The effects of zimeldine on voluntary ethanol consumption: Studies on the mechanism of action

Alcohol. Vol. 2, pp. 343-347, 1985. © Ankho InternationalInc. Printed in the U.S.A.

0741-8329/85 $3.00 + .00

The Effects of Zimeldine on Voluntary Ethanol Consumption: Studies on the Mechanism of Action K . G I L L , Z. A M I T A N D S. O. O G R E N *

Centre f o r Studies on Behavioral Neurobiology, Concordia University 1455 de M a i s o n n e u v e Blvd., Montreal, Quebec, H 3 G I M 8 *Astra L a k e m e d e l A B , Sodertalje, S w e d e n

GILL, K., Z. AMIT AND S. O. OGREN. The effects of zimeldine on voluntary ethanol consumption: Studies on the mechanism of action. ALCOHOL 2(2) 343-347, 1985.mResearch from our laboratory has shown that several specific serotonin uptake blockers (zimeldine, fluoxetine, sertraline) are effective in reducing voluntary ethanol consumption in rats. However, the mechanism of action of these drugs is not well understood. The series of experiments presented here examined whether zimeldine produces its effects on ethanol consumption via a serotonin mediated anorexic action. In addition the effects of chronic administration of zimeldine were examined in rats drinking a dextrose solution, as well as in ethanol-consuming animals following 5-HT depletion with p-chloroamphetamine. The results indicate that zimeldine reduces the consumption of ethanol, dextrose and saccharin solutions. However, these effects on fluid consumption are not blocked by prior serotonin depletion. The results are discussed in terms of serotonin's role in this process in general and the possibility that zimeldine's effects are not directly related to its capacity to block the reuptake of serotonin. Zimeldine

Serotonin

Serotonin uptake blockade

Anorexia

A great deal of evidence suggests that serotonin (5-HT) plays an inhibitory role in the regulation of consummatory behavior. Despite the many methodological variations employed in paradigms which measure anorexia (i.e., food deprivation vs. free feeding, liquid diets vs. solids and the use of various measures pertaining to the initiation, duration and frequency of feeding) there is a consensus that drugs which increase the levels o f cerebral serotonin (i.e., 5-HTP) or the release of serotonin (i.e., fenfluramine), as well as 5-HT agonists (i.e., quipazine) and uptake blockers (i.e., fluoxetine) produce " a n o r e x i a " in rats [3, 7, 10, 19, 24]. Within the body of literature pertaining to alcohol consumption there is considerable evidence that serotonergic manipulations such as 5-hydroxytryptophan (5-HTP) administration [8, 9, 25] or treatment with serotonin uptake blockers such as zimeldine or fluoxetine [13, 15, 17] reduce the consumption of ethanol. The interpretation of the effects of zimeldine on ethanol intake for example has rested on the hypothesis that this drug produced a blockade o f the positive reinforcing properties o f ethanol [16]. These authors reported that zimeldine had no effect on the consumption of a quinine-sucrose solution suggesting that the drug's effects were specific to alcohol. H o w e v e r more recent work (Gill, et al., in preparation) has shown that zimeldine appears to produce an unconditioned suppression o f saccharin consumption when given as a pretreatment. In addition, reports that fluoxetine has a potent, short duration anorectic activity [ 10,23] have raised the question whether zimeidine operates on ethanol consumption via a similar " a n o r e c t i c " mech-

Voluntary ethanol consumption

anism. The following experiments were carried out to test this hypothesis. EXPERIMENT I Wortman and Wurtman [23] have shown that fluoxetine appears to specifically suppress the consumption of carbohydrates while sparing protein consumption. These authors suggested that the consumption of carbohydrate accelerates serotonin synthesis (by altering plasma amino acid patterns and increasing circulating levels of tryptophan) which would in turn reduce further elective carbohydrate consumption via a negative feedback mechanism. Therefore, when synaptic levels of 5-HT are increased by drugs such as fenfluramine or fluoxetine animals would tend to reduce their selection o f carbohydrates. If one considers voluntary ethanol consumption as a source of elective carbohydrates it is possible that zimeldine may be acting via the mechanism proposed by Wurtman and Wurtman [23]. We have shown that zimeidine pretreatment produces an unconditioned suppression of saccharin intake (unpublished observations) however, this study was carried out in fluid deprived animals in a forced choice paradigm. Therefore, in the following study the effects o f zimeldine on voluntary free-choice consumption of the carbohydrate dextrose as well as saccharin and ethanol solutions were examined.

