treatment on baseline levels of 30% sucrose sham intake (in Experiment Two). Similar baselines were achieved and maintained irrespective of whether animals received daily saline or naloxone treatment. As mentioned earlier, chronic antagonist-induced up-regulation is associated with a supersensitivity to agonist effects. A marked increase in receptor number might, therefore, be predicted to increase susceptibility to endogenous ligands (29). This, in turn, could lead to an enhanced feeding response as a result of increased food palatability. It is, of course, possible that some enhancement of palatability might have resulted from the chronic naloxone treatment, but remained undetected due to the already maximal levels of sham intake with very concentrated sucrose solutions. However, a similar lack of effect of daily naloxone on control intake was found in tests with the palatable wet mash in Experiment Three. In the absence of conditions likely to provoke down-regulation, the tendency for repeated naloxone to induce up-regulation (rather than stabilization) should have been unopposed in this test. For reasons discussed earlier, wet mash intake in this model would have been sensitive to intake-enhancing, as well as -attenuating, effects of specific treatments. Possibly, a within-groups design might have detected some change. Thus, although these latter data suggest that repeated naloxone treatments fail to exert any significant influence upon the response of rats to palatable foods, it is important to reexamine this possibility, using other paradigms, before more concrete conclusions can be made. More generally, this account of the effects observed in the present studies must be considered as largely speculative. It is imperative, therefore, to replicate these results in combination with a detailed analysis of the effects of sham sucrose feeding and chronic naloxone treatment on opioid binding. This form of analysis is especially important to the hypothesized stabilization of receptor number following chronic naloxone, Although up-regulation with chronic antagonist treatments is now well documented, it has been argued (21) that continuous exposure to high drug concentrations (using implanted pellets or osmotic minipumps) is
425
required. However, it has been reported that C57 mice display a marked increase in brain opiate receptor density after bi-daily naloxone (1 mg/kg, IP) injections, with significant elevations apparent after only 2 days (5). Similarly rapid modification with this type of treatment cannot, therefore, be discounted in the rat. Nevertheless, bi-daily naloxone injections, in this study, were clearly effective in altering the anorectic response of sham-feeding animals. It will be interesting to see whether similar behavioural effects can be obtained with continuous naloxone infusion. As the dose-schedule in this experiment was chosen to avoid any suppression of intake during acquisition of a stable sham-feeding response, great care will be required in the design of such experiments to control for similar confounding factors. In conclusion, these experiments have confirmed the utility of the sham-feeding paradigm in the analysis of drug effects on palatability factors. Naloxone (at doses known to act stereospecifically at opioid receptors) attenuated sham intake of 10% and 20% in a manner which mimicked the behavioural effects of sucrose dilution. Emphasizing the importance of palatability to naloxone's effects, animals sham feeding 30% sucrose exhibited an enhanced (doubled) anorectic response to naloxone. This increased anorectic potency was prevented by pairing repeated naloxone administration with daily sham-feeding sessions: the degree of suppression after acute naloxone was restored to levels observed when animals sham fed weaker sucrose solutions. In intact rats, tolerance failed to develop to naloxone's effects on palatable food intake with daily drug treatment. It is hypothesized that repeated and prolonged ingestion of highly palatable solutions may induce sufficient release of endogenous opioids to cause down-regulation of opioid receptors. Further, repeated antagonist treatment may oppose any such ingestion-related adaptations. Although specific binding studies are necessary to assess the validity of this account, these data do provide further evidence for the involvement of opioidergic processes in the mediation of food palatability, and are indicative of neural plasticity in those systems responsible for the regulation of ingestive behaviours. AC~O~E~E~S The author is grateful to David Barber for assistance with surgery and data collection and to colleagues Jo Neill, Mike Clark and Steven Cooper for their help during preparation of the manuscript.
REFERENCES 1. Battistini, N.; Giardino, L.; Calza, L. Opiate receptor autoradiography in fasting rats. Int. J. Obes. 11($3):17-21; 1987. 2. Blanchard, S. G.; Chang, K.-J.; Cuatrecacas, P. Characterization of the association of tritiated enkephalin with neuroblastoma cells under conditions optimal for receptor down regulation. J. Biol. Chem. 258:1092-1097; 1983. 3. Blass, E. M.; Fitzgerald, E.; Kehoe, P. Interactions between sucrose, pain and isolation distress. Pharmacol. Biochem. Behav. 26:483-489; 1987. 4. Brady, L. S.; Herkenham, M. Dehydration reduces k-opiate receptor binding in the neurohypophysis of the rat. Brain Res. 425:212-217; 1987. 5. Brunello, N.; Volterra, A.; Di Giulio, A. M.; Cuomo, V.; Racagni, G. Modulation of opioid system in C57 mice after repeated treatment with morphine and naloxone: biochemical and behavioral correlates. Life Sci. 34:1669-1678; 1984. 6. 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. 7. 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; 1988:143-186.
8. Dum, J.; Gramsch, C.; Herz, A. Activation of hypothalamic [3endorphin pools by reward induced by highly palatable food. Pharmacol. Biochem. Behav. 18:443--447; 1983. 9. Kirkham, T. C. Investigation of opioid mechanisms in the control of feeding behaviour. Unpublished Ph.D. thesis, University of Leeds; 1985. 10. 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. 11. 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. Pharmacol. Biochem. Behav. 25:123-128; 1986. 12. Kirkham, T. C.; Cooper, S. J. The pyrazoloquinoline, CGS 8216, reduces sham feeding in the rat. Pharmacol. Biochem. Behav. 26:497-501; 1987. 13. Kirkham, T. C.; Cooper, S. J. Attenuation of sham feeding by naloxone is stereospecific: Evidence for opioid mediation of orosensory reward. Physiol. Behav. 43:845-848; 1988. 14. Kirkham, T. C.; Cooper, S. J. Naloxone attenuation of sham feeding is modified by manipulation of sucrose concentration. Physiol. Behav. 44:491-494; 1988. 15. Lahti, R. A.; Collins, R. J. Chronic naloxone results in prolonged
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16. 17. 18. 19. 20. 21. 22.
