Demonstrating morphine's potentiating effects on sucrose-intake

Brain Research BuUetin,

Vol.

17,

pp. 639-642,

1986. Q At&ho

0361~9230/t& $3.00 + .OO

International Inc. Printed in the U.S.A.

Demonstrating Morphine’s Potentiating Effects on Sucrose-Intake STEPHANIE

A. CZIRR AND LARRY D. REID’

Rensselaer

Polytechnic

Institute

CZIRR, S. A. AND L. D. REID. Demonstrating morphine’s potentiaring effects on sucrose-intake. BRAIN RES BULL 17(5) 63!%642, 1986.-Mildly deprived rats (18hr of deprivation of water) were given the opportunity to take a solution of sucrose daily for periods of either 10, 18,31,56, or 100 min. After daily intakes stabilized and prior to a session, rats were given an injection of either morphine sulfate, 1.0 mg/kg, or naloxone hydrochloride, 2.5 m&kg, an agonist and an antagonist, respectively, at the opioid receptors. Naloxone, as expected, decreased intakes regardless of the test-session’s length. Morphine decreased intakes of the shorter sessions, but increased intake of the longest session. Subsequently, injections of morphine were given 56 mitt into a MO-min session. These injections also increased intake. Morphine’s effects in potentiating intake seem to have special relevance with respect to the continuance of ingestion. Variations across experiments, in duration of test-sessions, could account for the variations in conclusions drawn about whether or not morphine and other agonists potentiate intake of ingesta. Feeding

Drinking

Opioids

Naloxone

Endogenous

opioid peptides

propensity to consume. MOR, for example, has been reported to potentiate and not potentiate intake of sweet solutions [l, 2, 4, 121. The resultant ambiguity about effects of agonists raises important issues concerning the roles both endogenous and exogenous opioids may play in the control of ingestion.

NALOXONE (NX), the prototypic antagonist at opioid receptors (opioceptors), reduces intake of nutrients across species with few exceptions and across many testing situations with many presented nutrients (see [ll] for a review). Morphine (MOR), an agonist, sometimes increases intakes. It can be inferred, therefore, that endogenous opioid systems (EOSs) modulate ingestion [8,13]. There are, however, reports of neither NX nor MOR affecting ingestion [ 111. Also, NX and MOR affect other behaviors besides ingestion [ 131. These latter fmdings question the generality of the summary that opioids and EOSs are involved with ingestion and question the specificity of opioids’ effects with respect to ingestion. The issues of specificity and generality of opioids’ effects may involve an understanding of how opioids modulate ingestion. NX, for example, does not affect latency to begin or initial rate of ingestion, but does reduce the duration of ingestion [3,15]. MOR, in small doses, may evoke tendencies opposite to those of NX, i.e., MOR may extend a bout of ingestion. It follows, therefore, that a critical variable for demonstrating an effect of opioids on ingestion may be the usually arbitrarily selected period in which subjects are allowed to ingest. A brief period, although sustaining high levels of ingestion, may not be optimal for demonstrating opioid-involvement, since opioids may only modify amount taken when opportunities to ingest go beyond the time when ingestion would ordinarily cease. The reports of NX’s effects in rats consistently support the conclusion that NX attenuates consumption [ll]. NX, for example, dose-relatedly reduces the amount of solutions of sucrose or saccharin taken by rats [14, 16, 171. There is surely less consistency in reports of tests of MOR on

EXPERIMENT

1

As mentioned, there is ambiguity concerning the conclusion that MOR increases intake of sweetened solutions. This may be due to the fact that the test-periods of the various experiments varied. So, with this experiment, a dose of MOR that is apt to potentiate ingestion was given to rats having differing amounts of time to consume a sucrosesolution (SS). METHOD

Subjects

The subjects were 60 Sprague-Dawley, male rats purchased from Taconic Farms (Germantown, NY) when they weighed from 150 to 175 g. Each was housed individually in a stainless-steel hanging cage, which remained its home throughout the procedure. Food (standard laboratory blocks) was always available throughout the procedures. Housing was in a windowless colony room maintained at 24°C and having 12 hr of light daily beginning at O!WOhr. Drugs and Solutions

The test-solution was 5% sucrose in tap water, i.e., 5 g of sugar to 100 g of total solution. SSs were presented in glass bottles equipped with ball-point sipping tubes.

‘Requests for reprints should be addressed to Larry D. Reid, Department of Psychology, Rensselaer Polytechnic Institute, Troy, NY 12180.

639

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Injections of morphine sulfate (MOR) were given in doses mg/kg and injections of naloxone hydrochloride (NX) were 2.5 mgikg (doses in terms of the salt). Placeboinjections were saline, the vehicle of MOR and NX. All injections were given subcutaneously, 15 min prior to presentation of SS, and were in volumes of 1 ml/kg. of

AND KCifl!

