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Selected opioids, ethanol and intake of ethanol
Alcohol, Vol. 1, pp. 43-46, 1984. ©AnkhoInternationalInc. Printed in the U.S.A.
0741-8329/84$3.00 + .00
Selected Opioids, Ethanol and Intake of Ethanol G E O R G E A. H U N T E R , C A R O L M. B E A M A N L A U R A L. D U N N A N D L A R R Y D. R E I D 1
Department o f Psychology Rensselaer Polytechnic Institute, Troy, N Y 12181 R e c e i v e d 19 S e p t e m b e r 1983 HUNTER, G. A., C. M. BEAMAN, L. L. DUNN AND L. D. REID. Selected opioids, ethamd and intake ofetham~l. ALCOHOL t(1) 43-46, 1984.--Rats were given an opportunity to drink tap water or a sweetened ethanol solution once a day. Across initial days of opportunity, rats increased their intake of the ethanol solution. Prior to some days' sessions with presented fluids, rats received either an injection of placebo (the carrier of drugs) or doses of ethylketocyclozocine, diprenorphine, or ethanol. Diprenorphine increased rats' intake of the ethanol solution compared to placebo. The other agents did not reliably modify intakes. These findings support a conclusion that selected activity in opioid systems of brain increase the propensity to drink alcoholic beverages. Opioids
Ethanol
Diprenorphine
Opiates
Ethylketocyclazocine
Ingestion
Alcoholism
cumstances for more ethanol intake. There has been little or no study of this phenomenon in the experimental laboratory. Consequently, we asked the question of how do injections of ETOH modify ingestion of a palatable ethanol solution among rats that have developed a taste for an ethanol solution.
WE [ 1,16] have recently shown that morphine increases rats' avidity toward sweetened ethanol solutions. This effect has been shown to be relatively specific, occurring with morphine, but not with a benzodiazepine. Also, morphine does not increment intake of a sucrose solution containing no ethanol. From these limited observations, it was inferred that any stimulus increasing endogenous opioid activity, including ethanol intake, may set the circumstances for more avid intake of ethanol [16]. In this report, we relate the effects of injections of ethylketocyclazocine (EKC), diprenorphine (DIP) and ethanol (ETOH) on rats' intakes of sweetened ethanol solutions using procedures similar to those showing morphine increases intake of ethanol solutions. EKC was tested because it is a prototypic agonist of the kappa opioid receptor, one of the proposed types of opioid receptors [12]. The actions of EKC and related compounds are thought to mimic the actions of dynorphin and similar fragments of the prepromolecule of dynorphin [5,10]. EKC injections do increase day-time feeding in nondeprived rats, but produces only limited increments in drinking [13]. I f E K C increments intake of ethanol solutions, it is reasonable to infer that the dynorphin-kappa opioid receptor system may be involved in ethanol reinforcement. If EKC does not increase avidity toward a palatable ethanol solution as does morphine, it is reasonable to infer that opioid actions other than those involving kappa opioid systems may be involved. DIP is a mixed opioid agonist-antagonist, having no analgesic potential and blocking other opioid's analgesia, but having the capability of increasing pressing for rewarding brain stimulation [15]. DIP's effects can be used as an unconditioned stimulus for the establishment a conditioned place preference [2], an index of the capability of an opioid to elicit positive affect I14,17]. For some individuals, ethanol intake seems to set the cir-
METHOD
Subjects Forty-two, male, Sprague-Dawley derived rats were obtained from Taconic Farms, Germantown, NY, weighing between 160 and 200 g. The rats were housed individually with food always available, and the colony room was maintained at 24°C, on a schedule of 12 hr of light/day (lights on at 1000 hr). These rats were, for 24 days prior to the beginning of these procedures, in a similar experiment [1] using the same regimen of deprivation and water ethanol solution presentation as used here. At the beginning of this study, the rats weighed between 230 and 350 g.