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increases in opiate binding sites in brain. Eur. J. Pharmacol. 51: 185-186; 1978. Lenoir, D.; Barg, J.; Simantov, R. Characterization and downregulation of opiate receptors in aggregating fetal rat brain cells. Brain Res. 304:285-290; 1984. Paden, C. M.; Krall, S.; Lynch, W. C. Heterogeneous distribution and upregulation of mu, delta and kappa opioid receptors in the amygdala. Brain Res. 418:349-355; 1987. Reid, L. D. Endogenous opioid peptides and regulation of feeding and drinking. Am. J. Clin. Nutr. 42:1099-1132; 1985. Reid, L. D.; Konecka, A. M.; Przewlocki, R.; Millan, M. H.; Millan, M. J.; Herz, A. Endogenous opioids, circadian rhythms, nutrient deprivation, eating and drinking. Life Sci. 16:909-913; 1982. Rockwood, G. A.; Reid, L. D. Naioxone modifies sugar-water intake in rats drinking with open gastric fistulas. Physiol. Behav. 29: 1175-1178; 1982. Schulz, R.; Wuster, M.; Herz, A. Supersensitivity to opioids following the chronic blockade of endorphin action by naloxone. Naunyn Schmiedebergs Arch. Pharmacol. 306:93-96; 1979. Smith, G. P.; Gibbs, J. Postprandial satiety. In: Sprague, J.; Epstein, A., eds. Progress in psychobiology, vol. 8. New York: Academic; 1979:179-242.
23. Tang, A. H.; Collins, R, J. Enhanced analgesic effects of morphine after chronic administration of naloxone in the rat. Eur. J. Pharmacol. 47:473--474; 1978. 24. Tempel, A.; Gardner, E. L.; Zukin, R. S. Neurochemical and functional correlates of naltrexone-induced opiate receptor up-regulation. J. Pharmacol. Exp. Ther. 232:439--444; 1985. 25. 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. 26. Wuster, M.; Schulz, R.; Herz, A. Inquiry into endorphinergic feedback mechanisms during the development of opiate tolerance/ dependence. Brain Res. 189:403-411; 1980. 27. Yoburn, B. C.; Nunes, F. A.; Adler, B.; Pastemak, G. W.; Inturrisi, C. E. Pharmacodynamic supersensitivity and opioid receptor upregulation in the mouse. J. Pharmacol. Exp. Ther. 239:132-135; 1986. 28. Yoburn, B. C.; Inturrisi, C. E. Modification of the response to opioid and nonopioid drugs by chronic antagonist treatment. Life Sci. 42:1689-1696; 1988. 29. Zukin, R. S.; Sugarman, J. R.; Fitz-Syage, M. L.; Gardner, E. L.; Zukin, S. R.; Gintzler, A. R. Naltrexone-induced opiate receptor supersensitivity. Brain Res. 245:285-292; 1982.
0031-9384/90 $3.00 + .00
Enhanced Anorectic Potency of Naloxone in Rats Sham Feeding 30% Sucrose: Reversal by Repeated Naloxone Administration T I M C. K I R K H A M 1
School o f Psychology, University o f Birmingham, P.O. Box 363, Birmingham B15 2TT, U.K. R e c e i v e d 12 June 1989
KIRKHAM, T. C. Enhanced anorectic potency of naloxone in rats sham feeding 30% sucrose: Reversal by repeated naloxone administration. PHYSIOL BEHAV 47(3) 419-426, 1990.--The time-course of naloxone-anorexia was monitored in gastric-fistulated rats sham feeding sucrose (10%, 20%, 30%) solutions. Naloxone reduced sham intake dose dependently without affecting feeding initiation and in a manner which resembled the effects of progressive sucrose dilution. However, when rats sham fed 30% sucrose there was a 2-fold increase in the anorectic potency of naloxone. This exaggerated response was prevented by prior repeated naloxone treatment (5 mg/kg IP, bi-daily), concurrent with the stabilization of sham intake levels (4 days). A further experiment ruled out the possibility that tolerance develops to naloxone effects on this treatment schedule, since intact rats showed a suppression of wet mash consumption following repeated naloxone treatment which was equivalent to an acute naloxone challenge. It is proposed that 1) repeated sucrose sham feeding enhances opioid release and leads to opioid receptor adaptation (down-regulation); 2) repeated (chronic) naloxone treatments have an opposite effect on opioid receptors (up-regulation); 3) the two manipulations, in combination, counteract each other's effects. These behavioural data demonstrate dynamic changes in sham-feeding performance as a function of sucrose concentration and naloxone treatments, reinforce the importance of palatability in naloxone-anorexia, and support opioid involvement in orosensory reward.
