Norepinephrine turnover and voluntary consumption of ethanol in the rat

Alcohol, Vol. 2, pp. 339--342, 1985. ~ Ankho International Inc. Printed in the U.S.A.

0741-8329/85 $3.00 + .00

Norepinephrine Turnover and Voluntary Consumption of Ethanol in the Rat S. M. SOCARANSKY, C. M. G. ARAGON, I. RUSK, Z. AMIT AND S. O. OGREN Center For Studies in Behavioural Neurobiology, Department o f Psychology, Concordia University 1455 de Maisonneuve Blvd., West, Montreal, Canada H3G I M B and Astra Research and Development Labs, Sodertalje, Sweden

SOCARANSKY, S. M.. C. M. G. ARAGON, I. RUSK, Z. AMIT AND S. O. OGREN. Norepinephrine turnover and voluntary consumption of ethanol in the rat. ALCOHOL 2(2) 339-342, 1985.--The effect of a centrally acting noradrenergic agonist on voluntary ethanol intake was investigated in the laboratory rat. Doses of 1.5, 7.5, 15 and 25 mg/kg of FLA-136 were administered to animals chronically exposed to ethanol on a free choice basis. Administration of the highest dose of the drug brought about a 40% reduction in voluntary ethanol intake with no significant effect on total fluid consumption. These results would seem to indicate a possible functional relationship between rate of norepinephrine turnover and amounts of ethanol voluntarily consumed by the laboratory rat. FLA-136

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T H E voluntary self administration of ethanol in animals has been well documented using a variety of routes of administration. Amit et al. [2] demonstrated that rats would readily consume unadulterated ethanol solutions when given in a free-choice with water. Several reports exist in the literature describing intragastric self administration [8, 9, 21] as well as intraveneous self administration [15,20]. Although a plethora of information exists with respect to the various actions of ethanol, few aspects have received as much attention as the influence of this substance on catecholamine (CA) function in the central nervous system (CNS). There is much evidence to suggest that ethanol may alter the synthesis, turnover and release of central CA in rats [13,17]. Borg et al. [5] showed increased norepinepherine (NE) turnover in alcoholics and healthy controls following acute administration of ethanol. Increased turnover of brain NE following an acute dose of ethanol has also been reported in rats [7, l l, 16]. What is most interesting about the apparent relationship between ethanol and CA function is its seemingly bidirectional character. In other words, not only does ethanol influence CA activity, but manipulations of CA systems may result in diminished affinity of the organism for ethanol consumption. It has been shown that neurochemical lesions of the CA system results in diminished voluntary oral consumption of ethanol [12]. Myers and Meichoir [13] demonstrated a reduction in preference for ethanol following intraventricular administration of the CA neurotoxin 6-OHDA. However, as this manipulation depletes both norepinepherine and dopamine levels, it was not until the subsequent experiment o f Brown and Amit [6] that the reduction in ethanol preference could be linked specifically to N E depletion. In another experiment, the same group demonstrated a similar effect by blocking the enzymatic conversion of

dopamine to norepinepherine through the use of the drugs FLA-63 and FLA-57 (inhibitors of dopamine-betahydrozylase, DBH), thereby reducing whole brain levels of NE [1,3]. Davis et al. [9] reported that rats decreased intragastric self administration of ethanol following injections of the drug FLA-57. The sum of these findings would appear to indicate that the NE system is an important neural substrate for the regulation of ethanol self administration in rats. However, it must be noted that manipulations which block N E synthesis (i.e., DBH inhibition) give rise to a generalized depletion o f all N E neurons [3]. Ideally, a drug which acts selectively to decrease presynaptic release of N E would be a more sophisticated and specific manipulation, as it has been shown that N E activity is regulated by presynaptic receptors [ 18]. Thus, the present experiment employed the drug FLA-136, a relatively new, centrally acting alpha-2 receptor agonist [10]. Given the specificity of action of this compound [4], it could serve as one of the most useful pharmacological tools yet developed in the investigation of the involvement of NE in the regulation of ethanol self administration. METHOD

