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Behavioral changes in rats on the day after acute ethanol intoxication
Alcohol, Vol. 4, pp. 503-507. e PergamonJournals Ltd., 1987. Printed in the U.S.A.
0741-8329/87$3.00 + .00
Behavioral Changes in Rats on the Day After Acute Ethanol Intoxication ' J. D. S I N C L A I R A N D K. G U S T A F S S O N Research Laboratories o f the Finnish Ah:ohol Company (Alko Ltd), Box 350, SF-O0100 Helsinki, Finland R e c e i v e d 23 April 1987; A c c e p t e d 26 M a y 1987 SINCLAIR, J. D. AND K. GUSTAFSSON. Behavioral changes in rats on the day after acute ethanol intoxication. ALCOHOL 4(6) 503-507, 1987.--Activity in computer-monitored running wheels, behavior in open-field tests, body temperature measured by rectal probe, and vocalization were examined in rats after a single injection of 2.5 g/kg ethanol or saline. A reduction in running-wheel activity occurred during intoxication, followed by a significant increase in running 20--24 hr after the injection. Also 24 hr after ethanol, among rats kept in normal cages, there was an increase in body temperature and vocalization and a decrease in ambulation in a dark, quiet open field. Each of these 3 aftereffects was reduced or abolished in the rats with access to a running wheel. The results confirm and extend previous findings of aftereffects of acute intoxication in rats with a time course similar to hangover in humans. Hangover
Acute ethanol intoxication
Activity
Exercise
Open-field test
Temperature regulation
peripheral changes accompanying hangover, such as increased blood lactate and free fatty acids, the central nervous system changes related to the preceding intoxication are believed to be more important [24]. Therefore, the relevance of animal models to human hangover might be expected to be directly proportional to the relevance of the animal models of intoxication. Three aftereffects of acute intoxication in rats have previously been reported that fit Rydberg's definition of hangover and are apparently related to central mechanisms. The susceptibility to kindled seizures is increased [10], body temperature is increased [6, 18-20], and rats vocalize more when handled [19,20]. The present study was designed to search for other possible aftereffects of ethanol in rats. Ethanol intoxication in rats is accompanied by a monotonically dose-dependent decrease of activity in a running wheel [2] and hyperactivity is commonly found as a withdrawal symptom in various species [5], so the aftereffects on wheel running were examined. Open-field behavior was also measured because mice have been reported to show reduced activity in an open field as an aftereffect of a single ethanol injection [15]. This was with an open-field test made in a dark, quiet room, in contrast to the usual procedure in our laboratory of using high illumination and loud white noise during the test [7], so both conditions were included in the present study. Finally, many people say that jogging or other forms of exercise help to reduce hangover symptoms [23]; consequently, in order to see any effects of wheel running on the other aftereffects, control rats were included that were treated identically to the running-wheel animals but remained in their home cages.
THERE are several well-established animal models of withdrawal from chronic ethanol (cf. [5]), but to our knowledge none has been proposed for the aftereffects of acute intoxication, commonly referred to as hangover. If hangover is defined, not in terms of subjective reports [5], but rather as Rydberg [14] does, "as a state following the acute alcohol stage, appearing about the time when the blood alcohol concentration approaches zero, and continuing for several hours," then hangover should be as amenable to research with animal models as are withdrawal and acute ethanol intoxication. Certain aspects of intoxication, such as the influence of social factors and the subjective reports of euphoria, would at best be very difficult to study in animals. Nevertheless, intoxication is believed to be the result of ethanol's actions on the nervous system that are essentially the same in humans and laboratory animals. Consequently, there are a variety of animal models believed to be relevant for the study of intoxication [1]. Along the same lines, one cannot expect animal models of hangover to reflect all the components found in humans. For instance, the same social factors that affect intoxication in humans may also affect the subsequent hangover, and these would not be amenable to study with animal models. It should, however, be possible to develop animal models for the components of hangover that are caused by the aftereffects of ethanol on physiological mechanisms. The importance of such physiological factors, in contrast to social factors, is supported by the very high heritability estimate (0.62) for the number of hangovers that has been found in human twin studies (see [3]). Furthermore, although there are many
1portions of this material were presented at the XV Annual Nordic Meeting on Biological Alcohol Research, Helsinki, 1984 [18] and at the 3rd ISBRA Congress, Helsinki, 1986 [19].