Naloxone Anorectic
Sham feeding Opioids
Sucrose
Palatability
Up-regulation
OPIOID receptor antagonists, such as naloxone and naltrexone, will reduce food and water consumption in a wide range of species. These well-established effects, together with the ability of opioid receptor agonists to elevate consumption, indicate that endogenous opioids are crucially involved in the regulation of ingestive behaviours (6, 7, 18). More specifically, it is believed that opioidergic systems contribute to the mediation of orosensory reward, or palatability, of ingesta. According to this hypothesis, then, the anorectic effects of opioid antagonists may be considered to be due to a reduction in the pleasantness of food. A valuable instrument for investigating the influence of palatability on feeding motivation and, thereby, the effects of drugs on palatability, is the sham-feeding paradigm (22,25). By means of gastric fistulation, ingested food--typically, a highly palatable sucrose solution--is retrieved before it can enter and be absorbed from the intestinal tract. Under these conditions, and in the absence of significant deprivation, satiation processes are minimized and intake is motivated solely by the rewarding properties of the sucrose. Sucrose sham feeding is stereospecifically attenuated by low doses of ualoxone (13,20). Moreover, since sham intake is directly related to sucrose concentration, it has been demonstrated that
Down-regulation
Reward
naloxone's effects on sham-feeding motivation mirror those obtained by simple dilution of the test solution (14); i.e., naloxonetreated rats sham feeding a particular sucrose solution behave as if they were consuming a more dilute, less palatable solution. These effects have been replicated across a range of sucrose concentrations and naloxone doses. However, previous work in this laboratory has revealed a particularly interesting anomaly: that is, rats which have been trained to sham feed a highly palatable 30% sucrose solution develop an exaggerated sensitivity to the suppressive effects of naloxone. The reduction of 30% sucrose intake induced by naloxone in these rats (13) is double that obtained with identical doses in animals sham feeding weaker sucrose solutions (14). The studies reported here represent an attempt to investigate these phenomena in more detail. Thus, the first experiment was designed with two objectives. Firstly, to confirm that naloxone can induce behaviour that is identical to the effect of sucrose dilution; i.e., that sucrose sham-feeding patterns after increasing doses of naloxone can be matched to those obtained under control conditions with successively weaker, less palatable sucrose solutions. Secondly, to demonstrate that the apparent increase in naloxone sensitivity seen with very palatable (30%) sucrose solutions is
~Requests for reprints should be addressed to Tim C. Kirkham, E. W. Bourne Behavioral Research Laboratory, The New York Hospital-Comell Medical Center, White Plains, NY 10605.
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KIRKHAM
replicable, and to more precisely quantify the effect. Consumption of palatable foods or liquids has been associated with endogenous opioid release (3,8). It has also been reported that elevations of brain opioid levels, in vivo, produce concomitant decreases in specific opiate binding, suggesting down-regulation of opioid receptors (1,4). It is possible, therefore, that heightened opioid release induced by repeated consumption of highly palatable sucrose may bring about similar receptor adaptations. Such changes may account for the enhanced anorectic potency of naloxone, as described above. Fewer receptors could facilitate the ability of a given dose of antagonist to compete with endogenous ligands for binding, thus making antagonism more complete and the consequent behavioural response more pronounced. Conversely, chronic administration of opioid antagonists can rapidly induce opioid receptor up-regulation (15, 24, 28, 29). For example, chronic naltrexone treatment causes a maximal (80%) increase in opiate receptor number in rat brain after about 8 days. Half-maximal increases occur after approximately four days; significant elevations are evident as early as the second day of treatment (29). Thus, any receptor adaptation induced by sucrose ingestion could occur as rapidly, within the period required for the facilitation of naloxone-anorexia in the sham-feeding model (approximately 3 days). Moreover, the ability of opioid antagonists to induce receptor up-regulation permits an indirect test of the putative down-regulating consequences of prolonged sucrose ingestion. Thus, in the second experiment, repeated naloxone administration was paired with daily sham feeding of 30% sucrose to determine whether the increase in sensitivity to acute naloxone could be reversed. In Experiment Three, the effects of daily naloxone treatments on the anorectic response to acute naloxone were tested in intact animals feeding a palatable wet mash diet. This test was included to determine whether any changes in sensitivity to naloxone following repeated administration of the drug in the sham-feeding model could be ascribed to factors, such as tolerance, which might occur independently of the ingestion of sucrose solutions.
returned to home cages and, after 30 min, food was restored. Criteria for successful sham-feeding tests were that ingested fluid should drain freely at all times and that the total volume of liquid recovered should not be less than the volume of sucrose ingested.
Drug Naloxone hydrochloride (Du Pont de Nemours & Co.) was dissolved in isotonic saline and injected IP in a volume of 1 ml/kg body weight. Vehicle and naloxone injections were made 30 min prior to testing. In Experiment One, doses of 0.25, 0.5, 1.0 and 2.0 mg/kg naloxone were tested. In Experiment Two, the effects of acute doses of 1.0 and 5.0 mg/kg naloxone on sham feeding were tested after several naloxone (5.0 mg/kg bi-daily) or saline treatments. A single dose of 5.0 mg/kg was tested in Experiment Three. Previous analyses (7, 13, 14) indicated that a dose of 1.0 mg/kg naloxone will reduce 1-hr sham intake of relatively dilute sucrose solutions (_-<20%), equivalent to the difference in intake produced by halving sucrose concentration. Those experiments also showed that doubling the dose of naloxone has an effect on intake akin to a further halving of concentration. For these reasons, in Experiment One, 1.0 mg/kg naloxone was administered to rats sham feeding 10%, 20% and 30% sucrose. Additionally, 20% sham intake was tested after injection of 2.0 mg/kg naloxone. In the 30% sucrose condition, because of the possibility of increased naloxone sensitivity, the effects of 0.25 and 0.5 mg/kg were also assessed. In Experiment Two, the acute dose of 5 mg/kg naloxone was chosen because in a previous experiment (13) it had produced a maximal suppression of 30% sucrose sham feeding and would, therefore, provide "floor" intake values against which the effects of repeated naloxone treatments on the drug's acute anorectic potency could be easily judged. An acute dose of 1.0 mg/kg was used to provide a standard by which the anorectic potency of naloxone could be compared between different conditions, within the experiment, and also with the data from Experiment One, obtained using lower concentrations of sucrose.
GENERALMETHOD
Animals Sixty-four male hooded rats (Birmingham General Strain), weighing between 200-245 g at time of acquisition, were used. Animals were housed singly and maintained under a 12:12 hr light-dark cycle (lights on at 0830 hr). Except for brief pretest food deprivation (in sham-feeding experiments only) and during test periods, rats had free access to the laboratory diet and drinking water.
