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Further study of induced behavioral dependence on ethanol in rats
Alcohol, Vol. 1, pp. 269-273. 1984. " Ankho InternationalInc. Printedin the U.S.A.
0741-8329/84$3.00 + .00
Further Study of Induced Behavioral Dependence on Ethanol in Rats J. LE M A G N E N A N D P. M A R F A I N G - J A L L A T
Laboratoire de Neurophysiologie Sensorielle et Comportementale, Collkge de France 11, Place Marcelin Berthelot, 75231 Paris-Cedex 0 5 France
LE MAGNEN, J. AND P. MARFAING-JALLAT. Further study ~finduced behavioral dependence on ethanol in rats. ALCOHOL 1(4) 26%273, 1984.--This study extends previous work from our laboratory on the induction of a high consumption of alcohol in rats, following a chronic treatment by high doses of ethanol. Thirty-three rats exhibited voluntary consumption of ethanol averaging I 1 g/kg/24 hr after the chronic treatment. Twenty two controls submitted to the same procedure of testing drunk 6 g/kg/24 hr. The results are interpreted as a basis for discussing the possible mechanisms involved in alcohol addiction. Ethanol dependence
Conditioned preference
Rat
forced administration, and by a semi-quantitative assessment of the symptoms [3,5]. In this condition, it is difficult to study the various parameters of chronic treatment or intake, such as initial sensitivity to ethanol, dose, duration and periodicity of chronic intake, which are involved in the induction of the true dependence, i.e., the behavioural one. This difficulty has been recently overcome in various laboratories by the development of procedures which now permit a high voluntary intake to be induced in rats following chronic forced administration, similar to the behavior spontaneously exhibited by human alcoholics [1, 8, 15]. In the present report, we extend preliminary data previously obtained and described with our original procedure [151. The purpose of this report is methodological. In addition, we present the results obtained in a large number of rats as a basis for discussing the mechanisms involved in voluntary alcohol intake in ethanol naive and dependent rats.
SINCE animals have no occasion to find and to drink alcohol in the wilderness, a study of alcohol dependence on animal models has no sense if it is not a means for understanding human alcoholism. It is trivial and nevertheless useful to recall that human alcoholism or alcohol addiction is represented: (1) by a voluntary intake of alcohol inducing severe disturbances of brain functions and therefore of behaviour, (2) by the induction and maintenance of a permanent craving for ethanol or dependence due to chronic intake and supporting it. The aim and the sole interest of animal models are the possibility to reproduce these symptoms experimentally in order to identify various determinants of acute and chronic alcohol intake. With this purpose, experimenters using the rat as a model, have encountered two series of difficulties. Rats spontaneously refuse to drink ethanol. The very small amount they drink is not sufficient to produce a substantial and sustained blood ethanol level and therefore to produce the acute deleterious effects of ethanol. In addition their low intake in a permanent ethanol-water choice situation or even in single bottle presentation is not efficient to induce human-like dependence. In order to overcome this difficulty, various techniques of forced administration have been developed which allow experimenters to attempt to establish a state of dependence in their experiments of animals by a chronic ethanol treatment [2, 4, 8, II, 14, 19]. But a second difficulty then appears in that rats do not consume large amounts of alcohol, in other words there are no signs of alcohol addiction following chronic forced intake, as human alcoholics do, after repeated high voluntary intake. For this reason, the state of the CNS created by the chronic action of high doses of ethanol designated "physical dependence" which apparently underlies the high reinforcing property of ethanol in humans, could not be directly studied in the rat model. The existence of this CNS state and its degree of severity could only be studied indirectly from examining the withdrawal syndrome at the cessation of
METHOD Fifty-five male adult Wistar rats, weighing 285_+6 g at the beginning of the experiment, were used. They were ascribed to either one of two groups: ethanol treated (N=33) or untreated control (N=22). Rats were implanted with a chronic gastric catheter according to the technique described elsewhere [18]. After six days of recovery from surgery, they were placed in individual Plexiglas cages equipped for automatic and programmed delivery of infusions via the gastric catheter by means of a motor driven syringe. Rats of the first group were intragastrically infused with 10 g/kg of ethanol per day for 15 days. A 16% v/v solution of 95% ethanol diluted in physiological saline was used. The daily dose was infused in five 2 g/kg pulses distributed from 2 a.m. to 7.30 p.m. A gap between 7.30 p.m. and 2 a.m. allowed the rat to eat and drink without any disturbarlce due to the acute effect of ethanol. During treatment, rats had free access to food and water. Twelve hours after the last infusion, oral ethanol intake
269
270
i.F MAGNEN AND M A R F A I N G - J A L L A T i~
EXPERIMENTALS
(n=33)
Alcohol
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FIG. I. Mean+_SEM (in m] and g a]c/kg) consumption of alcohol ( IE:~ v/v) and water
during the 3.8 hr presentations along the 6 days of alternate presentation in experimental and control rats. Experimental rats were previously administered I.G. with 1~ g/kg/day of ethanol for 15 days. Control rats were submitted to the same schedule of alternate presentation without a previous chronic treatment. Consumption of ethanol between control and ethanol groups was significantly different in the 3.8 hr presenta tions. P~, p<0.01; P~, p<0.01; P:~,p<-~0.01.