DruL~s Diprenorphine (DIP, Reckitt and Colman), ethytketocyclazocine methanesulfonate (EKC, Sterling-Winthrop), and ethanol (ETOH) were used. DIP was dissolved in 1% acetic acid and then further diluted with saline to 0.005, 0.025 and 0.05 mg/ml. EKC was mixed with saline at doses of 0.02, 0.04, and 0.08 mg/ml expressed as a base. DIP and EKC were administered subcutaneously at a volume of 1 ml/kg. ETOH was mixed with saline to a concentration of 25, 50 and 100 mg/ml and administered intraperitoneally at a volume of 10 ml/kg, yielding doses of 250, 500, and 1000 mg/kg. Physiological saline, in appropriate volumes, was used for placebo injections.
'Requests for reprints should be addressed to L. D. Reid.
43
44
HUNTER, BEAMAN. DUNN AND REID 12
Pro(!t'dure
........
hi
Every day at 1 hr into the light cycle, the rats were given a choice to drink from two bottles, one containing tap water and the other containing sweetened water, with 6% ethanol. The ethanol-sucrose-solution (ESS) was mixed by combining 6% ethanol (wt/wt), 5% sucrose (wt/wt), and tap water. After 1 hr, both bottles were removed and weighed to the nearest 0.1 g. The water bottle was then returned for 3 more hours. Then, the rats were deprived of all fluids until the next daily session. The rats were divided into 3 groups (low, middle, or high dose), 14 rats/group, with subjects assigned factorially on the basis of their history. Each group was divided in half. Half the rats got placebo on one day, the other half got placebo the next day (as a control for day-effects). On test days, the rats were given either a drug or placebo injection 30 rain before they received the bottles. The rats received a placebo injection and on the following day a drug. The procedure is divided into 3 consecutive blocks of sessions (one for DIP. EKC, and ETOH in that order) of 3 days each, with 3 days in between.
l ) , t a Reduction and Statistical Analy.~i.~ There are four measures of rats' performance: g of water taken, g of ESS taken, total fluid taken, and preference ratio (g of ESS/total fluid taken). Here we report two of those measures, g of ESS and preference ratio. We did a factorial analysis of variance (ANOVA) for repeated measures using data (g of ESS or preference ratios) across all drug injections and that ANOVA indicated that the factor associated with kind of injections was a reliable source of variance as well as certain interactions indicating the appropriateness of more specific analyses. Consequently, an ANOVA for repeated measures was done for each measure for each drug. That ANOVA was a 3 (three groups getting different doses) by 2 (placebo versus drug) factorial for repeated measures. When the ANOVA yielded a reliable main effect associated with placebo versus drug injections, or a reliable interaction between kind of injections by dose level, then t-tests for dependent measures were used to assess the reliability of differences between placebo and drug scores. RESUI,TS
Figure 1 presents a tabulation of ethanol solution and water taken across days at selected times throughout the rats' histories of opportunity to take ESS. Notice that initial experience with the ethanol solution leads to a rapid esculation of ethanol intake. After about two weeks, intakes increased only gradually. A summary of the results following injections are depicted in Fig. 2. As can be seen (left panels), DIP reliably increased amount of ESS taken and preference ratios. With the data of ESS-intakes associated with DIP injections, the ANOVA also yielded a reliable interaction term, F(2,39)=3.79, p=0.03. Tests for differences at each dose indicated that DIP reliably incremented intakes at only the two smaller doses, (ps<0.05), thereby accounting for the reliable interaction. DIP at the highest dose tends to decrease fluid intake, but still induces a preference for ethanol (lower left panel). With the ANOVA of preference ratios, only the factor associated with the drug-placebo main effect was a reliable source of variance. With the ANOVA of the EKC-data, none of the resulting
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DFIY5 FIG. 1. Mean grams of sweetened ethanol solution and water taken by rats. The rats were given an opportunity to take ethanol solution daily for one hour, along with water, and their daily intakes measured periodically. Data points are the means of baseline measures (days of no injections). Rats were given doses of drugs (placebos. benzodiazepines, or opioids) on days between baseline measures.