Statistical Analysis The effects of acute and chronic naloxone treatments on cumulative sham sucrose intake (at each measurement interval) and wet mash consumption were assessed using analysis of variance or, where appropriate, the Student's t-test. Post hoc comparisons, following significant F values, were made with the Newman-Keuls test. EXPERIMENTONE
Surgery and Sham-Feeding Procedure
Method
Twenty-four rats were selected for sham-feeding tests and, after habituation to housing conditions, handling and injection procedures, acrylic gastric cannulas were surgically implanted according to a previously described method (12). Following recovery from surgery, sham-feeding tests began. To facilitate stomach clearance, rats were deprived of food from 1000 hr on each test day. At 1330 hr cannulas were unsealed and stomachs emptied by repeated flushing with tepid water. Animals were then placed in test cages (identical to home cages). To ensure cannulas were free of obstruction, stomachs were rinsed again immediately before sucrose presentation at 1400 hr. Sucrose solutions (w/v, in distilled water) were contained in calibrated drinking tubes. In all tests intake was measured volumetrically, to the nearest 0.5 ml, at 5-min intervals for 1 hr. On completion of each test, stomachs were rinsed once more and the cannulas closed. Animals were
In all stages of this experiment a single group of 8 gastricfistulated rats was tested, receiving all treatments and acting as their own controls. Using the general procedure described above, the rats were successively trained to sham feed 5%, 10%, 20% and 30% sucrose solutions. For each concentration, dally shamfeeding tests were performed to obtain stable baseline intake rates (typically after 3 days). At least 2 nontest days separated trials with different sucrose concentrations. Having achieved stable intake levels of 10% sucrose, rats were retested on 2 consecutive days following saline (day 1) and 1 mg/kg naloxone (day 2). Similarly, sham intake of 20% sucrose was measured after vehicle and 1 mg/kg naloxone. Following a further test with this concentration (to ensure response stability) the procedure was repeated, over a further 2 days, with saline and 2 mg/kg naloxone. As sham intake after each vehicle treatment did not differ significantly, data
ENHANCED NALOXONE ATTENUATION OF SHAM FEEDING
o
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421
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FIG. 1. Cumulative intake (ml) of sucrose solutions over 1-hr sham-feeding tests, displaying (a) comparison of baseline intakes for each sucrose concentration tested in Experiment One, and the effects of naloxone pretreatment on sham intake of (b) 30%, (c) 20% and (d) 10% sucrose solutions. All values are means for 8 rats (bars indicate S.E.M.). *p<0.05, **p<0.01, difference from respective vehicle condition.
from these days were averaged to provide control values. Sham intake of 30% sucrose was subsequently measured, over a 2-week period, following successive administration of 1.0, 0.5 and 0.25 mg/kg naloxone. As before, each drug treatment was preceded by a vehicle day and intakes averaged to provide control data. After each drug treatment, rats were retested, without injection, on at least 1 day to detect any response deficit and, if necessary, to restore intake to baseline levels. Results and Discussion As can be seen from the comparison of baseline cumulative sucrose intakes (Fig. la), all concentrations of sucrose generated avid sham feeding. It is also apparent that the rate of intake, and hence the volume ingested, increased as a function of increasing sucrose concentration. Total control intakes for the different solutions (mean___ S.E.M.) were: 76.81 --- 10.13 ml (5%); 104.81 ---6.93 ml (10%); 127.81±4.68 ml (20%) and 130.25---6.83 (30% sucrose). The very small difference between intakes of the 20% and 30% solutions suggests that, at these concentrations, animals were ingesting near to their physical (motor) capacity. It is probable that intake patterns would be more easily distinguishable were the test to be prolonged, with high rates of 30% sucrose ingestion persisting for longer than with the weaker solution. In rats sham feeding 10% sucrose, 1 mg/kg naloxone produced a marked attenuation of intake. As in previous experiments (13,14), initial intake rates were unaffected (Fig. ld); significant suppression did not occur until after 15 min (p<0.05) of continuous ingestion. Thereafter, naloxone-treated rats continued to sham feed at a reduced rate, so that 1-hr intake was reduced by 23% to 80.56±8.38 ml, t(7)=3.93, p<0.01. As expected, this reduction was similar in magnitude to the difference (27%) between baseline totals of 10% and 5% sucrose. Moreover, confirming previous observations (14), the cumulative intake pattern of 10% sucrose after naloxone was equivalent to that for the 5% solution under control conditions (Fig. la, d). One-hour sham intake of 20% sucrose was reduced in a
dose-dependent manner by naloxone, F(2,14) = 20.76, p<0.001. Total intake after 1.0 and 2.0 mg/kg was reduced to 98.44___ 8.77 (p<0.01) and 82.94---7.81 ml (p<0.01), respectively. Again, the drug failed to affect intake rate between 0-5 min, F(2,14) = 1.13, NS, or from 5-10 min, F(2,14)=0.74, NS (Fig. lc). Significant suppression became apparent at 15 min, F(2,14) = 6.32, p<0.025, with both 1.0 and 2.0 mg/kg doses reducing intake (p<0.05). Although, after naloxone treatment, rats continued to sham feed throughout the remainder of the test, intake rate was much reduced by the drug and slowed progressively over time. That this effect was more marked with the higher dose is evident from the fact that not only was total intake after 2.0 mg/kg significantly less than the control value, but also reliably less than the 1.0 mg/kg total
(p<0.05). The 1.0 mg/kg dose of naloxone produced a net (23%) reduction of 20% sucrose intake, close to the reduction obtained with this dose when the rats sham fed the 10% solution. In effect, this dose had reduced total intake of 20% sucrose to the 10% control level. Moreover, as can be seen from Fig. 1, the pattern of 20% sucrose intake after 1.0 mg/kg naloxone closely matched that of 10% sucrose--in the same rats--after vehicle. Thus, the behavioural effect of this dose of naloxone was equivalent to that of halving the sucrose concentration. Additionally, the effect of doubling the dose of naloxone was equivalent to the effect of a further halving of the concentration of the test solution (i.e., to a 5% solution). After 2.0 mg/kg naloxone total intake was reduced by 35%, comparable to the difference (39%) between total baseline intakes of 20% and 5% sucrose. Furthermore, the cumulative intake pattern of 20% sucrose following this dose, although less linear, resembled the 5% control curve.