was examined in these previously treated rats and in ethanol naive controls. The purpose of the procedure adopted for testing alcohol intake in previously treated compared to untreated rats was: (1) to measure the level of intake during a single bottle presentation of ethanol solution; (2) nevertheless to compare ethanol intake to water intake used as a reference; (3) to test single bottle intake of alcohol or water during a relatively short period of time, i.e., a few hours. This was necessary because of the later application of the procedure to test the effect of short action drugs; (4) to test intake during this short daily period, the effects of the well-documented circadian cycle of fluid intake being eliminated. Rats were placed in cages equipped for programmed alternate presentations of a graded drinking tube containing either water or an ethyl-alcohol solution 10% v/v. A 12/12 dark-light cycle (6 a.m. light-6 p.m. dark) was monitored. The schedule of single bottle presentations was as follows: On the first day, 24 hr water deprived rats were continuously presented with an ethanol solution as the only source of fluid for 24 hr (starting at 6 p.m.). Rats were then presented with alternate presentations of eight hour duration of ethanol solution or water, for 6 days. On the first
day, alcohol was presented from 6 p.m. to 2 a.m. (P1), water from 2 a,m. to 10 a.m. (P~), and alcohol again from 10 a.m. to 6 p.m. (P:0. On the second day, water, alcohol and water were successively presented for the three 8 hr periods. In each of the three successive couples of days. it was thus possible to compare the amounts of ethanol and of water drunk during the same 8 hr period of the diurnal cycle and to compare the respective intake of the two fluids over 48 hours. All results are given as mean values-+SEM. Student's test Mann and Whitney comparisons and the Analysis of Variance were used for statistical analysis. R E S U L.TS
During the first 24 hr of continuous single bottle alcohol presentation, treated rats drank 38.3 + 1.6 ml [10.58_+_0.4 g per kg) while controls drank 28.2-+ 1.2 ml (7.4+0.3 g p e r kg). The difference was highly significant. Student t =4.6. p<0.01 (6.95, p<0.01L During the 3 subsequent couples of days, as illustrated in Fig. 1. previously treated rats drank different amounts of ethanol according to the time of presentation. This consumption was 18.2-+0.5:14.1+-0.7 and 9.1-+0.5 ml in P~, P.,
BEHAVIORAL D E P E N D E N C E ON E T H A N O L
271
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HG. 2. Mean±SEM (in ml and g alc/kg) of alcohol and water consumption during 3 subsequent pairs of days of alternate presentation. Experimental conditions as in Fig. I. In control group, ethanol consumption was significantly lower than water on the alternate day, p
and P:, respectively. This effect of the time of day on the 8 hr consumption was highly significant, U(P, PO=3.2, p<0.01 U(P~P:~)--3.6, p<0.01. Water intakes of 18.3_+0.7; 14_+0.6 and 6.8+0.5 ml during the corresponding periods on alternate days also were significantly different, U(P.Pz)=3.2, p<0.01, U(P=,P:~)=4.9,p<0.01. During P~, P~, and P:~ alcohol intakes were not significantly different from the amount of water drunk on the alternate day, P:, F(1,32)=3.4, n.s., Fig. 2. In previously treated rats, the sum of the three 8 hr ethanol intakes, i.e., the 24 hr intake, averaged over these 6 days, reached 41.4_+ 1.4 ml (10.7 g per kg). The 24 hr water intake (39.3_+ 1.5 ml) was not significantly different from that of the ethanol solution, F(1,32)=0.3, n.s. In previously untreated controls, the 8 hr ethanol and water intakes also were different according to the time of day: alcohol 11.5_+0.4; 8.8+0.4 and 3.4_+0.4 ml tor P~, P2 and P= respectively. U(P~P.4=3.1, p<0.01 and U(P..,P0=5.2, p<0.01 water 18.3_+0.7:14_+0.7 and 5.4_+0.4 ml, U(P~PO-3, p,::0.01, U(P~P:0=4.7, p<0.01. In each of the three 8 hr periods, ethanol intake was significantly lower than water intake on the alternate day, P,. F(I,21)=28.8, p<0.01. P.,, F(1,21)=15.1, p<0.01. P:, FI 1,21)=5.9, p<0.01. Averaged over the 3 pairs of days the 24 hr alcohol intake 23.7_+0.9 ml (6 g/kg) was significantly lower than the 24 hr water intake (37.6_+4 ml) F(1,21)=21, p"0,01.