values meet the standards for statistical significance. With the data associated with ETOH injections, the factor associated with groups and dose of injections was a reliable source of variance but this is due to the different baseline intakes and not the experimental variables. No other factor was a reliable source of variance. The analysis indicates that ETOH injections do not reliably modify subsequent ethanol intake. DISCUSSION
There was considerable variation among subjects in their propensity to consume the ESS. Some rats took large quantities of ESS while others took only small amounts under placebo even after many days of opportunity to drink (the range of g of ESS-intake across all subjects under the last placebo injection was between 0.7 g to 17.7 g, range 0f preference ratios was between 0.05 to 0.94). This considerable variability across subjects randomly assigned to groups with small numbers resulted in groups having differing mean mtakes under placebo conditions. Since groups and drugdosage are confounded in these procedures, caution should be exercised in drawing conclusions about the effects of dose on subsequent intake. This is particularly the case with the trials associated with ETOH-injections. This is the third of a series of studies associated with testing the idea that enhanced occupation and activation of opioid receptors increases rats" avidity for ~weetened ethanol solutions. These data confirm that injections of opioids can increase rats' drinking of the ethanol solution. and also begins the process of specifying the type of opioid receptor that may be involved. There is no basis in ~these data to support the idea of involvement of the kappa opioid receptor system. A receptor type for which MOR and DIP are agonists may be critical to opioids" capability to enhance ethanol intake. This receptor type is probably not the one which mediates analgesia, since DIP is clearly an antagonist with respect to opioid analgesia |I51.
OPIOIDS AND E T H A N O L I N T A K E
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.02 .Oq .08 ETHYLKETOCYCLAZOCINE
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FIG. 2. The top row depicts mean g of sweetened ethanol solution taken after an injection of placebo or drug. The bottom row depicts mean preference ratios (g of ESS/total fluid) after injections. The F-values and p-values are for the factor of an ANOVA associated with placebo versus drug injections. An asterisk indicates that a t-test between scores of placebo and a dose of a drug yields a wdue associated with/7< 0.05.
It is doubtful that DIP increases intake of the ethanol solution because it contains sucrose. DIP, at the doses used here, either had no effect or decreased intake of sucrose solutions having no ethanol [16]. With only the data showing that morphine will enhance avidity toward ethanol solutions, it is possible to conclude that morphine may be enhancing intake of the sweetened ethanol solution by merely being analgesic. Morphine could blunt any pain or discomfort associated with high concentrations of ethanol thereby allowing rats to take more sucrose solution. This is, however, an unlikely reality in light of the results of these tests. EKC is an analgesic and it does not increment intake of ethanol solutions. DIP is an antagonist toward opioid analgesia and it does increment opioid activity. So, it can be concluded that some other property of opioids, other than their potential to induce analgesia (also see [4]), is responsible for opioids' ability to increase rats" avidity for palatable ethanol solutions. Recently, Berridge and Grill [3] have acquired evidence supporting a two-dimensional model of palatability, with one dimension corresponding to a negative continuum and the other a positive continuum. Different opioid systems may be involved with the expression of each continuum, one opioid system related to analgesia dampening the negative continuum and one opioid system related to euphoria potentiating the positive continuum [3]. The data presented here, indicating no relationship between analgesic potential and increments in avidity toward palatable ethanol solutions and a relationship between opioids that can enhance signs of posilive affect and the enhancement of ethanol intake, lead to the conclusion that opioid modulation of ethanol intake is by way of mechanisms of the positive continuum of Berridge and Grilrs model. Intuitively, one might presume that injections of ethanol would decrease ethanol ingestion. In general, the rats seem to regulate their intake of ethanol, taking less ethanol solution the higher the concentration of ethanol. From another