30

1 .O

i * Placebo 25__

n Morphine

0 Naloxone

$4 0

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PiWC&i'rrW After a week of habituation to conditions of the laboratory, all subjects began a daily regimen of 18 hr of deprivation of water followed by presentation of SS. This regimen continued for 7 days, after which, injections began. Subjects were divided into 5 groups of 12 each, with each group having an opportunity to drink SS for one of five periods. Group 1 had access to SS for 10 min, Group 2 for 18 min, Group 3 for 31 min, Group 4 for 56 min, and Group 5 for 100 min. Following the end of the lOOmin test, all groups were presented with water for 4.3 hr. With the regimen occurring day after day and with each rat’s intake being similar day after day, injections were programmed to occur before some daily opportunities to consume. Toward the end of controlling for effects that may be associated with any given day rather than an injection, each group was further subdivided into two subgroups based on order of drug-injections. All subjects in one subgroup received injections in the order of P M P P N P P M P P N P, where P stands for placebo, M for morphine, and N for naloxone. Subjects in the other subgroup received injections intheorderofPNPP~PPNPP~P. Measures,

Data-Reduction

and Statistics

All bottles were weighed to the nearest 0.1 g before and after presentation, with the resulting intake-scores corrected for spillage [5,9]. Each subject had scores on 8 days under placebo, 2 days with MOR, and 2 days with NX, Initial analyses indicated that a groups’ scores did not vary reliably across the 8 days of placebo, the F(28,38S) for the inte~ction term (group by days)= 1.O9, p=O.34. Consequently, the means of the 8 scores of placebo, the 2 scores of MOR, and the 2 scores of NX were used as indices of the effects of different kinds of injections, yielding a single score for each rat for the effects of placebo, MOR, and NX. The resultant scores conform to the design of a S by 3 factorial analysis of variance (ANOVA) having repeated measures with a factor for the five groups (test-session lengths) and the three kinds of injections. RESULTS AND DISCUSSION

Figure 1 depicts the results. More SS was taken the longer the period, F(4,55)=35.0. NX decreased intake of SS regardless of period of testing. MOR decreased intakes when the period was short (30 min or less), but increased intakes when the period was long. The differential effects of drugs on intake are manifest in the reliable interaction term (type of injection by length of test) of the overall ANOVA, F(8,11~)~19.S, p
OL

’

10

I

I

,

I

16

31

56

100

Durations

of Test Sessions

(Min)

FIG. 1. Means are depicted which reflect the amount of sucrosesolution taken under the influence of placebo, morphine sulfate (I mg/kg), or naloxone hydrochloride (2.5 mgikg) as a function of length of oppo~unity (min of test-session) to take the solution. Morphine only increases intake with the longer op~~u~ities.

min @
MORPHINE

AND SUCROSE

641

INTAKE TABLE

Group 1 2 3 4

Injection P/P MoriP PiMor Mor/Mor

Pl

Tl

P2

26.6 28.3 26.3 26.2

26.4 27.6 27.3 26.7

9.6 10.9 10.9 11.1

1

* *

T2

Total P

9.6 14.3 13.7 15.7

36.2 39.2 37.2 37.3

TotalT

Diff

36.0 41.9 41.0 42.4

0.2 2.7 3.8 5.1

* * *

Pl refers to the amount consumed during the placebo day during the 1st segment of the session whereas P2 refers to the 2nd segment of the session. Tl and T2 refer, respectively, to the 1st and 2nd segments of the test-session. Total P and Total T refer to total amounts taken across the 1st and 2nd segments on day of placebo and day of test, respectively. An asterisk between two means indicates that t-test for dependent means yields a value indicative of statistically significant differences (p
length probably must be longer than that which taps the subjects’ initial propensity to respond. In other words, opioids affect continuance of ingestion rather than other aspects of ingestion, an issue also addressed in Experiment 2. The dose of MOR sulfate used in this demonstration is relatively small, 1 mg/kg, a dose producing no signs of behavioral depression. Obviously, larger doses producing catatonia would interfere with ingestion across short testsessions if for no other reason than catatonia is incompatible with ingestion. We suspect that the initial impact of even this small dose of MOR diverts the rats’ attention from ingesting and this accounts for the decreases seen with the IO-min session. EXPERIMENT

2

With Experiment 1, it was shown that MOR potentiated intake only with long test-sessions. This experiment addressed this question: If MOR were given after initial intake was over, would MOR still potentiate intakes? The interest, in other words, was whether or not MOR had to interact with initial processes of a bout for it to potentiate intake. METHOD