In the three 8 hr presentations, water intake of previously treated and untreated rats was almost identical and was not significantly different (P~ = 18.3 vs, 18.3 ml; P~= 14 vs. 14 ml; P:~=6.8 vs. 5.4 ml), Consequently ethanol intake by treated and control rats in each of the three 8 hr presentations could be compared directly. Alcohol intake in each 8 hr presentation and over 24 hr in previously treated rats was significantly higher than alcohol intake in untreated controls. P j - 1 8 . 2 v s . l l . 5 m l , U=4.8, p<0.01;P~ 14.1 vs. 8.8ml, U=4.25, p<0.01; P:~=9.1 vs. 3.4, U=4.3, p~0.01; 24 h r - 4 1 . 4 vs. 23.6 ml, U=4.7, p<0.01. In the 3 successive pairs of days no evolution of ethanol or water intake in treated or untreated rats was apparent. DISCUSSION
Thus ethanol naive rats presented with a 10% ethanol solution as the only source of fluid ingest 6 g/kg ethanol per 24 hr. When compared to their water intake during alternate presentations this demonstrates a strong aversion to the ethanol solution. Rats pretreated with a daily administration of 10 g/kg of ethanol for 15 days and presented with the ethanol solution drink more than 10.7 g/kg per 24 hour. As compared to water intake, their drinking of the ethanol solution gives evidence that the ethanol solution has become as
272
I,E M A G N E N ANI) M A R F A I N G - J A I , I . A I
palatable as water, This high average intake of 10.7 g/kg maintains the daily dose of ethanol previously administered through a voluntary behaviour of self-intoxication. This daily intake represents a rate of alcohol acquisition higher than the rate of alcohol elimination, which, in naive rats, is about 300 mg/kg/hr [21]. On the contrary the daily intake of untreated rats remains below the rate of ethanol elimination from the body. On the basis of previous studies, it has been argued that voluntary intake of ethanol solutions by ethanol naive non dependent rats is determined and limited at two levels by different mechanisms. In a free choice with water, a limited daily intake as 1 to 2 g/kg per day is observed in rat strains not selected as high or low prefering rats. Such a low daily intake cannot be due to the post-ingestive aversive effect of ethanol. The acute effect or administration of at least 1.5 g/kg is required to induce a taste aversion to saccharin or ethanol solutions. The acute effect of this dose cannot be achieved by a spontaneous irJtake of 1 to 2 g distributed over 24 h o u r s At this level of intake, the genetically determined sensory aversion to ethanol, which is identical to that exhibited by rats offered a bitter solution determines and limits intake [7,131. The spontaneous low intake can be augmented by various procedures such as exposing the rat to ethanol solution as the only source of fluid, such as in control rats of the present experiment, or by genetic select,ion of high drinker rats_ Then, the daily voluntary intake can reach 5 to 6 g/kg. How ever it is limited at this level by the aversion induced by post-ingestive consequences [2,10]. In individual rats the ethanol induced taste aversion is positively correlated to the initial sensitivity as tested by motor impairment or hypothermia [221. The less sensitive the rat is, the lower the aversion induced by 1.5 g/kg ethanol IP. This is the basis for a relationship between initial sensitivity and the level of maximal intake in ethanol naive rats. Thus, the rate of elimination is the limiting factor of oral consumption beyond which a positive level acts to repress intake through the conditioned aversion. Since the rate of intake never exceeds the rate of elimination, a permanently elevated blood ethanol level is avoided, In this condition. physical dependence is never achieved through a spontaneous oral intake. ls the highest intake of about 10 g/kg per day exhibited by ethanol pretreated rats an evidence for the presence of a state of dependence identical to that observed in man': This