perspective, the effects of ethanol should increment ethanol intake, presumably because ethanol might engage opioid mechanisms. Our data do not provide strong support for either proposition. Ethanol injections did not modify ethanol intakes. It is, however, interesting to note that ETOHinjections did not diminish subsequent ESS intake. The injection and testing regimen probably lead to peak brain ethanol levels during the opportunity to drink the ESS [9]. Ethanol apparently does not engage satiation mechanisms in the same way other "nutrients" may. At least, circulating ethanol does not necessarily dampen rats' propensities to take ethanol. These experiments reflect a somewhat different way of asking questions about putative opioid involvement in factors controlling ethanol intake. Any underlying assumption of this approach is that the critical feature of the addiction syndrome known as alcoholism is the positively reinforcing features o f e t h a n o r s actions. The focus of concern, therefore, is with ethanors positively reinforcing features and, relatedly, loss-of-control. This approach does not deny the importance of withdrawal phenomena, the neurological damage and other health problems accompanying advanced stages of alcoholism, or the general effects of large doses of ethanol, such as fluidization of membranes [ 18], as being important factors in the global assessment of alcoholism. The approach does presume that the long-term consequences of ethanol consumption follow from the fact that the effects of ethanol intake are reinforcing and that the intake of ethanol leads to further intake. The question, then, is how does ethanol achieve the ability to be positively reinforcing without there being specific central neural receptors for ethanol'? Potentially important parts of the answer to the question of how ethanol reinforces ethanol intake is the finding that ethanol metabolites may be opioid in character [6,8] and the concomitant knowledge of the endogenous opioid systems [7]. Before concluding that an opioid metabolite is involved with the circumstances of
46
HUNTER, BEAMAN, DUNN AND REID
ethanol reinforcement, it seems reasonable to ask w h e t h e r k n o w n opioids will e n h a n c e ethanol intake. These experiments lead to the answer: Yes, certain opioids can increase a rat's propensity to take a palatable ethanol solution. Furt h e r m o r e , the opioids' actions are relatively specific. Also, r e m a r k a b l y small doses of diprenorphine, an opioid, are effective. This surely opens the possibility that ethanol may engage an opioid m e c h a n i s m and, thereby, set the circumstances for ethanol reinforcement and e n h a n c e d propensity 1o take ethanol. T h e r e are a n u m b e r of implications of this line of thinking. The kinds of stresses that have b e e n shown to engage opioid m e c h a n i s m s should enhance intake o f ethanol solutions w h e r e a s those that fail to engage opioid m e c h a n i s m s should be neutral with respect to ethanol intake. The idea that high circulating levels of opioids increase the proclivity to take alcoholic b e v e r a g e s has implications for m e t h a d o n e maintenance prol~rams. An alcoholic beverage would be taken m o r e
avidly if (a) the brewing or fermentation process produced, in addition to ethanol, a c o m p o u n d activating e n d o g e n o u s opioid mechanisms, or (b) an opioid was added to the brew. Since it has recently been shown 1hat saccharin solution intake will produce morphine-like states a m o n g certain rats 111], saccharin and ethanol containing beverages may be particularly likely to be c o n s u m e d in large quantities by certain individuals,
ACKNOWI.EDGE M['NTS This research was supported, in p:uL b~, New York State Health Research Council, Grant HRC No. 13-109. We would like to thank Jean Bestle and Betty Osganian for help in preparation of this manuscript. C. Beaman and L, Dunn were supported by the Federal Work-Study program for undergraduates administered by Rensselaer Polytechnic Institute. We thank the respective manufacturers of drugs for gifts of those drugs
REFERENCES 1. Beaman, C. M., G. A. Hunter, L. L. Dunn and L. D. Reid. Opioids, benzodiazepines and intake of ethanol. Ah.ohol I: 3%42, 1984. 2. Beaman, C. M., G. A. Hunter and L. D. Reid. Diprenorphine, an antagonist of opioid analgesia, elicits a positive affective state in rats. In editorial review. 3. Berridge, K. C. and H. J. Grill. Alternating ingestive and aversive consummatory responses suggest a two-dimensional analysis of palatability in rats. Behav Neurosci 97: 563-573, 1983. 4. Bozarth, M. A. and R. A. Wise. Dissociation of the rewarding and physical dependence-producing properties of morphine. In: Problems o f Drug Dependence, edited by L. S. Harris. Rockville, MD: National Institute on Drug Abuse, 1982. 5. Chavkin, C. and A. GoIdstein. Dynorphin is a specific endogenus ligand of the k opioid receptor. Science 215:413-415. 1982. 6. Cohen, G. and M. Collins. Alkaloids from catecholamines in adrenal tissue: possible role in alcoholism. Science 167: 174% 1751, 1970. 7. Cox, B. M. Endogenous opioid peptides: A guide to structures and terminology. Life Sci 31: 1645-1658, 1982. 8. Davis, V. E. and M. J. Walsh. Alcohol, amines and alkaloids: a possible biochemical basis for alcohol addiction. Science 167: 1005-1007, 1970. 9. Holloway, F. A. and R. A. Wansley. Differential sensitivity of different discrimination behaviors to the effects of ethanol. Bull Psychonom Soc 2: 15%162, 1973.