The effects of naloxone on sham intake of 30% sucrose are illustrated in Fig. lb. Total intake was markedly reduced by the two highest doses, F(3,21)=21.18, p<0.001. The lowest (0.25 mg/kg) dose produced only a slight (6%), nonsignificant reduction to 122.25---6.82 ml. However, the absence of more marked suppression may indicate a failure of detection, rather than a lack
422
KIRKHAM
of effect of this dose. Given the close similarity between baseline intakes of the highest sucrose concentrations over 1 hr, it is possible that any effect of this dose would be discerned only if the test were continued for a longer period. Total intakes after 0.5 and 1.0 mg/kg naloxone were reduced to 87.81_+15.62 ml (p<0.01) and 67.25___13.38 ml (p<0.01), respectively. It is clear, therefore, that 1.0 mg/kg naloxone induced a considerably greater reduction (48%) of 30% sucrose than of the 20% or 10% solutions. Indeed, the net (33%) reduction after 0.5 mg/kg, in this condition, exceeded the suppression obtained with 1.0 mg/kg when rats sham fed the weaker sucrose solutions. Thus, when total intake values are compared, the anorectic potency of naloxone in animals sham feeding the 30% sucrose is approximately double that seen with weaker, less palatable, solutions. As in the previous conditions, naloxone reduced total consumption by inducing a marked decline in the rate of sham feeding. Again, there was no effect of the drug at the beginning (0-5 min) of the test, F(3,21)= 2.39, NS. However, 1.0 mg/kg naloxone suppressed intake at an earlier point (10 min; p<0.05) and induced a far more rapid deceleration of intake rate than in the preceding tests. The effects of 0.5 mg/kg naloxone were less marked, with a more gradual decline of intake rate. Although some depression of cumulative intake was apparent as early as 10-15 min, significant (p<0.05) attenuation of sham feeding did not occur until 20-25 min. After each of these treatments, as in the other tests, naloxone did not produce any obvious behavioural disruption. The animals' behaviour was, therefore, entirely compatible with the response of fistulated rats ingesting a more dilute solution in the absence of naloxone, supporting the hypothesis that the drug effectively reduces the palatability of the test solution. Moreover, this experiment has clearly demonstrated that ingestion of a highly palatable, very concentrated sucrose solution can alter the sensitivity of sham-feeding rats to naloxone's anorectic action. The mechanism(s) underlying this alteration are unknown, but it is possible that, as discussed earlier, elevated central opioid activity may induce opioid receptor down-regulation. Several researchers have demonstrated that both naloxone and naltrexone will induce opioid receptor up-regulation, when administered chronically over several days [e.g., (23, 24, 29)]. In Experiment Two, daily antagonist treatment was paired with the acquisition of stable rates of 30% sucrose sham feeding. It was expected that, by assessing the extent to which such treatment affected the response of sham-feeding rats to acute naloxone challenge, some inference concerning the role of receptor changes in increased sensitivity to the antagonist seen in Experiment One could be made. EXPERIMENT TWO
Method
Sixteen naive rats (2 groups of N = 8) were trained to sham feed 30% sucrose, using the same general procedure described above. Daily 1-hr intake tests were performed on each of 5 successive days. Stable baselines were obtained by day 3 for both groups. On day 4 all rats received saline injection before the test to obtain control data. Typically, in studies where up-regulation has been obtained after chronic antagonist administration, the drugs were administered via IV infusion or implantation of osmotic minipumps, usually at a rate of 10 mg/kg/day (15,24). The same daily dosage of naloxone was adopted in this study. However, to avoid interference with the development of high baseline sham intake, the dally dosage was equally split and administered in bi-daily injections.
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FIG. 2. Enhanced anorectic potency of 5 (a) or 1 mg/kg (b) naloxone in rats sham feeding 30% sucrose solution under control conditions and the attenuated effects of these doses following repeated naloxone administration. Circles represent data from rats given repeated saline treatment; squares are values for repeated naloxone treatment. Solid symbols indicate saline pretreatment; open symbols indicate acute naloxone pretreatment. 1: ©
Thus, on days 1-4, rats in group 1 received 5 mg/kg (IP) naloxone at both 0900 and 1700 hr. Over the same period, and at the same times, control animals (group 2) were injected with saline. On day 5, the 0900 injection of naloxone or saline was omitted. Instead, sham intake of 30% sucrose was retested (at 1400) 30 min after pretreatment with 5 mg/kg naloxone. After 2 test-free days, the above procedure was repeated, with naloxone (group 1) or saline (group 2) administration on days 8-11. On day 12, the test of acute effects of naloxone on sham feeding were repeated (as for day 5), but on this occasion each rat was pretreated with 1.0 mg/kg naloxone. Results and Discussion