high intake could achieve and permanently maintain a higher blood ethanol level than that required to induce both the acute toxic effects of ethanol on the CN S and its conditioned repressive effects on voluntary consumption. Indeed it could be argued thai neither condition i~ ~ealized. The tolerance of rats is increased by the chronic p,eatment: the threshokl dose required to induce aversion m previously ethanol treated rats is approximately double t201. Thus it could be argued that these rats reach this high level of voluntary mtake after a chronic treatment onl} because they have been made more tolerant to the acute el'fec~ of a high blood alcohol level, Indeed, we have shown in another article that the least ethanol sensitive rats before lreatment twhich are also likely to be the most tolerant at the end ~f ,reatment) exhibited a maximal intake in the stale induced by the treatmeut j161. The relationships between acquired tolerance and dependence in ethanol and other drug dependence have been e',;tensively studied and are still ques'~ioned I17,23]. Possib b lhe acquired nervous tolerance i~ involved in establishing a new elevated ceiling of intake B~,d in as much ae~ the acquired metabolic tolerance is lower {han the acquired ner~-ous tolerance, this maximal inlakc ~Bould induce and mainlain a permanently elevated blood atcohol concentration, .:~!though it is lower than the new level needed 1,o reduce aversion, this chronically elewtted blood ethanol level might be lhe basis fl~r the maintenance ot !he altered stale of ~he CNS created by forced administratum ~)n the othe~ hand, this state would be the basis l\v ihe positively reinforcing effect of ethanol. A common characteristic of dependence to ethanol, and to other addiction drugs is that, ll'nough ils acute e['fect, lhe agenl becomes efficient to allevi~::e lhe neurologic conse.quences of abstinence. It remains, unknown how brain dysfunctions which are chronically introduced b3 an agent me act,tely and Iransiently corrected by ~he ,,ante aget-ii, BUt through this effecl, ethanol and o!be: dlugs become fcv,.a~d. ing at levels a~ which {hey are :~,.,,:r',ive h~ r~on-dcpend~'nt subjecls. The recovery from malaise or ilhtess i'a; been demonstrated as Ii.C.S. for co~ditioned !asle prel~trencc 16,81. Such a conditioned lame preference lot ethanol in previously treated rats has not ye~ been ascertained. BH! it is highly probable, and it presumably is a cause of the high intake exhibited by the rats tested in this study. As such their intake may be taken as an expression of lheir dependence and as a measure of its l e v e l
REFERENCES
1. Deutsch, J. A. and N. Y. Walton. A rat alcoholism model in a free choice situation. Behav Biol 19: 34%360, 1977. 2. Eckardt, M. J. Conditioned taste aversion produced by the oral ingestion of ethanol in the rat. Physiol Psy¢.hol 3:317-321, 1975. 3. Friedman, H. J. Assessment of physical dependence on and withdrawal from ethanol in animals. Alcohol 7oleram'e and l)ependenee, edited by H. Rigter and J. C. Crabbe, Jr. Amsterdam: Elsevier 1980. 4. Goldstein, D. B. Relationship of alcohol dose to intensity of withdrawal signs in mice. J Pharmaeol g\vp Ther 180: 203-215, 1972. 5. Goldstein, D. B. Animal studies of alcohol withdrawal reactions. Res Adv Alcohol Drug Probl 4: 77-109. 1978. 6. Green, K. F. and J. Garcia. Recuperation from illness: flavor enhancement for rats, Science 173: 74%751, 1971. 7. Kahn, M. and E. Stellar. Alcohol preference in normal and anosmic rats. ,I Comp Physiol Psychol 53: 571-575. 1960.
8. Le Bourhis, B., G. Auhere and D. Claoquart. Stud): ol alcohol dependence in the rat. Ad Lxp Med Bi,}l 126: 339-358, 1980. 9. Ire Magnen, J., P. Marfaing-Jallat and I), Miceli. A bioassay ol ethanol-dependence in rats. Pharmac{~l Biochem Bclla~ 12: 707-709, 1980. 10. Lester, D., M. Nachman and J~ Le Magnen. Aversiveconditioning by ethanol in the rat. Q J Stud Ah.ohol 31: 578-586, 1970. 11, Lieber, C. S. and L. M. De Carti. Ethanol dependence and tolerance. A nutritionally controlled experimental model in the rat. Res ('ommun Chem Pathol Pharmaco! 6: 983--991, 1973. 12. Majchrowicz, E. Induction of physical dependence upon ethanol and the associated behavioral changes in rats. PS),cllopharmacologia 43: 245-254, 1975. 13. Marfaing-Jallat, P. and J. Le Magnen. Nouvelles donnO.es sur h: r61e des aff~rences buccales darts le determinisme des consommations spontanees d'alcool chez le rat. CR Soc Biol 158: 204%21}53. 1964.
