Opiate blockade inhibits saccharin intake and blocks normal preference acquisition

Pharmatoh~gv Bmthemt~tr3 & Behavmr, Vol 24. pp 833-836, 1986 o AnkhoInternationalInc Pnnted ~nthe U S A

0091-3057/86$3 00 + 00

Opiate Blockade Inhibits Saccharin Intake and Blocks Normal Preference Acquisition W E S L E Y C. L Y N C H D e p a r t m e n t o f P s y c h o l o g y , M o n t a n a S t a t e Universtty, B o z e m a n , M T 59717 R e c e i v e d 21 J u n e 1985 LYNCH. W C Optate blocl,ade mhtbtts sa~tharm retake and blotl~s normal preference atqut~ttton PHARMACOL BIOCHEM BEHAV 24(4) 833-836, 1986 --Recent evidence mdtcates a close connectmn between oral sensory funcuon and op~o~d effects on feeding Not only msgustatory mot~vatmn influenced by opmte drugs [1 I] but apparently gustatory st~muh can also acttvate central opmte receptor systems [3] In 3 experiments we stud~ed the effect of opmte receptor blockade on dnnkmg mottvated by lhe sweet taste of sacchann. Experiment 1 estabhshed a dose-response functmn for mh~bmon of retake by naloxone (NAL) m short (60 ram) 2-bottle tests. Th~s experiment demonstrated the extreme sens~ttv~tyof nondepnved, nonstressed ammals to NAL and estimated the MED~oat less than 0 1 mg/kg (SC), well below the threshold for effects due to illness or general motor dtsturbance Experiment 2 further demonstrated that NAL's effecttveness depends on sacchann concentratmn. In particular, the lowest NAL dose stud~ed was effecttve near the threshold for sacchann preference but not at h~gher concentranons. These data suggest that endogenous op~o~d systems may be actmvated by taste st~muh m a graded fashion. Finally, experiment 3 showed that the typical acqms~tmn of preference for a moderate sacchann concentration can be effectively blocked by dmly pre-test NAL mject~on Together these experiments further demonstrate the close funcnonal relanonsh~p between opto~d systems and gustatory sensory systems Taste preference

Feeding

Dnnkmg

Appente

Op~o~ds

Naloxone

tatory stimulation on endogenous opioid function has also been reported. L~eblich et al. [9] compared female rats genetically selected for high versus low retake of saccharin (LC2-H~ vs. LC2-Lo). When LC2-H~ rats were given daily access to 3 mM sacchann they showed a lack of analgesic response (cross-tolerance) to a moderate dose of morphine (2.5 mg/kg), whereas LC2-Lo rats given daffy saccharin or LC2-H~ rats g~ven no sacchann showed a typical (nontolerant) analgesic response These data ~mply that, in th~s selected group of rats, sacchann intake somehow modifies the functional status of endogenous opioid systems. The same conclusion was more forcefully dlustrated by Dum et al [3] who reported an ~ncreased release of/~-endorphin and a corresponding decrease in m v~vo binding of 3H-etorphine w~thin the hypothalamus of nondepnved, nonstressed ammals g~ven a highly palatable food or drink (chocolate candy or chocolate mdk) immedmtely before assays were performed. The decreased etorphine binding, m particular, suggests that palatable foods somehow activate endogenous opio~ds which m turn compete w~th etorphine for available binding s~tes. The present experiments examined further the influence of endogenous opio~d systems on fluid intake motivated by taste. All experiments were carded out in nondepnved, nonstressed adult male rats tested m their home cages. In this s~tuation the primary incentive to d n n k is provided by the gustatory quahty of solutions presented during short (60 nun) 2-bottle test sessions. Experiments 1 and 2 determined

A number of recent studies suggest a connection between oral sensory stimulation and opiate receptor function. Indirect ewdence comes from studies of feeding and dnnkmg m which taste prowdes the primary incentive for retake By far the most numerous of these have employed opiate antagomsts which suppress intake and in which retakesuppression ~s enhanced by flavor [2, 8, 14]. For instance Locke et al [ 10] showed that various opmte antagomsts suppress ~ntake of sweetened condensed milk m non-deprived monkeys and rats, and Rockwood and Reid [13] found that sucrose retake was suppressed by the antagonist naloxone even in nondepnved rats sham dnnkmg w~th open gastric fistulas. They reasoned that naloxone's effect must be due to modification of the "affect~ve reactlwty to palatable solutions, and not due to feedback from postabsorpt~onal events" (p 1175). Other studies have shown that retake motivated by sweet taste ~s highly sensitive to opiate antagomsm [l 1,18]. More d~rect ewdence for a connection between gustatory stimulation and opmte system function comes from a study by Herman and Novin [6] which demonstrated a naloxone-revers~ble inhibition by morphine of gustatory-evoked umt act~wty recorded from the parabrach~al (gustatory) nucleus. Umt activity evoked by a I 8% salt solution applied to the tongue was inhibited by morphine but tactde-evoked activity from the tongue, recorded from the tngeminal nucleus, was unaffected While the above studies suggest that opiate drugs can influence gustatory sensation, the converse influence of gus-