503
504
S I N C L A I R AND G U S T A F S S O N METHOD 40
Animals
after saline
30
Male Long Evans rats (n= 31) of approximately 3 months of age were used. They were individually housed for 1 week before the experiment.
20 10 ...~-~va f t e r ®thanol
Equipment Four 36 cm running wheels with affixed living cages were monitored with a Commodore Vic 20 computer. The 83 cm diameter open field had 1830 luxes at floor level and 78 dB white noise in the light-noisy condition and only dim red light in the dark-quiet condition. Both the room containing the running wheels and the room with the suspended-wire home cages rooms were maintained at 21-23°C and illuminated from 6 p.m. to 6 a.m. by 4 overhead 40 watt fluorescent tubes. A TE 3 thermister (Ellab Instruments, Copenhagen) was used for temperature measurements. Water and standard powdered rat food (Astra-Ewos, Sweden) were available in both the home cages and the running wheels.
4
8 12 16 HOURS AFTER INJECTION
\~ 20
24
HOURS AFTER INJECTION 1
4
8
12
i~i~:l
.t
_> ,(
z
Procedure The general design was to measure the aftereffects of alcohol on running-wheel activity, body temperature, vocalization, and open-field behavior, plus the effect of the exercise in the running wheel on the other 3 measures. Each rat was tested twice, once after alcohol and once after saline, in a balanced order, and thus served as its own control. The rats were divided into two groups matched for body weight: one group (n = 16) was put into running wheels and the other (n= 15) remained in the home cages. After the first group had been in the running wheels for 24 hr, they were removed and had their temperature measured (6 cm anal insertion, recording after 40 sec). The number of audible vocalizations made during handling and temperature measurement were also recorded. Then they were injected with either 2.5 g/kg ethanol (as a warmed 12.5% w/v solution in saline, n = 8) or a similar volume of warmed saline (n = 8), and were returned to the running wheel. At the same time the home-cage rats also had their temperature and vocalization recorded and were injected with either ethanol (n=7) or saline (n=8). All of the rats had their temperature and vocalization again measured 24 hr after the injections, and then were tested in either the light-noisy or dark-quiet open field. They were then all placed in home cages, except when with temperature and vocalization was again recorded 48 hr after the injections. The procedure was repeated in exactly the same manner 13 days later except the animals previously given saline now had ethanol and the rats that previously had ethanol received saline. The experiment was run sequentially, with batches of 4 running-wheel rats and 4 home-cage rats started every 3 days, and with the other c o n d i t i o n . b a l a n c e d in each batch. The running-wheel results were combined into 6 segments of 4 hr each (except the first which was only 3 hr long because the data from the first hr were discarded).