10. James, 1. F.. C. Chavkin and A. Goldstein. Selectivity of dynorphin for/, opioid receptors. LiJe Sci 31: t331-1334, 1982 11. Lieblich, I., E. Cohen. J. R Granchrow. E M. Blass and F. Bergman. Morphine tolerance m genetically selected rats induced by chronically elevated saccharin intake St ieme 221: 871-873. 1983. 12. Martin, W. R., C. G. Eades..I ,x. lhompson, R. E. Huppler and P. E. Gilbert. The effects of morphine and nalorphine-like drugs in the non-dependent and morphine-dependent chronic spinal dog. J Pharmacol Exp Ther 197: 517-532. 1976. 13. Morely, J. E., A. S. Levine. M. Grace and J. Kniep. An investigation of the role of kappa opiate receptor agonists in the initiation of feeding. Life Sci 31: 2617-2626, 1982. 14. Mucha, R. F., D. van der Koov. M. O'Shaughnessy and P. Bucenieks. Drug reinforcement studied by the use of place conditioning in rats. Brain Res 243: 91-105. 1983. 15. Pollerberg, G. E., T. Costa. G. T. Shearman. A. Herz and L. D. Reid. Opioid antinociception and positive reinforcement are mediated by different types of opioid receptors. Lifb Sci 33: 154%1559, 1983. 16. Reid, L. D. and G. A. Hunter. Morphine and naloxone modulate intake of ethanol. Alcohol 1: 33-37. 1984. 17. Rossi, N. A. and L. D. Reid. Affective states associated with morphine injections. Phvsiol Psvchol 4: 26%274. 1976. 18. Tabakoff, B. Current trends in biologic research on alcoholism. Drug Alcohol Depend !1: 33-37. 1983.
0741-8329/84$3.00 + .00
Selected Opioids, Ethanol and Intake of Ethanol G E O R G E A. H U N T E R , C A R O L M. B E A M A N L A U R A L. D U N N A N D L A R R Y D. R E I D 1
Department o f Psychology Rensselaer Polytechnic Institute, Troy, N Y 12181 R e c e i v e d 19 S e p t e m b e r 1983 HUNTER, G. A., C. M. BEAMAN, L. L. DUNN AND L. D. REID. Selected opioids, ethamd and intake ofetham~l. ALCOHOL t(1) 43-46, 1984.--Rats were given an opportunity to drink tap water or a sweetened ethanol solution once a day. Across initial days of opportunity, rats increased their intake of the ethanol solution. Prior to some days' sessions with presented fluids, rats received either an injection of placebo (the carrier of drugs) or doses of ethylketocyclozocine, diprenorphine, or ethanol. Diprenorphine increased rats' intake of the ethanol solution compared to placebo. The other agents did not reliably modify intakes. These findings support a conclusion that selected activity in opioid systems of brain increase the propensity to drink alcoholic beverages. Opioids
Ethanol
Diprenorphine
Opiates
Ethylketocyclazocine
Ingestion
Alcoholism
cumstances for more ethanol intake. There has been little or no study of this phenomenon in the experimental laboratory. Consequently, we asked the question of how do injections of ETOH modify ingestion of a palatable ethanol solution among rats that have developed a taste for an ethanol solution.