BEHAVIORAL
DEPENDENCE
ON ETHANOL
14. Marfaing-Jallat, P., M. Pruvost and J. Le Magnen. La consommation d'ethanol par auto-administration intragastrique chez le rat. J Physiol (Paris) 68: 81-95, 1974. 15. Marfaing-Jallat, P. and J. Le Magnen. Induction of high voluntary ethanol intake in dependent rats. Pharmacol Biochem Behav 17: 60%612, 1982. 16. Marfaing-Jallat, P. and J. Le Magnen. Relationship between initial sensitivity to ethanol and the high alcohol intake in dependent rats. To be published. 17. Miceli, D. and J. Le Magnen. Relations between metabolic and nervous tolerance towards ethanol in naive and chronically intoxicated rats. Pharmacol Bio~'hem Behav 10: 32%334, 1979. 18. Nicolaidis, S., N. Rowland, M. J. Meile, P. Marfaing-Jallat and A. Pesez. A flexible technique for long-term infusion in unrestrained rats. Pharmacol Biochem Behav 2:131-136, 1974.
273 19. Numan, R. and A. M. Gilroy. Induction of physical dependence upon ethanol in rats using intravenous infusion. Pharmacol Biochem Behav 9: 279-282, 1978. 20. Pacteau, C., P. Marfaing-Jallat and J. Le Magnen. Thresholds of ethanol-induced taste aversion in naive compared to intoxicated rats. To be published. 21. Segovia-Riquelme, N., J. Vitale, R. M. Hegsted and J. Mardones. Alcohol metabolism in drinking and non-drinking rats. J Biol Chem 223: 399-403, 1956. 22. Sinden, J. D. Les determinants postabsorptifs des reponses de preference et d'aversion pour I'ethanol chez le rat. Th~se juin 1983, Paris. 23. Wood, J. M. and R. Laverty. Metabolic and pharmacodynamic tolerance to ethanol in rats. Pharmacol Biochem Behav 10: 871-874, 1979.
0741-8329/84$3.00 + .00
Further Study of Induced Behavioral Dependence on Ethanol in Rats J. LE M A G N E N A N D P. M A R F A I N G - J A L L A T
Laboratoire de Neurophysiologie Sensorielle et Comportementale, Collkge de France 11, Place Marcelin Berthelot, 75231 Paris-Cedex 0 5 France
LE MAGNEN, J. AND P. MARFAING-JALLAT. Further study ~finduced behavioral dependence on ethanol in rats. ALCOHOL 1(4) 26%273, 1984.--This study extends previous work from our laboratory on the induction of a high consumption of alcohol in rats, following a chronic treatment by high doses of ethanol. Thirty-three rats exhibited voluntary consumption of ethanol averaging I 1 g/kg/24 hr after the chronic treatment. Twenty two controls submitted to the same procedure of testing drunk 6 g/kg/24 hr. The results are interpreted as a basis for discussing the possible mechanisms involved in alcohol addiction. Ethanol dependence
Conditioned preference
Rat
forced administration, and by a semi-quantitative assessment of the symptoms [3,5]. In this condition, it is difficult to study the various parameters of chronic treatment or intake, such as initial sensitivity to ethanol, dose, duration and periodicity of chronic intake, which are involved in the induction of the true dependence, i.e., the behavioural one. This difficulty has been recently overcome in various laboratories by the development of procedures which now permit a high voluntary intake to be induced in rats following chronic forced administration, similar to the behavior spontaneously exhibited by human alcoholics [1, 8, 15]. In the present report, we extend preliminary data previously obtained and described with our original procedure [151. The purpose of this report is methodological. In addition, we present the results obtained in a large number of rats as a basis for discussing the mechanisms involved in voluntary alcohol intake in ethanol naive and dependent rats.