Statistical Analyses The effects of ethanol on running-wheel activity were analyzed with matched-pair t-tests comparing each animal's activity after ethanol with its own activity at a comparable
W
O I/J O gl
10
0
2o
t
'
t
l
i
1 1 ii 3O
FIG. t. Effects of a 2.5 g/kg ethanol injection on activity in running wheels. The upper frame shows the mean number of revolutions/hr after ethanol and by the same rats after saline. The lower frame shows the mean (+_SE) of each rat's activity after ethanol minus its own activity after saline. **Significant difference between ethanol and saline results, p<0.01. time after saline. An analysis of variance was conducted on total ambulation, inner segment ambulation and defecation in the open field, and on the changes in body temperature and vocalization from the pre-injection measures. The significance of these changes themselves and the between cell comparisons of ambulation were analyzed with matched-pair t-tests. Since rather few animals vocalized but Some d i d so frequently, the vocalizations over all three measurements were ranked and statistical tests performed upon the ranks. RESULTS
Activity Ethanol significantly reduced activity during the first 12 hr (Fig. I). An increase in activity w a s then found during the last 8 hr, with the activity 20--24 hr after ethanol being significantly higher than after the saline injection, t(14)=2.95, p =0.0098. There were no significant differences in the hyperactivity results between the animals receiving ethanol first and saline first and there were no significant group differences on the activity on the days before the injections. The reduction in
HANGOVER IN RATS
505
TABLE 1 CHANGES IN BODYTEMPERATUREAFTER2.5 g,/kgETHANOLOR SALINE RELATIVETO PRE-INJECTION(°C, MEAN _+SE) 24 hr
48 hr
After Ethanol Home cage Ethanol first (n=7) Saline first (8) Running wheel Ethanol first (8) Saline first (8) All rats (31)
+0.90 _+ 0.20¢ +0.32 _+ 0.13" +0.59 _+ 0.13~t§#
+0.49 ± 0.18" +0.11 _~ 0.35 +0.29 _+ 0.20
+0.04 +0,30 +0.17 +0.37
-0.16 -0.11 -0.14 +0.07
_+ 0.09 _+ 0.17 _+ 0.10 _+ 0.09~t¶
_+ 0.12 + 0.24 _+ 0.13 _+ 0.12
After Saline Home cage Ethanol first (7) Saline first (8) Running wheel Ethanol first (8) Saline first (8) All rats (31)
+0.10 ± 0.23 -0.18 _+ 0.27 -0.05 _+ 0.18
+0.41 _+ 0.27 -0.21 _+ 0.18 +0.08 _+ 0.17
+0.22 -0.15 +0.04 -0.00
-0.03 -0.25 -0.14 -0.03
± 0.17 _+ 0.20 _+ 0.15 ± 0.11
_+ 0.22 ± 0.16 _+ 0.14 ± 0.11
*Significant increase over pre-injection p<0.05; ~p<0.01; ~p<0.001. §Significantly higher than increase in running wheel rats, p<0.01. ¶Significantly higher than 24 hr after saline, p<0,05; #p<0.01.
ethanol was first. The home-cage rats receiving ethanol first also had a significant increase at 48 hr over their preinjection temperatures, but showed a similar tendency after saline.
Open Field The aftereffect of ethanol on open-field performance differed in the home-cage rats and the running-wheel rats. The home-cage animals showed significantly less total ambulation and less activity in the inner segments of the dark-quite open field on the day after ethanol than on the day after saline (Table 2). This reduction was not seen, however, in the animals that had been running in the wheels prior to testing; the interaction between injection (ethanol vs. saline) and prior housing location was significant, F(1,27)---6.27, p =0.019. Activity in general was lower in the light-noisy open field than in the dark-quiet one, and no aftereffect of ethanol was seen under the light-noisy condition; the interaction between injection and open-field condition was significant (p =0.042). There were no significant results related to the order of injection and the only significant difference with defecation scores (not shown) was a higher value for the running-wheel rats (p =0.043), regardless of injection and other conditions.
Vocalization The changes in vocalization 24 hr after ethanol (Table 3) also differed according to the prior housing location. The home-cage rats vocalized significantly more than prior to the injection, the running-wheel rats tended (p =0.10) to vocalize less, and the difference between groups was significant. At 48 hr after ethanol, when the running-wheel rats had been back in their home cages for 24 hr, the difference between them and the home-cage group was no longer significant. There was, however, a significant increase over both the pre-injection vocalization and the vocalization 48 hr after saline in both the home-cage rats and in all animals combined. There also was a significant interaction between the order of injection, the substance injected, and time order of injection, the substance injected, and time (p =0.043). DISCUSSION
activity seen both 4--7 hr and 8--11 hr after ethanol was, however, significantly (p<0.05) greater in the rats receiving ethanol first.