WE [ 1,16] have recently shown that morphine increases rats' avidity toward sweetened ethanol solutions. This effect has been shown to be relatively specific, occurring with morphine, but not with a benzodiazepine. Also, morphine does not increment intake of a sucrose solution containing no ethanol. From these limited observations, it was inferred that any stimulus increasing endogenous opioid activity, including ethanol intake, may set the circumstances for more avid intake of ethanol [16]. In this report, we relate the effects of injections of ethylketocyclazocine (EKC), diprenorphine (DIP) and ethanol (ETOH) on rats' intakes of sweetened ethanol solutions using procedures similar to those showing morphine increases intake of ethanol solutions. EKC was tested because it is a prototypic agonist of the kappa opioid receptor, one of the proposed types of opioid receptors [12]. The actions of EKC and related compounds are thought to mimic the actions of dynorphin and similar fragments of the prepromolecule of dynorphin [5,10]. EKC injections do increase day-time feeding in nondeprived rats, but produces only limited increments in drinking [13]. I f E K C increments intake of ethanol solutions, it is reasonable to infer that the dynorphin-kappa opioid receptor system may be involved in ethanol reinforcement. If EKC does not increase avidity toward a palatable ethanol solution as does morphine, it is reasonable to infer that opioid actions other than those involving kappa opioid systems may be involved. DIP is a mixed opioid agonist-antagonist, having no analgesic potential and blocking other opioid's analgesia, but having the capability of increasing pressing for rewarding brain stimulation [15]. DIP's effects can be used as an unconditioned stimulus for the establishment a conditioned place preference [2], an index of the capability of an opioid to elicit positive affect I14,17]. For some individuals, ethanol intake seems to set the cir-
METHOD
Subjects Forty-two, male, Sprague-Dawley derived rats were obtained from Taconic Farms, Germantown, NY, weighing between 160 and 200 g. The rats were housed individually with food always available, and the colony room was maintained at 24°C, on a schedule of 12 hr of light/day (lights on at 1000 hr). These rats were, for 24 days prior to the beginning of these procedures, in a similar experiment [1] using the same regimen of deprivation and water ethanol solution presentation as used here. At the beginning of this study, the rats weighed between 230 and 350 g.
DruL~s Diprenorphine (DIP, Reckitt and Colman), ethytketocyclazocine methanesulfonate (EKC, Sterling-Winthrop), and ethanol (ETOH) were used. DIP was dissolved in 1% acetic acid and then further diluted with saline to 0.005, 0.025 and 0.05 mg/ml. EKC was mixed with saline at doses of 0.02, 0.04, and 0.08 mg/ml expressed as a base. DIP and EKC were administered subcutaneously at a volume of 1 ml/kg. ETOH was mixed with saline to a concentration of 25, 50 and 100 mg/ml and administered intraperitoneally at a volume of 10 ml/kg, yielding doses of 250, 500, and 1000 mg/kg. Physiological saline, in appropriate volumes, was used for placebo injections.
'Requests for reprints should be addressed to L. D. Reid.
43
44
HUNTER, BEAMAN. DUNN AND REID 12
Pro(!t'dure
........
hi
Every day at 1 hr into the light cycle, the rats were given a choice to drink from two bottles, one containing tap water and the other containing sweetened water, with 6% ethanol. The ethanol-sucrose-solution (ESS) was mixed by combining 6% ethanol (wt/wt), 5% sucrose (wt/wt), and tap water. After 1 hr, both bottles were removed and weighed to the nearest 0.1 g. The water bottle was then returned for 3 more hours. Then, the rats were deprived of all fluids until the next daily session. The rats were divided into 3 groups (low, middle, or high dose), 14 rats/group, with subjects assigned factorially on the basis of their history. Each group was divided in half. Half the rats got placebo on one day, the other half got placebo the next day (as a control for day-effects). On test days, the rats were given either a drug or placebo injection 30 rain before they received the bottles. The rats received a placebo injection and on the following day a drug. The procedure is divided into 3 consecutive blocks of sessions (one for DIP. EKC, and ETOH in that order) of 3 days each, with 3 days in between.
l ) , t a Reduction and Statistical Analy.~i.~ There are four measures of rats' performance: g of water taken, g of ESS taken, total fluid taken, and preference ratio (g of ESS/total fluid taken). Here we report two of those measures, g of ESS and preference ratio. We did a factorial analysis of variance (ANOVA) for repeated measures using data (g of ESS or preference ratios) across all drug injections and that ANOVA indicated that the factor associated with kind of injections was a reliable source of variance as well as certain interactions indicating the appropriateness of more specific analyses. Consequently, an ANOVA for repeated measures was done for each measure for each drug. That ANOVA was a 3 (three groups getting different doses) by 2 (placebo versus drug) factorial for repeated measures. When the ANOVA yielded a reliable main effect associated with placebo versus drug injections, or a reliable interaction between kind of injections by dose level, then t-tests for dependent measures were used to assess the reliability of differences between placebo and drug scores. RESUI,TS
Figure 1 presents a tabulation of ethanol solution and water taken across days at selected times throughout the rats' histories of opportunity to take ESS. Notice that initial experience with the ethanol solution leads to a rapid esculation of ethanol intake. After about two weeks, intakes increased only gradually. A summary of the results following injections are depicted in Fig. 2. As can be seen (left panels), DIP reliably increased amount of ESS taken and preference ratios. With the data of ESS-intakes associated with DIP injections, the ANOVA also yielded a reliable interaction term, F(2,39)=3.79, p=0.03. Tests for differences at each dose indicated that DIP reliably incremented intakes at only the two smaller doses, (ps<0.05), thereby accounting for the reliable interaction. DIP at the highest dose tends to decrease fluid intake, but still induces a preference for ethanol (lower left panel). With the ANOVA of preference ratios, only the factor associated with the drug-placebo main effect was a reliable source of variance. With the ANOVA of the EKC-data, none of the resulting
Z
--J ¢2~
I
T IIC
i Z W ~
,
0 0
7
I~
Zl
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$S
Ji t~
DFIY5 FIG. 1. Mean grams of sweetened ethanol solution and water taken by rats. The rats were given an opportunity to take ethanol solution daily for one hour, along with water, and their daily intakes measured periodically. Data points are the means of baseline measures (days of no injections). Rats were given doses of drugs (placebos. benzodiazepines, or opioids) on days between baseline measures.