833

834

LYNCH -16 -14

~ -la

F~sher s L S D b c d e . . . . . ..

-14 1'7"71Sacc

*.*

I--1H20 /

~" _1~

• Id • .-p<

~

-~

• -

P<

OI

|

p,cO5 ns

002

p,~

00$ oo

~ -'~o /

•

~_ .~ I~

o',% Sacc

~

~o%

[--]

~0% o,o

.oo

!~

~1

,

I

•

,

~

,

~

~-6 r,,, ¢~

p -4 ~, ~-

~

r-1

_..._ ~ -$ e.

~.

~

"'"

001

p,~

•

O

001

-

p.(

.*

-"/

~-2

0

o

I ;

:~

.01 0 a

.03 b

NALOXONE

.10 c

.30 d

1.0 e

0.1

1.0

N A L O X O N E DOSE (mg/kg)

DOSE (mg/kg)

FIG I Dose-effect ofnaloxone-HCl on tntake ofa h~ghly preferred (0 1%, w/v) solution of Na-sacchann versus water Group mean d~fferencc scores (Days 18-17) ±SEM Negattve values tndtcate suppression of dnnk~ng Mean (±SEM) pre-drug intake (ml) of saccharm and water, respectively, was 15 3__.0 89 and 1 6±0 38 Inset shows stattst~cally stgnlficant group dtfferences (Ftsher's LSD test)

FIG 2 Dose-effect of naloxone-HCI on retake of 3 sacchann solutions spanning the peak preference range (0 03-0 30%. w/v) Mean retake d~fference scores (±SEM) are from a single group of rats (n= 10) tested repeatedly over 10 weeks, calculated by subtracting scores for the week ~mmed~ately preceding (SAL) and dunng drug (NAL) treatment Mean (_+SEM) pre-drug tntake volumes (ml) for the 3 doses (0 01, 0 i and I 0 mg/kg) were respecttvely, 15 1± I 53, 15 1±2 I1 and 9 2±2 47 The lower volume assocmted wtth the I mg/kg dose reflects the fact that th~s dose was g~ven earher m training

the lowest effective doses of naloxone (NAL) that acutely suppress Intake of highly preferred solutions of sacchann Experiment 3 examined the effects of moderate daffy doses of N A L given throughout testing, on sacchann preference acqms]tlon.

concentration Ten rats selected from a larger group of 30 on the bas~s of their consistent (3 day) consumption of 0 I% sacchann, were given repeated 2-bottle preference tests over 10 weeks All ammals were tested 3 days/week once w~th each of 3 sacchann concentrations selected to cover the peak preference range (0 03, 0.10, and 0 30% w/v) Solutions were each paired w~th water and the positions and solution concentrat]ons randomized. Injecuons began on week 3 w~th each week of drug InJections preceded by one or two weeks of S A L rejections Over the following 7 weeks 3 N A L doses were given in descending dose order: 1 0, 0.1, 0 01 mg/kg (SC). In experiment 3, three groups of nmve rats (n=8) were each g~ven 15 days of 1 hr dady access to 0 1% sacchann vs water (5 days/week) Group I (SAL) r e c o v e d an injection of S A L (1 ml/kg) 15 mm before each dady session Group 2 (NAL-pre) received an injection of N A L (! mg/kg) also 15 m]n before daily testing Group 3 (NAL-post) received the same N A L dose (1 mg/kg) immediately after each dady session. Group 3 was included to control for the possibility that the retake suppression by N A L might be due to condmoned taste aversion (CTA). By assomatmg the taste of sacchann (CS) with the presentation of N A L (US), an aversion to the taste of sacchann might gradually develop thus r e duong overall retake However, if the primary effect of N A L was to act as a US for aversion condtt]onmg, the retake suppressant effect should be greater m group 3 than m group 2 since group 3 represents the optimal condition for CTA learning, namely CS (taste) preceding US (drug)