Bo@ Temperature The temperature, for all 31 rats combined, was significantly higher on the day after ethanol than just prior to the injection: the increase was also significantly different from what they showed 24 hr after saline (Table 1). The mean temperatures (+SE) with ethanol were 37.20+0.10 ° (preinjection), 37.57__0.06 ° (24 hr), and 37.26_+0.08 ° (48 hr); the corresponding temperatures with saline were 37.10_+0.09 °, 37.10_+0.10 ° and 37.07_+0.08 °. The hyperthermia 24 hr after ethanol was significantly larger in the home-cage rats than in the running-wheel animals (F=8.94, p =0.0059). The injection order (ethanol vs. saline first) did not have a direct significant effect on the hyperthermia, but it did interact with location (F=7.97, p=0.0089): the home-cage rats showed more hyperthermia on the day after ethanol when ethanol was the first injection but the running-wheel rats had less hyperthermia when
Several aftereffects of acute ethanol were observed: hyperactivity in the running wheel, hyperthermia, under certain conditions a decrease in open-field activity, and an increase in vocalization. The activity records from the running wheel (Fig. 1) have a strong resemblance to the time course for body temperature changes observed previously when measuring by rectal probe [20]. Both running and temperature were reduced during intoxication; the decrease disappeared at about the time the ethanol would be expected to have been eliminated [10]; and thereafter a change opposite to that produced by ethanol was seen, reaching maximum significance 20--24 hr after alcohol. The hyperthermia now seen 24 hr after ethanol among the home-cage animals was similar in magnitude to that previously observed [20]. Their increase in vocalization at 24 hr is also in agreement with the earlier study, but the significant increase at 48 hr was not seen previously; the only major procedural differences are that temperature was measured every 4 hr in the previous study and the open-field test was not given. The 48 hr increase could be related to the hyper-
506
H A N G O V E R IN R A T S
TABLE 2 AMBULATION IN THE OPEN FIELD 24 HR AFTER AN ETHANOL OR SALINE INJECTION (NUMBER OF SEGMENTS ENTERED. MEAN ~ SE fN))
TABLE 3 CHANGES IN VOCALIZATION AFTER 2~5g~kgETHANOL OR SAHNE RELATIVE TO PRE-INJECTION (MEAN +:_SE) 24 hr
48 hr
Open-Field Condition Injection
Dark-Quiet
Light-Noisy
After Etham~l Home cage Ethanol first (n:-71 Saline first (8)
Total Activity in All Segments Home cage Ethanol Saline
17.4 ___3.6*(7) 28.0 -+ 2.1 (7)
16.9 ± 3.7 (8) 16.0 ± 3.0 (8)
Running wheel Ethanol Saline
24.1 ± 3.2 (8) 22.2 ± 4.2 (8)
20.6 ± 4.6 (8) 16.8 ± 4.1 (8)
Running wheel Ethanol first (8) Saline first (8) All rats
Activity in Inner Segments Home cage Ethanol Saline
3.4 ± 2.5*(7) 6.9 ± 1.7 (7)
2.9 ± 1.6 (8) 2.1 ± 1.5 (8)
Running wheel Ethanol Saline
5.0 + 1.7 (8) 5.5 + 1.7 (8)
5.8 ± 2.4 (8) 3.4 ± 1.6 (8)
*Significantly less than after saline, p<0.05. thermia on the s e c o n d day after ethanol r e p o r t e d by Gallaher and E n g e r [6]; this late aftereffect on t e m p e r a t u r e had not been seen in our p r e v i o u s study but s o m e indication for it was found n o w a m o n g the rats receiving ethanol as their first injection. T h e d e c r e a s e in open-field activity 24 hr after ethanol is in a g r e e m e n t with the results with mice [15] which were also obtained with a dark-quiet o p e n field. Since suppression of open-field ambulation, and particularly o f exploration of the inner segments, is supposedly a mark o f emotional reactions [16], the result could be due to a greater reaction on the day after ethanol to stress or fear elicited by the open-field situation. The light-noisy o p e n field may h a v