values meet the standards for statistical significance. With the data associated with ETOH injections, the factor associated with groups and dose of injections was a reliable source of variance but this is due to the different baseline intakes and not the experimental variables. No other factor was a reliable source of variance. The analysis indicates that ETOH injections do not reliably modify subsequent ethanol intake. DISCUSSION
There was considerable variation among subjects in their propensity to consume the ESS. Some rats took large quantities of ESS while others took only small amounts under placebo even after many days of opportunity to drink (the range of g of ESS-intake across all subjects under the last placebo injection was between 0.7 g to 17.7 g, range 0f preference ratios was between 0.05 to 0.94). This considerable variability across subjects randomly assigned to groups with small numbers resulted in groups having differing mean mtakes under placebo conditions. Since groups and drugdosage are confounded in these procedures, caution should be exercised in drawing conclusions about the effects of dose on subsequent intake. This is particularly the case with the trials associated with ETOH-injections. This is the third of a series of studies associated with testing the idea that enhanced occupation and activation of opioid receptors increases rats" avidity for ~weetened ethanol solutions. These data confirm that injections of opioids can increase rats' drinking of the ethanol solution. and also begins the process of specifying the type of opioid receptor that may be involved. There is no basis in ~these data to support the idea of involvement of the kappa opioid receptor system. A receptor type for which MOR and DIP are agonists may be critical to opioids" capability to enhance ethanol intake. This receptor type is probably not the one which mediates analgesia, since DIP is clearly an antagonist with respect to opioid analgesia |I51.
OPIOIDS AND E T H A N O L I N T A K E
d o
45
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0
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.02 .Oq .08 ETHYLKETOCYCLAZOCINE
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tO00
FIG. 2. The top row depicts mean g of sweetened ethanol solution taken after an injection of placebo or drug. The bottom row depicts mean preference ratios (g of ESS/total fluid) after injections. The F-values and p-values are for the factor of an ANOVA associated with placebo versus drug injections. An asterisk indicates that a t-test between scores of placebo and a dose of a drug yields a wdue associated with/7< 0.05.