METHOD In each of the following experiments adult male Hoitzman albino rats were housed individually with ad hb access to food and water except dunng test sessions. All tests were carned out m home cages dunng the hght portion of a 14' l0 hr hght/dark cycle (on at l0 a m.) Room temperature was mamtmned at approximately 25°C. Naloxone-HC1 (Endo Laboratories) was dissolved m 0.9% bactenostat]c NaCI (SAL). All ]n.lect]ons were given subcutaneously at the nape of the neck m l ml/kg volumes. Experiment ! estabhshed a dose-response function for N A L ' s effect on sacchann retake m fifty rats randomly assigned to 1 of 5 dose groups (n=10) For II days prior to 2-bottle tesung all ammals were allowed l hr dady access to a highly preferred (0.1% w/v) sacchann solution. Two-bottle presentations (sacchann vs water) began on day 12 with each session preceded (15 m]n) by a vehicle injection (days 11-17) or by I of 5 N A L doses (day 18). Doses for the 5 groups were. 0 0, 0.03, 0.10, 0.30, and I 0 mg/kg (SC). Experiment 2 determined whether or not the intakesuppressant effect of N A L vaned as a function of sacchann

OPIOIDS A N D TASTE

835

g

i

Z

~,o -r o

I

~

Iul

~o ~

~, N A L - pre /I~N ~_

~

4/

/,

~

~'"T- '~

.~

nn solution is effectively blocked by daffy pre-treatment with N A L . A trend analysis based on the data in Fig. 3 [5] confh~ned a highly significant main effect of treatment on group mean intake, F(2,21)=12.91, p<0.001, as well as a highly s~gnificant overall difference m linear trends, F(2,21)= 13.70, p<0.001 In addition, individual between-group trends were also significant for NAL-pre vs. NAL-post, F(1,14)=8.84, p<0.025, and for NAL-pre vs. SAL, F ( ! , 1 4 ) = 3 7 9 8 , p<0.001. Thus pre-test N A L mject~on more strongly inhibited the acqms~t~on of sacchann preference than e~ther posttest N A L or than pre-test S A L Whde the group mean difference between NAL-post and S A L was s~gmficant, F(l,14)=7 29, p < 0 025, the hnear trend d~fference between these groups fell just short of s~gmficance, F(l,14)=4 40, p ~ 0 10

--

0

| I

,

, 3

,

, $

DAYS

,

,

•

7

of

,

|

9

|

I1

|

,

13

,

¶

IS

TESTING

FIG 3 Da~lygroup mean ~ntake of 0 1% sacchann (w/v) for 3 groups tested 5 days per week for 3 weeks. SAL" daily pretreatment with 0 9% NaCI, NAL-pre daffy pretreatmenI with naloxone-HCI (I 0 mg/kg, SC), NAL-post dady post-test treatment w~th the same dose of naloxone-HCI See text for detads and statistics

RESULTS

Figure 1 dlustrates the dose-effect of N A L on ~ntake of 0 1% saccharin vs. water Group mean difference scores were calculated by subtracting volumes on day 17 (pre-drug) from volumes on day 18 (drug) for each animal. Negative difference scores (upward) indicate suppression. It ~s clear that saccharin consumption varies with N A L m a dosedependent manner. Statistical analys|s (one-way ANOVA) confirmed a highly s~gnificant main effect of dose, F(4,45):8.945, p ~ 0 001. Multiple comparisons among the 5 groups showed the 3 highest N A L doses were s~gnificantly more effective than the zero dose and that the highest dose (1 0 mg/kg) was significantly more effective than any of the 4 lower doses (Fisher's LSD test) The M E D ~ for N A L was estimated to be 0 065 mg/kg by calculating the N A L dose at which half the animals showed a 20% or greater reduction ~n ~ntake (pre/post). The results of experiment 2 are shown m F~g. 2. Intake difference scores were calculated by subtracting the volumes consumed of each solution during the week of S A L injections from those dunng the ~mmed~ately following N A L week, y~elding 9 d~fference scores for each ammal (3 solutions x 3 N A L doses) The results dlustrated m F~g. 2 suggest that N A L dose interacts w~th sacchann concentration such that the lowest N A L dose (0.01 mg/kg, SC) ~s a less effective retake-suppressant at the 2 highest sacchann concentrat~ons than at the lowest concentration. When t-tests were used to evaluate the effect of each treatment separately (a-levels adjusted for multiple tests, rejecting the nullhypothes~s only ifp~0.005) tt was found that N A L was uniformly effective except when the lowest N A L dose was g~ven m the presence of the 2 higher sacchann concentrations This suggests that near the threshold of palatabihty, a very low N A L dose (0.01 mg/kg) effectively suppresses mtake, whereas at higher (super-threshold) sacchann concentrations, retake ~s unaffected by this low dose The results of experiment 3 (F~g 3) show that acquisition of preference for an otherwise highly preferred 0.1% saccha-