SINCE animals have no occasion to find and to drink alcohol in the wilderness, a study of alcohol dependence on animal models has no sense if it is not a means for understanding human alcoholism. It is trivial and nevertheless useful to recall that human alcoholism or alcohol addiction is represented: (1) by a voluntary intake of alcohol inducing severe disturbances of brain functions and therefore of behaviour, (2) by the induction and maintenance of a permanent craving for ethanol or dependence due to chronic intake and supporting it. The aim and the sole interest of animal models are the possibility to reproduce these symptoms experimentally in order to identify various determinants of acute and chronic alcohol intake. With this purpose, experimenters using the rat as a model, have encountered two series of difficulties. Rats spontaneously refuse to drink ethanol. The very small amount they drink is not sufficient to produce a substantial and sustained blood ethanol level and therefore to produce the acute deleterious effects of ethanol. In addition their low intake in a permanent ethanol-water choice situation or even in single bottle presentation is not efficient to induce human-like dependence. In order to overcome this difficulty, various techniques of forced administration have been developed which allow experimenters to attempt to establish a state of dependence in their experiments of animals by a chronic ethanol treatment [2, 4, 8, II, 14, 19]. But a second difficulty then appears in that rats do not consume large amounts of alcohol, in other words there are no signs of alcohol addiction following chronic forced intake, as human alcoholics do, after repeated high voluntary intake. For this reason, the state of the CNS created by the chronic action of high doses of ethanol designated "physical dependence" which apparently underlies the high reinforcing property of ethanol in humans, could not be directly studied in the rat model. The existence of this CNS state and its degree of severity could only be studied indirectly from examining the withdrawal syndrome at the cessation of
METHOD Fifty-five male adult Wistar rats, weighing 285_+6 g at the beginning of the experiment, were used. They were ascribed to either one of two groups: ethanol treated (N=33) or untreated control (N=22). Rats were implanted with a chronic gastric catheter according to the technique described elsewhere [18]. After six days of recovery from surgery, they were placed in individual Plexiglas cages equipped for automatic and programmed delivery of infusions via the gastric catheter by means of a motor driven syringe. Rats of the first group were intragastrically infused with 10 g/kg of ethanol per day for 15 days. A 16% v/v solution of 95% ethanol diluted in physiological saline was used. The daily dose was infused in five 2 g/kg pulses distributed from 2 a.m. to 7.30 p.m. A gap between 7.30 p.m. and 2 a.m. allowed the rat to eat and drink without any disturbarlce due to the acute effect of ethanol. During treatment, rats had free access to food and water. Twelve hours after the last infusion, oral ethanol intake
269
270
i.F MAGNEN AND M A R F A I N G - J A L L A T i~
EXPERIMENTALS
(n=33)
Alcohol
r - - I Water
40
_10
2o
_s c
._z
3
I
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2,~t;
CONTROLS (n=22) 40
.10
20
.5
.~-
o3 ~I ~ >
0
o
o 18h 2h
2h 10h
l o b 18h
-)Zlh
FIG. I. Mean+_SEM (in m] and g a]c/kg) consumption of alcohol ( IE:~ v/v) and water
during the 3.8 hr presentations along the 6 days of alternate presentation in experimental and control rats. Experimental rats were previously administered I.G. with 1~ g/kg/day of ethanol for 15 days. Control rats were submitted to the same schedule of alternate presentation without a previous chronic treatment. Consumption of ethanol between control and ethanol groups was significantly different in the 3.8 hr presenta tions. P~, p<0.01; P~, p<0.01; P:~,p<-~0.01.
was examined in these previously treated rats and in ethanol naive controls. The purpose of the procedure adopted for testing alcohol intake in previously treated compared to untreated rats was: (1) to measure the level of intake during a single bottle presentation of ethanol solution; (2) nevertheless to compare ethanol intake to water intake used as a reference; (3) to test single bottle intake of alcohol or water during a relatively short period of time, i.e., a few hours. This was necessary because of the later application of the procedure to test the effect of short action drugs; (4) to test intake during this short daily period, the effects of the well-documented circadian cycle of fluid intake being eliminated. Rats were placed in cages equipped for programmed alternate presentations of a graded drinking tube containing either water or an ethyl-alcohol solution 10% v/v. A 12/12 dark-light cycle (6 a.m. light-6 p.m. dark) was monitored. The schedule of single bottle presentations was as follows: On the first day, 24 hr water deprived rats were continuously presented with an ethanol solution as the only source of fluid for 24 hr (starting at 6 p.m.). Rats were then presented with alternate presentations of eight hour duration of ethanol solution or water, for 6 days. On the first
day, alcohol was presented from 6 p.m. to 2 a.m. (P1), water from 2 a,m. to 10 a.m. (P~), and alcohol again from 10 a.m. to 6 p.m. (P:0. On the second day, water, alcohol and water were successively presented for the three 8 hr periods. In each of the three successive couples of days. it was thus possible to compare the amounts of ethanol and of water drunk during the same 8 hr period of the diurnal cycle and to compare the respective intake of the two fluids over 48 hours. All results are given as mean values-+SEM. Student's test Mann and Whitney comparisons and the Analysis of Variance were used for statistical analysis. R E S U L.TS
During the first 24 hr of continuous single bottle alcohol presentation, treated rats drank 38.3 + 1.6 ml [10.58_+_0.4 g per kg) while controls drank 28.2-+ 1.2 ml (7.4+0.3 g p e r kg). The difference was highly significant. Student t =4.6. p<0.01 (6.95, p<0.01L During the 3 subsequent couples of days, as illustrated in Fig. 1. previously treated rats drank different amounts of ethanol according to the time of presentation. This consumption was 18.2-+0.5:14.1+-0.7 and 9.1-+0.5 ml in P~, P.,
BEHAVIORAL D E P E N D E N C E ON E T H A N O L
271
O Alcohol [ ] Water
EXPERIMENTALS ( n : 33) 20.