At the conclusion of Experiment 1, all subjects were removed from the schedule of deprivation for a period of 18 days. Subjects were then factorially arranged, taking into account their previous group-membership, to form four new groups of 15 each. The 18-hr schedule of deprivation of water, followed by presentation of SS, was then reinstated, with all subjects having access to SS for 100 min. Following 13 days under the regimen, a 2-day series of injections began. On the 1st day, all groups received 2 injections of saline, the 1st given 15 min prior to presentation of SS, and the 2nd given 56 min into the session. As the 2nd injection was being given, a rat’s bottle was removed from its cage and weighed. Fifteen min later, all subjects were given an additional 44 min to drink SS, followed by the usual period of water only. On test-day, Group 1 again received 2 saline-injections. Group 2 was given MOR with the 1st injection and saline with the 2nd; Group 3 had saline first and MOR second; and Group 4 received MOR in both injections. Group 4 was given a dose of 0.5 mg/kg with each injection, so that its total dose was equivalent to the 1.0 m&g dose of Groups 2 and 3.

RESULTS AND DISCUSSION

Table 1 depicts mean intakes during the 1st and 2nd segments on placebo- and test-days. Please note that means for Group 1 (those getting placebos both days) on placebo- and test-days are not reliably different from one another, therefore, any effects seen with MOR with other groups are apt to be the result of MOR. Group 2, i.e., those getting MOR prior to the 1st session, took more sucrose across the entire session than taken previously on placebo-day, as well as taking more than Group 1 (the controls). This replicates the results from similar procedures of Experiment 1, i.e., MOR increases SS-intake during a lOO-min session. Notice, however, that increased intake occurred only during the 2nd segment. The lack of a reliable increment in intake during the 1st segment for Group 2 is concordant with the small increment of Experiment 1 and previous experiments showing MOR does not increase intake in a 1-hr session [ 121. This is further confvmed by the results of Group 4. It seems that an hour is at the border for observing a small-dose-MOReffect. Group 3, those receiving no MOR until the 2nd segment of the test, increased their intakes during the 2nd segment compared to placebo-day, but the increase does not meet standards for statistical reliability, t( 14)= 1.79, p
DISCUSSION

It can be inferred from a variety of tests with MOR and ingestion, and MOR and other indices of positive reinforcement, that the dose-response relationship between MOR and

642

propensity to engage in behavior resulting in positive reinforcement is complex [13]. The effects of MOR are characterized by a triple interaction of dose, days of daily dosing and time after dosing. These data indicate that there is a further complexity, i.e., the test-session must be of sufftcient length for effects of opioids to be manifest. When the locus of opioids’ effects (toward the end of ingestion) and when the dose-response relationships of MOR are taken into account, there may be few instances in which MOR and NX do not, respectively, increase and decrease ingestion. MOR and NX, however, produce changes in other behaviors as well as ingestion [ 131. Consequently, MOR and NX’s effects on ingestion may reveal a general modulatory effect on behavior sustained by positive reinforcement. There is, however, a possibility for specificity within the EOSs with respect to ingestion, There are important implications to be drawn from the conclusion that MOR and other agonists affect the duration of an ingestive bout. When assessing MOR’s effects on intake of different kinds of ingesta, false conclusions could be derived with short testing sessions. If a rat, for example, only ingests fat for a brief period but ingests carbohydrates for a long period, and if a test-session is longer than the typical end of ingestion of fat but shorter than the typical end of ingestion of carbohydrates, then it would be expected that MOR will potentiate intake of fat, but not carbohydrates. Such a finding may be relevant to understanding differential opioid-involvement in selection of foods, but it may also merely reflect that rats sustain intake of one kind of food more than another.

There are problems with drawing conclusions from only an antagonist’s effects [ 131, and, therefore, data demonstrating that agonists have opposite effects are a critical standard for supporting a conclusion of a relationship betvveen a neurochemical system and a specific behavior [ 1I]. MOR and NX, an agonist and an antagonist at the opioceptor. do seem to produce opposite effects on ingestion. Given. however, the possibility for there to be more than one type ofopioceptor (in doses administered in behavioral studies). and given that not all antagonists at the opioceptor act as NX [ IO]. then there is a possibility that only one type of opioiceptor is involved with ingestion or a specific type of ingestion. The available evidence from tests involving laboratory subjects do support the conclusion that opioids are involved with sustaining consumption once begun ] II]. Susrained consumption is characterized colloquially as gluttony. Gluttony is manifest in some forms of obesity and in ingestion of alcoholic beverages. Failures to sustain ingestion could lead to anorexia. Perturbations in EOSs, therefore. may be involved in certain eating disorders. At the very least, it seems reasonable to conclude that the endogenous opioids are candidates deserving serious inspection in the search for factors involved in gluttony and its converse.

We thank Christopher Bensinger. Wjlljarn Hyatt. Jay Volanth. and Jean Bestle for help with the production of this research. This work was supported, in part. by a grant from the Paul Beer Trust, administered by R.P.1.

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