DISCUSSION

Taste is a strong incentive to eat and dnnk in most species ~ncluding man. The above results suggest that in the absence of other motives to drink (deprivation, stress, etc.) the ~ncentare motivation provided by sweet taste depends upon an adequately functmning endogenous optoid system. These results cannot easily be accounted for m terms of nonspecific motor d~sturbance since effective suppressant doses of N A L demonstrated m experiments 1 and 2 are nearly an order of magnitude below most estimates for effects on general activity or motor coordination ~n rodents [1, 7, 12, 15] These effects also are not hkely to be due to malaise or to cond~tioned taste aversion, since the effective doses reported ~n experiments 1 and 2 are well below those typically required to produce dlness or CTA [4,17] and since a d~rect comparison between pre- and post-test N A L treatments (experiment 3) showed that post-test N A L (an arrangement more likely to produce CTA) was actually less effective than pre-test N A L which v|rtually ehm~nated preference acquisition. The present results, then, suggest the following conclusions F~rst, that fluid-retake motivated exclusively by sweet taste ~s extremely sensmve to the disruptive effect(s) of opmte receptor blockade and that N A L inhibits fired intake m a dose-dependent fashion over nearly a 100~fold range with an approximate MED.~ of 0.065 mg/kg (SC) Second, that the effectiveness of N A L varies wRh saccharin concentration such that near the preference threshold low N A L doses are effective but these become less so (or entirely ineffective) above threshold. Th~s ~s a tentative conclusion but one that may shed light on the interaction between opiate system and sensory system functions. It suggests that when external ~ncentives are weak, a fully functioning op~o~d system ~s essentml to the maintenance of goal-d~rected behavior Dum et al I3] have convincingly demonstrated that h~ghly palatable foods (primarily sweets) activate endogenous op~o~d pools leading to opiate receptor occupancy Assuming th~s ~s a graded response to sweetness (or palatab|hty or perhaps some general motivational quality of food), weaker activation by weaker or less palatable flmds would perhaps be more easdy blocked by low doses of op~ate antagomsts It remains to be determined exactly which qualrues of oral stimulation are responsible for opio~d system activation and exactly which elements of the central and/or peripheral op~o~d systems are revolved. Finally, experiment 3 suggests that d~uly opmte receptor blockade prior to preference acquismon effectively ehm~nares the normal preference for sacchann. Whether or not preference would recover once antagonist treatment was

836

LYNCH

e n d e d is u n k n o w n A n i n t e r e s t i n g q u e s t i o n in hght o f the r e s u l t s o f D u m et al [3] is w h e t h e r the N A L - p o s t effects are d u e to C T A o r to a b l o c k a d e o f r e w a r d i n g a f t e r effects o f o p l o l d a c t t v a t m n . I f p a l a t a b l e foods a c t i v a t e oplold pools a n d t h e effects o u t l a s t the s t i m u l u s , s u c h a f t e r effects m a y cont r i b u t e to t h e p o s l t w e i n c e n t i v e to d n n k T h u s t h e r e are 2 a p p a r e n t l y c o n t r a d i c t o r y i n t e r p r e t a t i o n s o f the N A L - p o s t effects" E i t h e r N A L ~s itself a n a v e r s l v e s t i m u l u s that w h e n p a i r e d with a n o v e l s w e e t t a s t e leads to C T A a n d s u p p r e s sion o f s u b s e q u e n t d n n k l n g or, a l t e r n a t i v e l y , N A L antagorazes a n o t h e r w i s e e m o t i o n a l l y p o s i t i v e ",ffter effect o f s w e e t taste, t h e r e b y r e d u c i n g the i n c e n t i v e to drank T h e d i f f e r e n c e b e t w e e n t h e s e i n t e r p r e t a t i o n s lies m the r e l a t i o n s h i p bet w e e n s e n s o r y e v e n t s a n d oplold a c t i v a t i o n In the first case,

no s p e o a l role ~s g~ven to the g u s t a t o r y e v e n t C T A is ass u m e d to be a d~rect r e s p o n s e to a v e r s l v e classical condlt~onmg In the s e c o n d case, g u s t a t o r y s t i m u l a t i o n is seen as activating e n d o g e n o u s op~old pools leading to positive "after effects w h i c h are t h e n b l o c k e d by n a l o x o n e