It is clear from Fig. 2a that naioxone treatment, over days 1-4, did not impair the acquisition of a vigorous sham-feeding response to the 30% sucrose. Total control intakes after daily naioxone (group 1) and saline (group 2) treatments were 108.5_+4.69 and t 11.5 _+8.61 ml, respectively. Naloxone (5 mg/kg) pretreatment on day 5 produced a marked attenuation of 1-hr sham intake in both groups, F(1,14) = 67.27, p<0.001. Moreover, there was a significant interaction between chronic treatment and the magnitude of intake suppression after acute naloxone, F(1,14)= 4.903, p<0.05, with control animals being more sensitive. Total sham intake of the control group was reduced by 70% to 32.56_+ 11.77 ml (p<0.01). This reduction was due to a very marked slowing of intake rate, with a significant reduction already apparent at 10 min (p<0.05). Only 2 out of 8 subjects continued to sham feed for longer than 40 min. Termination of consumption was associated with bouts of grooming and exploratory ambulation, which continued sporadically through the remainder of the test. Although these behaviours are components of the behavioural satiety sequence (22), in this situation there was no occurrence of the resting or sleeping typical of normally satiated rats. It is notable that a similar degree of suppression and pattern of 30% sucrose intake was induced by this dose of naloxone in a previous study (13). In contrast to those extreme effects, total intake of rats in group
ENHANCED NALOXONE ATTENUATION OF SHAM FEEDING
1 was reduced by only 42%, to 63.13+-11.31 ml (p<0.01). Control rates of intake continued for 15 min before any suppression was evident, and cumulative intake was not reliably affected until 30 min (p<0.05). In accordance with the more gradual decline of intake rate in this test, group 1 animals were observed to ingest for longer; 7 out of the 8 rats were still sham feeding after 40 min. The pattern of cumulative intake induced by 5.0 mg/kg naloxone, in these chronically treated rats, was thus more similar to that of 30% sucrose generated in the previous experiment after 1.0 mg/kg naloxone (Fig. lb). Similar, although less-pronounced, results were obtained during the second week of testing (Fig. 2b). Both groups maintained high levels of sham feeding, with control intakes on day 11 being of the same order as on day 4; again there were no differences between total control intake for group 1 (107.63---4.19 ml) and group 2 (115.69+-9.4 ml). On day 12, 1.0 mg/kg naloxone significantly attenuated 1-hr intake of each group, F(1,14)= 25.80, p<0.001. However, the animals which had received daily saline treatment (group 2) were again more sensitive to the actions of the drug. Generally, in group 2, 1.0 mg/kg naloxone produced behavioural effects which resembled those induced by the same dose when rats sham fed 30% sucrose in Experiment One (Fig. la). Total intake was reduced by 45% to 64.0__. 13.75 ml (p<0.01); again, a result of reduced intake rates. Control intake rate was maintained over 0-10 min, with a significant (p<0.05) reduction obtained after 25 min of continuous ingestion. The majority of rats (6 out of 8) were still ingesting at 50 min and half continued to sham feed for the full hour. As before, repeated naloxone administration successfully attenuated the anorectic effect of the 1.0 mg/kg dose. Total 30% sucrose intake of group 1 rats on day 12 was reduced by only 25% to 81.25+- 11.17 ml (p<0.01). Importantly, this degree of suppression was equivalent to that obtained with 1.0 mg/kg in animals sham feeding 20% and 10% sucrose solutions (Fig. lc, d) in Experiment One. Once more, intake suppression involved a gradual decline of intake rate following a period (0-25 min) of normal ingestion: significant (p<0.05) suppression was not evident until after 45 min. This experiment has demonstrated once again that relatively brief periods of sham feeding with 30% sucrose (1 hour on each of 4 successive days) are sufficient to induce extreme sensitivity to the acute anorectic effect of the drug. The potency of naloxone under these present conditions far exceeds that observed in the same paradigm, with weaker sucrose solutions, or in other reports of antagonist effects on food intake. Indeed, in intact, mildly food-deprived rats doses of 60-80 mg/kg of naloxone (which are unlikely to be opioid receptor specific) are required to produce comparable reductions of 1-hr food consumption [unpublished data (9)]. Perhaps more important is the observation that the development of enhanced naloxone sensitivity, as animals stabilize their sham intake of 30% sucrose, can be prevented by concurrent, bi-dally naloxone administration. The present data indicate that this manipulation acts to normalize the suppressive effect of naloxone, so that the acute effects of naloxone are of a similar magnitude to those obtained with animals sham feeding more dilute sucrose solutions. Repeated naloxone administration appears, therefore, to oppose whatever processes induce the increased sensitivity of control rats to acute naloxone. Before these data can be properly considered in terms of the current receptor regulation hypothesis, it is necessary to obtain some further indication that repeated antagonist administration does actually counteract adaptations specifically provoked by ingestion of the 30% sucrose. It is possible that prolonged exposure to naloxone would render animals less sensitive to its
423
TABLE 1 LACKOF EFFECTOF REPEATEDNALOXONEADMINISTRATIONON WETMASHINTAKEAFTERSALINEOR NALOXONEPRETREATMENT Repeated Treatment (Day 1-4)
Acute Treatment(Day 5) Saline
Naloxone
Saline
17.98 (0.93)
9.72 (1.47)
<0.01
Naloxone
16.56 (0.61)
10.45 (1.16)
<0.01
p
N.S.
N.S.
All values are mean (S.E.M.) 30-rain intake (g) for 10 rats (separate groups for each treatment). Significance levels were determined by Newman-Keulstest.