Procedure Male Long Evans rats (Charles River, Canada) weighing

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G I L L . AMIT A N D OGREN

125-150 grams at the start of the experiment were used. Rats were individually housed in standard stainless steel cages and were maintained in a temperature and humidity controlled environment on a 12 hour dark/light cycle. Fluids were presented in two Richter tubes mounted on the front of the cages. F o o d and water were available ad lib. Following 5 days acclimatization to the animal colony a screening procedure was initiated where rats received ethanol, dextrose or saccharin solutions presented in an ascending series of concentrations on alternate days in a free choice with water. Initially rats were presented with either a 2% (v/v) ethanol, 3% (w/v) dextrose or 0.1% (w/v) saccharin solution. On each presentation the concentration of the ethanol and dextrose solutions were increased by 1% and the saccharin solution by 0.05%. Once final concentrations of 10% ethanol, 14% dextrose and 0.6% saccharin were reached rats were switched to an everyday presentation schedule and quinine sulphate (0.015%, w/v) was added to both the dextrose and saccharin solutions in order to reduce consumption to a level comparable to that of the ethanol consuming group. The concentration of 14% dextrose was chosen to be approximately isocaioric to the 10% ethanol solution. Once consumption had stabilized rats in each group were matched for baseline intake and were assigned to either a saline (2 ml/kg) or zimeldine (20 mg/kg) group and were injected IP for 5 consecutive days.

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The data shown in Fig. 1 are expressed as the mean reference ratio (calculated as % of total daily fluid intake) for the dextrose, saccharin and ethanol consuming groups during the three periods of the experiment. During the injection period the consumption of all three fluids was reduced in those rats treated with zimeldine (ZIM) compared to saline injected controls. Analysis of Variance (with repeated measures) yielded significant drug x days interactions as follows: ethanol, F(14,140)=2.63, p<0.002; dextrose, F(14,140)= 5.14, p<0.001 ; saccharin, F(14,140)=4.5, p<0.001. Post hoc (Tukey) analysis indicated that zimeidine treatment significantly (,o<0.05) reduced preference for all three solutions compared to saline treated controls. Discussion

These data indicate that zimeldine produced a significant reduction in voluntary consumption of ethanol as well as dextrose and saccharin solutions. Given the non-caloric nature of saccharin, it appears that the consumption of highly preferred solutions in general rather than only those solutions having caloric content were reduced following zimeldine treatment. As discussed above the " a n o r e c t i c " effects of a diverse number of serotonin manipulations are well documented. However there are some inconsistencies within this body of literature which raise questions whether " a n o r e x i a " can in fact be considered a unitary phenomenon mediated by central serotonin [4,6]. The development of tolerance to the effects of various anorectic agents is an example o f one inconsistency [2,18]. This is considered in the following experiment. EXPERIMENT

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fluoxetine on ethanol consumption has been shown to occur [1], however tolerance to the inhibitory effects on food consumption have not been demonstrated [18]. Rapid development of tolerance to the effects of other anorectic agents such as fenfluramine or quipazine has been exhibited in a number of paradigms, however these agents do not appear to produce cross-tolerance [2,18]. These findings imply that the mechanisms underlying the anorectic potency of these various agents may be different and several authors have raised the possibility that " a n o r e x i a " may involve non-serotonergic [2] and/or peripheral systems [4,6]. As shown in the previous experiment tolerance to the administration of zimeldine did not appear to develop during the 5 day injection period and in fact consumption remained low in the post-injection phase. Previous work using Sprague-Dawley rats has typically demonstrated tolerance to the effects of zimeldine and norzimeldine after 4--7 daily injections [ 1]. The following experiment was carried out to further examine the effects of chronic administration of zimeldine, in the Long Evans strain of rats. Procedure

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FIG. 2. Mean dextrose preference for those animals treated with zimeidine (20 mg/kg) or saline for a 10 day period. trose consumption and were maintained on a 14% (w/v) dextrose solution which contained 0.015% (w/v) quinine sulphate. Once baseline intake had stabilized animals were matched for dextrose comsumption, divided into two groups and were injected for 10 consecutive days with either saline (2 ml/kg) or zimeldine (20 mg/kg).