ACKNOWLEDGEMENTS Thanks to Drs R~ck Block and Barbara Fernandez for stattsucal assistance and to Ms Leshe L~bby and Mr Bob Jones for laboratory assistance Th~s work was supported ~n part by MONTS-NSF project ISP-801149, MSU Research Creativity Award and a grant from Procter and Gamble Naloxone-HCI was a gift from Endo Laboratories

REFERENCES I Amlr, S , H Gahna, R Blair, Z W Brown and Z Am~t Opiate receptors may mediate the suppressant but not the excitatory action of ACTH on motor activity of rats Eur J Pharmat ol 66: 307-313, 1980 2 Apfelbaum, M and A Mandenoff Naltrexone suppresses hyperphag~a reduced m the rat by a h~ghly palatable d~et Pharmacol B,~t hem Behav 15: 89-91, 1981 3 Dum, J , C H Gramsch and A Herz Act~vatmn ofhypothalam~c/3-endorph~n pools by reward ~nduced by h~ghly palatable food Pharmacol Btochem Behav 18: 443-447, 1983 4 Frenk, H and G H Rogers The suppressant effects of naloxone on food and water intake m the rat Behav Neural Bml 26: 23-40, 1979 5 Grant, D A Analysts-of-Variance tests m the analys~s and comparison of curves Psythol Bull 53: 141-154, 1956 6 Herman, G and D Nov~n Morphine ~nh~b~tton ofparabrachtal taste umts reversed by naloxone Brain Res Bull 5: Suppl 4, 169-173, 1980 7 Katz, R J Nallrexone antagomsm of exploration m the rat lnt J Neurosct 9: 49-51, 1979 8 Levme, A S , S S Murray, J Kne~p. M Grace and J E Morley Flavor enhances the ant~d~psogemc effect of naloxone Phystol Behav 28: 23-25, 1982 9 L~ebhch, I , E Cohen, J R Ganchrow. E M Blass and F Bergmann Morphine tolerance tn genetically selected rats reduced by chromcaily elevated sacchanne retake St tence 221: 871-873. 1983 10 Locke, K W , D R Brown and S G Holtzman Effects of opmte antagomsts and putative mu- and kappa-agomsts on mdk intake m rat and squirrel monkey Pharmacol Btot hem Behav 17: 1275-1279, 1982

I1 Lynch. W C and L Ltbby Naloxone suppresses ~ntake of h~ghly preferred sacchann solutions m food deprived and sated rats Ltfi" St~ 33: 1909-1914, 1983 12 Pert, A.. L A DeWald. H Lmo and C Siv~t Effects ofop~o~d pept~des on motor behawors s~tes and mechanisms of action In Endorphms m Mental Health Researt h. edited by E Usdm. W E Bunney. Jr and N S Khne New York Oxford U Press, 1979, pp 45-61 13 Rockwood, G A and L D Reid Naloxone modifies sugarwater retake m rats dnnk~ng w~th open gastric fistulas Phv.~,d Behav 29:1175-1178, 1982 14 Slvly, S M , D J Calcagnettl and L D Re~d Oplolds and palatabd~ty in lhe Neural Bast.~ o f Feeding and Reu ard. edited by B G Hoebeland D Novm Brunswick. ME Haerlnst~tute. 1982, pp 517-524 15 Ukal. M and T Kameyama Naloxone speofically blocks the hnear locomotion m mice Brain Res 328: 378-380, 1985 16 Walker, J M , G G Bernston, T S Paulucc~ and T C Champney Blockade of endogenous opiates reduces activity m the rat Pharma~ol Bmthem Behav 14:113-I 16, 1981 17 Wu, M - F , S E Cruz-Morales. J R Qmnan, J M Stapleton and L D Reid Naloxone reduces fired consumption Relat~onsh~p of th~s effect to conditioned taste aversion and morphine dependence Bull Psythonom Sot 14: 323-325. 1979 18 Wu, M - F , M D L~nd, J M S t a p l e t o n a n d L D Reid Doserelationship between naloxone rejections and retake of sucrose solutmn Bull Psyt honom Sot 17: 101-103, 1981