Subjects Subjects were male Long Evans rats (Canadian Breeding Farm Laboratories Ltd., St. Constant, Quebec) weighing approximately 150-175 g at the start of the experiment. Rats were housed individually in stainless steel cages in a room regulated for constant temperature and humidity, on a 12 hr light/dark schedule. Drinking solutions were presented in glass Richter tubes mounted on the front of the cages. Standard lab chow was freely available throughout the experi-

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ment. The numbers of animals in the differing groups are given in the figures. Drug and Injections FLA-136 (4-Amino-3-(2,6-dichlorobenzylidenehydrozino)1,2,2-triazole) was prepared for administration by mixing the drug in a warm 1:1 mixture o f 1.2 polypropelene glycol and water. This solution was then diluted by 50% with water just prior to administration. Dose levels administered were 1.5, 7.5, 15, and 25 mg/kg body weight. The drug was administered intraperitoneally using a small bore needle. Control animals received injections of approximately equal volume to experimental animals, consisting of a polypropelene glycol and water mixture. Procedure All subjects were handled for seven days prior to the start of the experiment and were given free access to two Richter tubes filled with water. Rats were weighed and fluid consumption measured daily throughout the experiment. Phase 1. Acquisition Pha.~c. (Days 1-20). Animals were given ethanol on alternate days in a free-choice paradigm modified from the procedure of Amit et al. [2], where one Richter contained an ethanol-tap water solution and the other Richter tube contained tap water only. On intervening days only water was given in both tubes. On ethanol presentation days ethanol solutions were presented in increasing concentrations ranging from 2% to 10% (v/v). Two presentations were given at each concentration. On the day after the

second presentation of 10% ethanol animals were switched to an everyday presentation of ethanol at this concentration. In order to control for position bias, the tubes containing the ethanol solution and water only were switched with each presentation. Phase !1. Baseline Perh~d. (Days 21-30). Following stabilization of ethanol intake at 10% concentration animals were randomly assigned on day 30 to one of the four drug levels or the control group. Phase HI. Injection Period. (Days 31-36). During this period animals received one intraperitoneal injection of FLA-136 (Astra Pharmaceuticals, Sweden) which was administered daily at 17:00 hr. The control group received injections of the vehicle solution. Phase IV. Post lnjecthm Perh~d. (Days 37-45). F o r ten days subsequent to the injection period ethanol and water consumption continued to be recorded and body weight taken. RESULTS A two-way analysis of variance was performed on total fluid intake over a 26 day period comprising the baseline, drug and post drug phases of the experiment, which showed no significant differences, F(25,70)=1.54, p>0.05. A twoway analysis of variance was performed over the same period for ethanol intake which showed a significant effect of drug, F(4,70)=4.67, p<0.05. In addition, a significant effect of days and a significant drug by day interaction was detected at the p < 0 . 0 5 level (see Fig. l). A two-way analysis of variance was performed for

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FIG. 2. Ethanol preference calculated as percentage of total fluids consumed as ethanol for control and experimental animals, during baseline, drug and post drug periods. ethanol preference over the 26 day experimental period which showed a significant difference existed between drug groups, F(4,70)=5.27, p<0.05. In addition, a significant effect of days as well as a significant drug by days interaction was observed at the p<0.05 level (see Fig. 2). Post Hoc analysis employing the Tukey HSD test showed that at the highest dose level (25 mg/kg) ethanol intake was significantly less than the control group at p<0.05. Furthermore, the ethanol preference of the group receiving 25 mg/kg was significantly less than that of the control group p<0.05. In general, animals receiving 25 mg/kg showed a decrease in ethanol preference of approximately 40c; (Fig. 2).

DISCUSSI()N

FLA-136, at the 25 mg/kg do~e significantly decreased ethanol consumption in rats as compared to controls. In addition, levels of drinking within these groups was significantly reduced as compared to their own baseline intake levels. Rats that received either 1.5. 7.5. and 15 mg/kg showed no significant differences in ethanol intake from controls. Furthermore, the mean group ethanol intake levels for these animals was not significantly different during drug treatment as compared to their own baseline levels. As FLA-136 is a relatively new drug and has not been used in behavioural studies, the doses presently employed were chosen according to their differential effects upon NE release [4]. The findings of this experiment seem to suggest that the significant attenuation of ethanol consumption at the