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As shown in Fig. 2 mean dextrose preference during the injection phase was reduced in the zimeidine treated group. Analysis of Variance (with repeated measures) yielded a significant drug × days interaction, F(19,152)=2.91, p<0.001). Post hoc (Tukey) tests indicated that the dextrose preference was significantly reduced during the entire injection phase (0<0.05). Absolute consumption of dextrose (mi) in the ZIM treated group was also significantly reduced (0<0.05) during the injection phase compared to saline treated controls. EXPERIMENT

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In order to further characterize the role of 5-HT in mediating the consummatory response to zimeldine this study was carried out in which central serotonin was depleted with the neurotoxic agent p-chloroamphetamine (PCA) prior to chronic treatment with zimeldine. Previous studies examining the effects of 5-HT depletion on anorexia produced by agents such as fenfluramine or quipazine have yielded conflicting results [2, II, 19, 22]. The various procedures employed to deplete 5-HT (lesions vs. neurotoxins), strain differences and the varying time intervals employed between producing the depletion and testing for anorexia may have contributed to these discrepancies. In a previous report [1] we presented preliminary data indicating that prior 5-HT depletion with PCA did not attenuate the effectiveness of zimeldine in reducing ethanol intake. However the Sprague-Dawley rats used in that study displayed rapid tolerance to the effects of zimeldine and it was not clear whether 5-HT depletion blocked this tolerance or whether in fact it potentiated the effects of zimeldine. The dose of PCA used to deplete 5-HT was 2 x l 0 mg/kg which has been demonstrated to reduce brain levels of 5-HT, 5-HIAA, tryptophan hydroxylase and the high affinity uptake of 5-HT [12,21].

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FIG. 3. Mean ethanol consumption (grams absolute ethanol/kg/day) during the baseline, injection and post-injection periods for the PCA and saline pretreated animals subsequently treated with zimeidine (20 mg/kg) or saline. periment 1. Animals were screened for alcohol consumption and were maintained on a 10% (v/v) ethanol solution until baseline intake had stabilized. At this time the animals were divided into two groups which received either PCA (10 mg/kg) or saline (2 ml/kg) on two consecutive days. For the f'wst 3 days following the PCA injections animals exhibited a large drop in body weight and decreases in both water and ethanol consumption. However, by the 4th day animals had resumed drinking alcohol and were gaining weight. Following a 10 day recovery period during which time baseline intake restabilized the PCA and saline pretreated groups were further divided into two groups and were injected with either saline (2 ml/kg) or zimeidine (20 mg/kg) IP for I0 consecutive days. At the termination of the experiment rats were sacrificed by decapitation and brains were rapidly extracted, washed with saline and frozen over dry ice. Determinations of whole brain 5-HT and 5-HIAA content were made by HPLC with electrochemical detection. Brains were weighed, homogenized in five volumes of 0.1 M HCIO, and centrifuged at 10,000 rpm for 10 min. The clear supematant was rdtered (Bioanalytical Systems Microfilter) and an aliquot was injected directly onto a reverse phase Ct~-u Bondapac Column (Waters Scientific Ltd). The mobile phase consisted of a 0.1 M sodium phosphate buffer, pH 3.8 containing 0,1 mM EDTA and 10% methanol (v/v). The flow rate was set at 1.0 ml/min. The electrochemical detector (Bioanalytical Systems) employed a glassy carbon electrode which was maintained at +0.78 V with respect to the reference electrode. Results

Procedure

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Mean ethanol consumption expressed as grams of absolute ethanol/kg/day during the three phases of the experiment is shown in Fig. 3. It appears from these data that prior 5-HT