It is doubtful that DIP increases intake of the ethanol solution because it contains sucrose. DIP, at the doses used here, either had no effect or decreased intake of sucrose solutions having no ethanol [16]. With only the data showing that morphine will enhance avidity toward ethanol solutions, it is possible to conclude that morphine may be enhancing intake of the sweetened ethanol solution by merely being analgesic. Morphine could blunt any pain or discomfort associated with high concentrations of ethanol thereby allowing rats to take more sucrose solution. This is, however, an unlikely reality in light of the results of these tests. EKC is an analgesic and it does not increment intake of ethanol solutions. DIP is an antagonist toward opioid analgesia and it does increment opioid activity. So, it can be concluded that some other property of opioids, other than their potential to induce analgesia (also see [4]), is responsible for opioids' ability to increase rats" avidity for palatable ethanol solutions. Recently, Berridge and Grill [3] have acquired evidence supporting a two-dimensional model of palatability, with one dimension corresponding to a negative continuum and the other a positive continuum. Different opioid systems may be involved with the expression of each continuum, one opioid system related to analgesia dampening the negative continuum and one opioid system related to euphoria potentiating the positive continuum [3]. The data presented here, indicating no relationship between analgesic potential and increments in avidity toward palatable ethanol solutions and a relationship between opioids that can enhance signs of posilive affect and the enhancement of ethanol intake, lead to the conclusion that opioid modulation of ethanol intake is by way of mechanisms of the positive continuum of Berridge and Grilrs model. Intuitively, one might presume that injections of ethanol would decrease ethanol ingestion. In general, the rats seem to regulate their intake of ethanol, taking less ethanol solution the higher the concentration of ethanol. From another
perspective, the effects of ethanol should increment ethanol intake, presumably because ethanol might engage opioid mechanisms. Our data do not provide strong support for either proposition. Ethanol injections did not modify ethanol intakes. It is, however, interesting to note that ETOHinjections did not diminish subsequent ESS intake. The injection and testing regimen probably lead to peak brain ethanol levels during the opportunity to drink the ESS [9]. Ethanol apparently does not engage satiation mechanisms in the same way other "nutrients" may. At least, circulating ethanol does not necessarily dampen rats' propensities to take ethanol. These experiments reflect a somewhat different way of asking questions about putative opioid involvement in factors controlling ethanol intake. Any underlying assumption of this approach is that the critical feature of the addiction syndrome known as alcoholism is the positively reinforcing features o f e t h a n o r s actions. The focus of concern, therefore, is with ethanors positively reinforcing features and, relatedly, loss-of-control. This approach does not deny the importance of withdrawal phenomena, the neurological damage and other health problems accompanying advanced stages of alcoholism, or the general effects of large doses of ethanol, such as fluidization of membranes [ 18], as being important factors in the global assessment of alcoholism. The approach does presume that the long-term consequences of ethanol consumption follow from the fact that the effects of ethanol intake are reinforcing and that the intake of ethanol leads to further intake. The question, then, is how does ethanol achieve the ability to be positively reinforcing without there being specific central neural receptors for ethanol'? Potentially important parts of the answer to the question of how ethanol reinforces ethanol intake is the finding that ethanol metabolites may be opioid in character [6,8] and the concomitant knowledge of the endogenous opioid systems [7]. Before concluding that an opioid metabolite is involved with the circumstances of
46
HUNTER, BEAMAN, DUNN AND REID
ethanol reinforcement, it seems reasonable to ask w h e t h e r k n o w n opioids will e n h a n c e ethanol intake. These experiments lead to the answer: Yes, certain opioids can increase a rat's propensity to take a palatable ethanol solution. Furt h e r m o r e , the opioids' actions are relatively specific. Also, r e m a r k a b l y small doses of diprenorphine, an opioid, are effective. This surely opens the possibility that ethanol may engage an opioid m e c h a n i s m and, thereby, set the circumstances for ethanol reinforcement and e n h a n c e d propensity 1o take ethanol. T h e r e are a n u m b e r of implications of this line of thinking. The kinds of stresses that have b e e n shown to engage opioid m e c h a n i s m s should enhance intake o f ethanol solutions w h e r e a s those that fail to engage opioid m e c h a n i s m s should be neutral with respect to ethanol intake. The idea that high circulating levels of opioids increase the proclivity to take alcoholic b e v e r a g e s has implications for m e t h a d o n e maintenance prol~rams. An alcoholic beverage would be taken m o r e
avidly if (a) the brewing or fermentation process produced, in addition to ethanol, a c o m p o u n d activating e n d o g e n o u s opioid mechanisms, or (b) an opioid was added to the brew. Since it has recently been shown 1hat saccharin solution intake will produce morphine-like states a m o n g certain rats 111], saccharin and ethanol containing beverages may be particularly likely to be c o n s u m e d in large quantities by certain individuals,
ACKNOWI.EDGE M['NTS This research was supported, in p:uL b~, New York State Health Research Council, Grant HRC No. 13-109. We would like to thank Jean Bestle and Betty Osganian for help in preparation of this manuscript. C. Beaman and L, Dunn were supported by the Federal Work-Study program for undergraduates administered by Rensselaer Polytechnic Institute. We thank the respective manufacturers of drugs for gifts of those drugs
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