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374 CONTROLS (n=22)
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374
]Oh~18h~
~18h~2h
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~76
HG. 2. Mean±SEM (in ml and g alc/kg) of alcohol and water consumption during 3 subsequent pairs of days of alternate presentation. Experimental conditions as in Fig. I. In control group, ethanol consumption was significantly lower than water on the alternate day, p
and P:, respectively. This effect of the time of day on the 8 hr consumption was highly significant, U(P, PO=3.2, p<0.01 U(P~P:~)--3.6, p<0.01. Water intakes of 18.3_+0.7; 14_+0.6 and 6.8+0.5 ml during the corresponding periods on alternate days also were significantly different, U(P.Pz)=3.2, p<0.01, U(P=,P:~)=4.9,p<0.01. During P~, P~, and P:~ alcohol intakes were not significantly different from the amount of water drunk on the alternate day, P:, F(1,32)=3.4, n.s., Fig. 2. In previously treated rats, the sum of the three 8 hr ethanol intakes, i.e., the 24 hr intake, averaged over these 6 days, reached 41.4_+ 1.4 ml (10.7 g per kg). The 24 hr water intake (39.3_+ 1.5 ml) was not significantly different from that of the ethanol solution, F(1,32)=0.3, n.s. In previously untreated controls, the 8 hr ethanol and water intakes also were different according to the time of day: alcohol 11.5_+0.4; 8.8+0.4 and 3.4_+0.4 ml tor P~, P2 and P= respectively. U(P~P.4=3.1, p<0.01 and U(P..,P0=5.2, p<0.01 water 18.3_+0.7:14_+0.7 and 5.4_+0.4 ml, U(P~PO-3, p,::0.01, U(P~P:0=4.7, p<0.01. In each of the three 8 hr periods, ethanol intake was significantly lower than water intake on the alternate day, P,. F(I,21)=28.8, p<0.01. P.,, F(1,21)=15.1, p<0.01. P:, FI 1,21)=5.9, p<0.01. Averaged over the 3 pairs of days the 24 hr alcohol intake 23.7_+0.9 ml (6 g/kg) was significantly lower than the 24 hr water intake (37.6_+4 ml) F(1,21)=21, p"0,01.
In the three 8 hr presentations, water intake of previously treated and untreated rats was almost identical and was not significantly different (P~ = 18.3 vs, 18.3 ml; P~= 14 vs. 14 ml; P:~=6.8 vs. 5.4 ml), Consequently ethanol intake by treated and control rats in each of the three 8 hr presentations could be compared directly. Alcohol intake in each 8 hr presentation and over 24 hr in previously treated rats was significantly higher than alcohol intake in untreated controls. P j - 1 8 . 2 v s . l l . 5 m l , U=4.8, p<0.01;P~ 14.1 vs. 8.8ml, U=4.25, p<0.01; P:~=9.1 vs. 3.4, U=4.3, p~0.01; 24 h r - 4 1 . 4 vs. 23.6 ml, U=4.7, p<0.01. In the 3 successive pairs of days no evolution of ethanol or water intake in treated or untreated rats was apparent. DISCUSSION
Thus ethanol naive rats presented with a 10% ethanol solution as the only source of fluid ingest 6 g/kg ethanol per 24 hr. When compared to their water intake during alternate presentations this demonstrates a strong aversion to the ethanol solution. Rats pretreated with a daily administration of 10 g/kg of ethanol for 15 days and presented with the ethanol solution drink more than 10.7 g/kg per 24 hour. As compared to water intake, their drinking of the ethanol solution gives evidence that the ethanol solution has become as
272
I,E M A G N E N ANI) M A R F A I N G - J A I , I . A I
palatable as water, This high average intake of 10.7 g/kg maintains the daily dose of ethanol previously administered through a voluntary behaviour of self-intoxication. This daily intake represents a rate of alcohol acquisition higher than the rate of alcohol elimination, which, in naive rats, is about 300 mg/kg/hr [21]. On the contrary the daily intake of untreated rats remains below the rate of ethanol elimination from the body. On the basis of previous studies, it has been argued that voluntary intake of ethanol solutions by ethanol naive non dependent rats is determined and limited at two levels by different mechanisms. In a free choice with water, a limited daily intake as 1 to 2 g/kg per day is observed in rat strains not selected as high or low prefering rats. Such a low daily intake cannot be due to the post-ingestive aversive effect of ethanol. The acute effect or administration of at least 1.5 g/kg is required to induce a taste aversion to saccharin or ethanol solutions. The