anorectic effect independent of sucrose access, perhaps through the development of tolerance. This possibility was tested in Experiment Three, by subjecting intact rats to the same dally naloxone regime as before and subsequently testing acute effects of the drug on consumption of a highly palatable test diet. EXPERIMENTTHREE Me~od
Forty unoperated rats (as described in the General Method section) were randomly assigned to 4 groups of N = 10. Over 3 successive days all animals were habituated to a palatable wet mash diet. This diet consisted of 150 ml powdered food (rat and mouse expanded ground diet No. 1, Special Diet Services Ltd., Essex, U.K.) and 200 ml water. At 1430 on each day animals received approximately 40 g of freshly prepared mash, in their home cages. After 30 min, any remaining food (plus spillage) was recovered and reweighed. Animals were not deprived prior to these, or subsequent tests. After 3 test-free days the daily dose regime was commenced. Animals in groups 1 and 2 received 5 mg/kg naloxone IP at 0900 and at 1700 (i.e., a total of 10 mg/kg/day) on each of 4 successive days. Groups 3 and 4 were given saline injections at those times, over the same period. On day 5, no morning injection was made. At 1400 groups 1 and 3 were treated with 5 mg/kg IP naloxone. The remaining rats were injected with saline at the same time. At 1430, all rats were presented with a weighed portion of the wet mash. After 30 min the remaining food and spillage were collected and reweighed. Results and Discussion
Wet mash intake had stabilized by day 3 of the habituation stage, with all groups consuming similar quantities. Respective 30-min intakes for groups 1--4 were: 15.03- 1.08, 15.11 -+0.83, 14.6--- 1.07 and 16.38---0.59 g, F(3,36)=0.71, NS. The effects of the separate treatments on day 5 intake are summarized in Table 1. Statistical analysis revealed that naloxone pretreatment reliably reduced wet mash intake of both group 1 and group 3, F(1,36)= 43.66, p<0.001. However, there was no evidence of any interaction between treatment on days 1-4 and naloxone's suppressive effect on day 5, F(1,36)=0.98, NS: repeated naloxone treatment had no effect on either control intakes or acute effects of the drug, F(1,36) =0.10, NS. These data, therefore, provide no evidence that rats become tolerant to the anorectic effects of naloxone with repeated administration (at the dose and time-course used here). Moreover,
424
KIRKHAM
comparison of day 5 intake of the two vehicle groups (groups 2 and 4) failed to reveal any effect of chronic naloxone on normal feeding responses. It is worth noting that although control intakes of the palatable mash were high, these values do not represent the maximum limit to consumption. Thus, this feeding model (unlike the 1-hour sham-feeding tests) would be capable of detecting both positive and negative changes in acceptability (or palatability) of the diet, which might have been induced by multiple naloxone treatments. The absence of any reduction in the anorectic potency of naloxone implies that repeated administrationof the drug, alone, is not sufficient to account for the changes observed in Experiment Two (it will, of course, be necessary to similarly assess the long-term consequences of naloxone on the drug's acute suppressive effect in the sham-feeding model, using more dilute sucrose solutions). Overall, these results tend to consolidate the "normalization" hypothesis discussed above. In the absence of conditions (e.g., sham feeding 30% sucrose) which induce increased sensitivity to naloxone, bi-daily antagonist administration does not, by itself, bring about any alteration in an animal's anorectic response. The implications of this will be discussed more fully in the following section. GENERALDISCUSSION The results of the present experiments add to a growing literature which relates opioid processes to the control of ingestive behaviours and, more particularly, to the mediation of food palatability or orosensory reward (18). As discussed previously, it is believed that a reduction in the palatability of ingesta underlies the anorectic effects of opioid antagonists. The changes in shamfeeding behaviour (which is predominantly motivated by the rewarding properties of sucrose) following naloxone administration are wholly consistent with this hypothesis. Confirming the results of earlier studies (13,14), in Experiment One the effects of naloxone were behaviourally equivalent to those obtained by a dilution of the test solution. Thus, the cumulative sham intake curves in naloxone-treated rats were, qualitatively and quantitatively, very similar to those obtained with weaker sucrose solutions under control conditions. Moreover, in each case, naloxone failed to exert any influence on the initiation of sham feeding. This is an important observation as, in agreement with earlier reports (10,11), it indicates that naloxone interferes with neural processes activated by ingestion, rather than anticipation, of the sucrose. This, in turn, suggests that opioids are not directly involved in initiating ingestive behaviour, but may have a positive role in maintaining consumption once begun. As food palatability is a major determinant of the maintenance of consumption, the fact that motivational effects of naloxone occur only at later stages of testing again supports a role of opioids in the mediation of orosensory reward. The importance of palatability factors to the anorectic effects of naloxone is further indicated by the exaggerated response to the drug in animals sham feeding 30% sucrose. In Experiment One, baseline intake of 30% sucrose was only marginally greater than that of the 20% solution which suggests that concentration (stimulus intensity), rather than volume consumed, is the crucial factor. So far, this enhanced anorectic potency has only been obtained with 30% sucrose, although the absolute concentration threshold remains to be determined. The onset of this change occurs very rapidly; increased naloxone sensitivity can be obtained after only 2 days (1 hr/day) of sham feeding with the solution. It remains to be determined whether similar effects may be obtained with more dilute solutions over a longer period of training. In rats, ingestion of palatable, sweet solutions has been reported to stimulate opioid release within the brain, correlated
behaviourally with the induction of naltrexone-reversible analgesia (3,8). As the data in Fig. la demonstrate, the rate, duration and volume of sham sucrose intake is a direct function of the palatability of the test solutions. If, as is proposed, activation of opioid systems underlies the rewarding effects of ingestion, it is possible that opioid activity will also be directly related to sucrose concentration. In this light, the magnified response to naioxone in rats sham feeding the most palatable solution may be due to some neural adaptation induced by chronically elevated levels of endogenous opioids. It has been found that long-term exposure to opioid peptides, in vitro, induces a reduction in the number of opiate receptor-binding sites (2,16). There is also evidence to indicate that such downregulation may occur in vivo, following manipulations known to stimulate the release of endogenous ligands for these receptors. For example, 72-hr food deprivation, which has been associated with increased hypothalamic beta-endorphin levels (19), produced a significant reduction of 3H-naloxone-binding sites in rat hypothalamus and medulla oblongata (1). Also in rats, increased levels of neurohypophyseal dynorphin following chronic dehydration were correlated with marked reductions in specific 3H-bremazocine binding in the neurohypophysis, suggesting a down-regulation of kappa receptors (4). These data suggest that down-regulation of