346

GILL, AMIT AND OGREN TABLE 1 THE EFFECTSOF PCA ON WHOLE BRAIN 5-HT AND 5-HIAACONTENT

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Results are expressed as the mean (_+S.E.M.) in ng of 5-HT or 5-HIAA/g wet weight brain. *Significantly different from saline controls (p<0.001) by twotailed t-test. depletion with PCA does not attenuate the reduction of ethanol intake following zimeidine treatment. Analysis of Variance (with repeated measures) yielded significant drug x day interactions as follows: PCA pretreated, F(19,190)= 3.02, p<0.001; saline pretreated, F(19,190)=2.07, p<0.007. Table l reports the results of the brain amine assay. PCA produced a 65% reduction in the whole brain content of both 5-HT and 5-HIAA. GENERAL DISCUSSION In this series of experiments we have examined the "anorectic" property of zimeldine and its relevance to this drug's effects on voluntary ethanol intake. Zimeldine decreased the preference for and absolute consumption of a number of flavoured solutions. In the Long Evans strain of rats tolerance did not appear to develop during a 10 day injection period and prior 5-HT depletion did not attenuate the effectiveness of zimeldine in reducing ethanol intake. Under the broadest definition of "anorectic" as for example, a compound which reduces feeding or drinking, it is clear that zimeldine produces anorexia. It is also clear however that despite the fact that many anorectic drugs act on the

serotonergic system their mechanisms of action may be different and may be unrelated to their effects on the central 5-HT system [2, 4, 6, 11, 18], It is possible that fenfluramine for example acts at a peripheral locus of action by delaying gastric clearance [4]. An example of where this most clearly is important to the present argument is the article of Geller et al. [9] reporting that the decarboxylase inhibitor RO 4-4602 was capable of blocking the effects of 5-HTP in reducing ethanol intake. These authors attributed this blockade to a reduction in central 5-HT and stated that it provided further support for the involvement of brain serotonin in voluntary ethanol intake. However recent work [5] has provided evidence that in fact administration of RO 4-4602 increases brain levels of 5-HT while decreasing peripheral 5-HT, thus accounting for the blockade of the behavioral effects of 5-HTP. Another line of evidence also calls into question the role of serotonin in general and 5-HT uptake in particular as the mediator of the "anorexic" effects of zimeldine. Samanin et al. [20] have pointed out that in general much higher doses of 5-HT uptake blockers are necessary to produce anorexia than are needed to effectively block 5-HT uptake. In addition, they found that LM-5008 (a specific 5-HT uptake blocker) did not produce anorexia. This suggests that 5-HT uptake blockade per se may not be a sufficient factor to induce anorexia. Taken together there are many discrepancies within the body of literature pertaining to the role of serotonin in consummatory behavior in general which may be resolved by further examination of the properties--both central and peripheral--of the drugs that are used to manipulate this system. Zimeldine's action on the intake of alcohol and other flavoured solutions may not be directly related to this drug's effects on the serotonin system. As previously pointed out [l] there are several alternate explanations of this effect which include possible direct actions on other neurotransmitter systems. It is also possible that zimeldine alters taste sensitivity in a manner similar to that reported for opiate antagonists [14]. Further research will resolve these issues.

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ZIMELDINE AND ETHANOL CONSUMPTION 14. Levine, A. S., S. S. Murrary, J. Kneip, M. Grace and J. Morley. Flavor enhances the antidipsogenic effect of naioxone. Physiol Behav 28: 23-25, 1982. 15. Rockman, G. E., Z. Amit, G. Cart, Z. W. Brown and S. O. Ogren. Attenuation of ethanol intake by 5-hydroxytryptamine uptake blockade in laboratory rats. I. Involvement of brain 5-hydroxytryptamine in the mediation of the positive reinforcing properties of ethanol. Arch Int Pharmacodyn Ther 241: 245-259, 1979. 16. Rockman, G. E., A. Amit, G. Carr and S. O. Ogren. Attenuation of ethanol intake by 5-hydroxytryptamine uptake blockade in laboratory rats. II. Possible interaction with brain norepinephrine. Arch lnt Pharmacodyn Ther 241: 260-265, 1979. 17. Rockman, G. E., Z. Amit, Z. W. Brown, C. Bourque and S. O. Ogren. An investigation of the mechanisms of action of 5-hydroxytryptamine in the suppression of ethanol intake. Neuropharmacology 21: 341-347, 1982. 18. Rowland, N., S. M. Antelman and D. Kocan. Differences among "serotonergic" anorectics in a cross-tolerance paradigm: do they all act on serotonin systems? Eur J Pharmacol 81: 57-66, 1982.

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