e b e e n so stressful that it suppressed activity in all animals regardless of their prior injection. E a c h of the three effects seen 24 hr after ethanol in the h o m e - c a g e rats was significantly r e d u c e d in the runningw h e e l rats. T h e r e are various possible explanations that will have to be e x a m i n e d in future studies, but one possibility is that the exercise m a d e by the running-wheel rats r e d u c e d the o t h e r aftereffects o f ethanol. This suggests the importance o f having a controlled study in h u m a n s to see if vigorous exercise really does reduce hangover. O n e can e v e n speculate that a reduction in unpleasant aftereffects reinforced the running and thus caused the increase in this activity. T h e r e are, h o w e v e r , several o t h e r h y p o t h e s e s that might a c c o u n t for the increase in running. It could be (1) a direct aftereffect of p r e v i o u s ethanol, (2) an aftereffect o f the p r e v i o u s hypoactivity [13,21 (p. 364)], (3) a result of an alteration in normal circadian r h y t h m s [6], (4) a result of increased sensitivity or reactivity to stimulation on the day after ethanol, or (5) a c o m p e n s a t o r y reaction, opposite to the drug effect (i.e., hypoactivity), elicited by stimuli that had p r e v i o u s l y b e e n present w h e n e t h a n o l was administered or during the early phases of intoxication [17]. Essentially these h y p o t h e s e s w e r e evaluated previously
Home cage Ethanol first (7) Saline first (8) Running wheel Ethanol first (8) Saline first 181 All rats
! 7 , ~.!~ 0.6 : 0.6 1.1 ~ 0.5* 1.6 '_ 1,9 -0.4 ~: 0.4 1.0 :_~ 1.0 0 c 0.6 After Saline
1.3 r- 0.4 2.2 ± 2.2 1.8 +-. 1.2+ 2.6 -0.3 1.2 ~.5
:~ 1.2 ± 0.3 ± 0.7 ± 075
0~1 :-. 0.7 0,3 ~- 0.3 0.2 .t 0.3
0,9 _- 0.9 (1.4 ~c 0.4 --0.2 ± 0.5
0~3 ~ 0.5 0. ! :!: 0 t 0.2 0.2 0.2 - {).2
0. I 0 0.1 - 0.1
+ 0.3 :~ 0 = 0.1 - 0.2
*Significant increase over pre-injection, p =0.41, and significantly higher than change in runnmg wheel rats, p =0.043. tSignificant increase over pre-injection, p=0.004, and significantly larger increase than 48 hr after saline. # =0.022. ;Significant increase over pre-injection, p=0.001, and significantly larger increase than 48 hr after saline, p =0.012. with regards to r e b o u n d h y p e r t h e r m i a and its changes with tolerance and with isolation [20], and the e v i d e n c e s e e m e d to favor the last two. T h e r e is yet little basis for evaluating t h e m with regards to the increase in running, but the finding that the rats w e r e not also hyperactive in the open field may h a v e some relevance. This difference could h a v e b e e n caused by the running wheel but not the open field being c o n d u c i v e to the type o f vigorous exercise n e e d e d to reduce unpleasant aftereffects or by either of the last 2 hypotheses. W h e e l running differs f r o m open-field activity in that it p r o d u c e s a large a m o u n t o f stimulation and this stimulation apparently promotes further running (cf. [4]). An increased sensitivity or reactivity to stimulation might, therefore, produce m o r e wheel running, but it would increase the stress or fear reaction in the o p e n field and d e c r e a s e ambulation there. Alternatively, it can be noted that the increased wheel running occurred in the same situation present during the early phases of intoxication and nearly the s a m e situation as ethanol administration (same room. s a m e lighting conditions). The o p e n field, h o w e v e r , was a n o v e l situation which could not h a v e b e e n associated with the p r e v i o u s ethanol administration. Thus c o m p e n s a t o r y reactions, including hyperactivity, could h a v e been elicited in the running w h e e l but not in the o p e n field. Despite the high cost to society and the discomfort to the individual caused by h a n g o v e r [22], there has b e e n relative!y little research on the problem. O n e purpose of the present line of r e s e a r c h is, therefore, the d e v e l o p m e n t of an animal