acute effect of this dose cannot be achieved by a spontaneous irJtake of 1 to 2 g distributed over 24 h o u r s At this level of intake, the genetically determined sensory aversion to ethanol, which is identical to that exhibited by rats offered a bitter solution determines and limits intake [7,131. The spontaneous low intake can be augmented by various procedures such as exposing the rat to ethanol solution as the only source of fluid, such as in control rats of the present experiment, or by genetic select,ion of high drinker rats_ Then, the daily voluntary intake can reach 5 to 6 g/kg. How ever it is limited at this level by the aversion induced by post-ingestive consequences [2,10]. In individual rats the ethanol induced taste aversion is positively correlated to the initial sensitivity as tested by motor impairment or hypothermia [221. The less sensitive the rat is, the lower the aversion induced by 1.5 g/kg ethanol IP. This is the basis for a relationship between initial sensitivity and the level of maximal intake in ethanol naive rats. Thus, the rate of elimination is the limiting factor of oral consumption beyond which a positive level acts to repress intake through the conditioned aversion. Since the rate of intake never exceeds the rate of elimination, a permanently elevated blood ethanol level is avoided, In this condition. physical dependence is never achieved through a spontaneous oral intake. ls the highest intake of about 10 g/kg per day exhibited by ethanol pretreated rats an evidence for the presence of a state of dependence identical to that observed in man': This
high intake could achieve and permanently maintain a higher blood ethanol level than that required to induce both the acute toxic effects of ethanol on the CN S and its conditioned repressive effects on voluntary consumption. Indeed it could be argued thai neither condition i~ ~ealized. The tolerance of rats is increased by the chronic p,eatment: the threshokl dose required to induce aversion m previously ethanol treated rats is approximately double t201. Thus it could be argued that these rats reach this high level of voluntary mtake after a chronic treatment onl} because they have been made more tolerant to the acute el'fec~ of a high blood alcohol level, Indeed, we have shown in another article that the least ethanol sensitive rats before lreatment twhich are also likely to be the most tolerant at the end ~f ,reatment) exhibited a maximal intake in the stale induced by the treatmeut j161. The relationships between acquired tolerance and dependence in ethanol and other drug dependence have been e',;tensively studied and are still ques'~ioned I17,23]. Possib b lhe acquired nervous tolerance i~ involved in establishing a new elevated ceiling of intake B~,d in as much ae~ the acquired metabolic tolerance is lower {han the acquired ner~-ous tolerance, this maximal inlakc ~Bould induce and mainlain a permanently elevated blood atcohol concentration, .:~!though it is lower than the new level needed 1,o reduce aversion, this chronically elewtted blood ethanol level might be lhe basis fl~r the maintenance ot !he altered stale of ~he CNS created by forced administratum ~)n the othe~ hand, this state would be the basis l\v ihe positively reinforcing effect of ethanol. A common characteristic of dependence to ethanol, and to other addiction drugs is that, ll'nough ils acute e['fect, lhe agenl becomes efficient to allevi~::e lhe neurologic conse.quences of abstinence. It remains, unknown how brain dysfunctions which are chronically introduced b3 an agent me act,tely and Iransiently corrected by ~he ,,ante aget-ii, BUt through this effecl, ethanol and o!be: dlugs become fcv,.a~d. ing at levels a~ which {hey are :~,.,,:r',ive h~ r~on-dcpend~'nt subjecls. The recovery from malaise or ilhtess i'a; been demonstrated as Ii.C.S. for co~ditioned !asle prel~trencc 16,81. Such a conditioned lame preference lot ethanol in previously treated rats has not ye~ been ascertained. BH! it is highly probable, and it presumably is a cause of the high intake exhibited by the rats tested in this study. As such their intake may be taken as an expression of lheir dependence and as a measure of its l e v e l
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