receptors at opioidergic synapses may be a homeostatic, regulatory response to overstimulation by increased levels of endogenous ligand (4). It is possible, therefore, that the opioid release induced during repeated sessions of sham feeding with 30% sucrose may also be of sufficient magnitude to provoke opioid receptor down-regulation. This, in turn, may explain the enhanced anorectic potency of naloxone under these same conditions. A given dose of naloxone could more completely antagonize opioid actions in a downregulated system than might the same dose in an unadapted system, competing more effectively with an endogenous ligand for occupation of a reduced number of opioid receptor-binding sites; the net behavioural effect being equivalent to that of a higher dose of naloxone under other conditions. Such an account would be compatible with the established ability of chronic naloxone or naltrexone administration to induce opioid receptor up-regulation, and with the results of Experiment Two. Chronic antagonist treatment has been shown to increase the number of brain mu, delta and kappa opiate receptors [e.g., (17,29)]. These changes appear to be specific to opioid systems. Up-regulation is associated with the development of functional supersensitivity to the effects of opioid receptor agonists, both in vivo and in vitro (21, 23, 24, 27, 28), but without any apparent change in sensitivity to serotonin, epinephrine (21), benzodiazepines (28), or to muscarinic ligands (29). It is important to note that the doses of naloxone used in this experiment have also been shown to interact stereospecifically with opioid receptors (13). As reported above, in this study repeated naloxone treatment appeared to normalize the anorectic response of animals sham feeding 30% sucrose. When chronic antagonist administration was paired with daily 30% sucrose sham feeding, the magnified response to acute naloxone seen under control conditions was abolished. The degree of intake suppression was thus no greater than would be expected had these same doses been administered to animals sham feeding less palatable solutions. If the observed increase in naloxone sensitivity can be ascribed to a regulatory response to increased opioid levels, the restoration (or maintenance) of more typical levels of anorexia with repeated naloxone may represent the consequences of a counter-regulatory effect of the drug. According to this hypothesis, then, the up-regulation generated by prolonged naloxone blockade of opioid receptors could oppose the putative down-regulation produced by sucroseinduced opioid release. In line with the data from Experiment
ENHANCED NALOXONE ATTENUATION OF SHAM FEEDING
Two, these opposing influences could result in an effective stabilization of basal receptor number and, hence, a "normal" level of naloxone sensitivity. An effect of repeated naloxone administration to stabilize opioid receptor number, rather than to induce a net increase, may help to account for the lack of effect of this treatment on baseline levels of 30% sucrose sham intake (in Experiment Two). Similar baselines were achieved and maintained irrespective of whether animals received daily saline or naloxone treatment. As mentioned earlier, chronic antagonist-induced up-regulation is associated with a supersensitivity to agonist effects. A marked increase in receptor number might, therefore, be predicted to increase susceptibility to endogenous ligands (29). This, in turn, could lead to an enhanced feeding response as a result of increased food palatability. It is, of course, possible that some enhancement of palatability might have resulted from the chronic naloxone treatment, but remained undetected due to the already maximal levels of sham intake with very concentrated sucrose solutions. However, a similar lack of effect of daily naloxone on control intake was found in tests with the palatable wet mash in Experiment Three. In the absence of conditions likely to provoke down-regulation, the tendency for repeated naloxone to induce up-regulation (rather than stabilization) should have been unopposed in this test. For reasons discussed earlier, wet mash intake in this model would have been sensitive to intake-enhancing, as well as -attenuating, effects of specific treatments. Possibly, a within-groups design might have detected some change. Thus, although these latter data suggest that repeated naloxone treatments fail to exert any significant influence upon the response of rats to palatable foods, it is important to reexamine this possibility, using other paradigms, before more concrete conclusions can be made. More generally, this account of the effects observed in the present studies must be considered as largely speculative. It is imperative, therefore, to replicate these results in combination with a detailed analysis of the effects of sham sucrose feeding and chronic naloxone treatment on opioid binding. This form of analysis is especially important to the hypothesized stabilization of receptor number following chronic naloxone, Although up-regulation with chronic antagonist treatments is now well documented, it has been argued (21) that continuous exposure to high drug concentrations (using implanted pellets or osmotic minipumps) is
425
required. However, it has been reported that C57 mice display a marked increase in brain opiate receptor density after bi-daily naloxone (1 mg/kg, IP) injections, with significant elevations apparent after only 2 days (5). Similarly rapid modification with this type of treatment cannot, therefore, be discounted in the rat. Nevertheless, bi-daily naloxone injections, in this study, were clearly effective in altering the anorectic response of sham-feeding animals. It will be interesting to see whether similar behavioural effects can be obtained with continuous naloxone infusion. As the dose-schedule in this experiment was chosen to avoid any suppression of intake during acquisition of a stable sham-feeding response, great care will be required in the design of such experiments to control for similar confounding factors. In conclusion, these experiments have confirmed the utility of the sham-feeding paradigm in the analysis of drug effects on palatability factors. Naloxone (at doses known to act stereospecifically at opioid receptors) attenuated sham intake of 10% and 20% in a manner which mimicked the behavioural effects of sucrose dilution. Emphasizing the importance of palatability to naloxone's effects, animals sham feeding 30% sucrose exhibited an enhanced (doubled) anorectic response to naloxone. This increased anorectic potency was prevented by pairing repeated naloxone administration with daily sham-feeding sessions: the degree of suppression after acute naloxone was restored to levels observed when animals sham fed weaker sucrose solutions. In intact rats, tolerance failed to develop to naloxone's effects on palatable food intake with daily drug treatment. It is hypothesized that repeated and prolonged ingestion of highly palatable solutions may induce sufficient release of endogenous opioids to cause down-regulation of opioid receptors. Further, repeated antagonist treatment may oppose any such ingestion-related adaptations. Although specific binding studies are necessary to assess the validity of this account, these data do provide further evidence for the involvement of opioidergic processes in the mediation of food palatability, and are indicative of neural plasticity in those systems responsible for the regulation of ingestive behaviours. AC~O~E~E~S The author is grateful to David Barber for assistance with surgery and data collection and to colleagues Jo Neill, Mike Clark and Steven Cooper for their help during preparation of the manuscript.
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