25 mg/kg dose is due to its maximal effect on the rate of release of NE [4]. The exact nature of the relationship between N E function and self selection of ethanol has yet to be fully understood, but some evidence exists which suggesis a potential modulation of the reinforcing properties of ethanol by NE manipulations. The function of the N E system has been shown to be implicated in the positive reinforcing properties of ethanol [I.3]. Studies have shown that following decreases in NE synthesis by treatment with dopamine beta hydoxylase inhibitors, rats displayed significant decreases in their propensity to self administer ethanol [1, 3, 8, 9]. As the present findings extend only to the consumption of ethanol and not to any other solution with caloric and/or specific taste properties, it is still too early to conclude a direct effect of FLA-136 on the reinforcing properties of ethanol. However, this should not detract from the possible myriad of uses which potentially exist for FLA-136. Its similarity in CNS effects to cionidine make it a possible candidate for use in the treatment of alcohol withdrawal. As clonidine has recently been employed in the treatment of alcohol withdrawal [19], and because FLA-136 has lower hypotensive potential, perhaps FLA-136 might be seen as a successor drug to clonidine in alcohol withdrawal treatment. With respect to opiates, as cionidine is now considered a routine treatment in such cases, Rappoit and Lawrence [14] suggest yet another role for FLA-136 in a similar vane to its potential use in the treatment of alcohol withdrawal. Clearly the most exciting aspect of the present findings is the action of this drug on the voluntary intake o f ethanol. A

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40% reduction of ethanol c o n s u m p t i o n at doses which do not change total fluid or food intake suggest a possible antialcoholic agent is at hand. A s this drug acts n e u r o c h e m i c a l l y on the potential substrate for ethanol reward [1] and, in addition, appears to have only a minimal effect on blood pressure [4], it appears to hold s o m e promise as a c o m p o n e n t of a

new alcohol t r e a t m e n t program. Chemical intervention coupled with counselling m a y result in the most efficient t r e a t m e n t package yet devised. Studies are presently in progress which will attempt to delineate the m e c h a n i s m s by which this drug acts and its effectiveness in s o m e o f the areas described.

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11. Hunt, W. A. and E. Majchrowicz. Alterations in the turnover of brain norepinephrine and dopamine in alcohol dependent rats. J Neurochem 23: 549-553, 1974. 12. Mason, S., M. Corcoran and H. Fibiger. Noradrenaiine and ethanol intake in the rat. Neurosci Lett 12: 137-142, 1979. 13. Myers, R. D. and C. Melchoir. Alcohol drinking in the rat after destruction of serotonergic and catecholaminergic neurons in the brain. Res Commun Chem Pathol Pharmacol 10: 363-378, 1975. 14. Rappolt, R. T. and F. Lawrence. Outpatient detoxification of heroin addiction. Clin Toxicol 14: 141-142, 1979. 15. Siden, J. D. and J. Le Magnen. Parameters of low dose ethanol intravenous self-administration in the rat. Pharmacol Bh~chem Behav 16: 181-193, 1982. 16. Smith, B. R., C. M. G. Aragon and Z. Amit. A time dependent biphasic effect of an acute ethanol injection on 3methoxy-4-hydroxyphenylethylene glycol sulfate in rat brain. Biochem Pharmacol. in press, 1985. 17. Thadani, P. V. and E. B. Truitt. Effect of acute ethanol or acetaldehyde administration on the uptake, release, metabolism and turnover rate of norepinepherine in rat brain. Biochem Pharmacol 26: 1147-1150, 1977. 18. Westfall, T. C. Evidence that noradrenergic transmitter release is regulated by presynaptic receptors. Fed Proc 43: 1352-1357. 1984. 19. Wilkins, A. J., W. J. Jenkins and J. A. Steiner. Efficacy of Clonidine treatment of alcohol withdrawal state. Psychopharmacology (Berlin) 81: 78-80, 1983. 20. Winger, G. D. and J. H. Woods. The reinforcing property of ethanol in the rhesus monkey. I. Initiation maintainance and termination of intravenous ethanol reinforced responding. Ann N Y Acad Sci 215: 162-175, 1973. 2 I. Yanagita, T. and S. Takahashi. Dependance liability of several hypnotic agents evaluated in the monkey. J Pharmacol Exp Therp 185: 307-316, 1973.