SINCLAIR AND GUSTAFSSON
507
model that might be useful in hangover research. Perhaps one could study h a n g o v e r with the existing animal models of withdrawal, if h a n g o v e r is, in fact, merely a w e a k form of withdrawal [5], but this relationship has yet to be firmly established. Indeed, one apparent difference has already been noted. Physical d e p e n d e n c e and the severity of withdrawal generally increase in parallel with the d e v e l o p m e n t of tolerance, but h a n g o v e r severity in humans is decreased by tolerance (see [20]). F u r t h e r m o r e , e v e n if h a n g o v e r and withdrawal do differ only quantitatively, there may well be effective h a n g o v e r treatments that would do little to reduce the more severe withdrawal s y m p t o m s , and a specific animal model of h a n g o v e r might be useful in finding these treatments. As discussed elsewhere [20], the face validity of the known aftereffects o f acute intoxication in rats for human h a n g o v e r is rather good. The predictive validity of the animal model, which is of more practical importance, can be partially tested by seeing if the aftereffects in rats are decreased by factors or treatments reported to lessen h a n g o v e r in humans. So far the only evidence of this sort is that tolerance
reduces not only human h a n g o v e r but also rebound hyperthermia in rats [20], and perhaps the present finding that exercise apparently reduced hyperthermia, vocalization, and the open-field aftereffects. Other treatments that might be tested include low doses of ethanol, aspirin [12] or the prophylactic use of tolfenamic acid [8], pyritinol [9], and chlormethiazole [11]. (The validity of the testing is, of course, limited by the reliability of the data from humans establishing that these treatments are effective.) Increasing the n u m b e r of aftereffects known to o c c u r in rats greatly improves the possibility for testing the relevance. F o r instance, a finding that aspirin or tolfenamic acid reduced the rebound hyperthermia would not be very meaningful. Testing w h e t h e r they counteracted a wide variety of aftereffects could, h o w e v e r , help to show w h e t h e r the rat model has relevance to human hangover. ACKNOWLEDGEMENT We wish to thank Dr. Jouni Aalto for developing the system for computer monitoring of the running wheels.
REFERENCES I. Alkana, R. L. and R. D. Malcolm. Comparison of the effects of acute alcohol intoxication on behavior in humans and other animals. In: Animal Models in Ah'ohol Research, edited by K. Eriksson, J. D. Sinclair and K. Kiianmaa. London: Academic Press, 1980, pp. 193-269. 2. Duncan, P. D. and A. M. Baez. The effect of ethanol on wheel running in rats. Pharmaeol Biochem Behav 15: 819-821, 1981. 3. Eriksson, K. Inherited metabolism and behavior towards alcohol: Critical evaluation of human and animal research. In: Animal Models in Alcohol Research, edited by K. Eriksson, J. D. Sinclair and K. Kiianmaa. London: Academic Press, 1980, pp. 3-20. 4. Finger, F. W. Measuring behavioral activity. In: Methods in Psychobiology. vol 2, edited by R. D. Myers. New York: Academic Press, 1972. 5. Freund, G. Comparison of alcohol dependence, withdrawal and hangover in humans and animals. In: Animal Models in Alcohol Research. edited by K. Eriksson, J. D. Sinclair and K. Kiianmaa. London: Academic Press, 1980, pp. 293-308. 6. Gallaher, E. J. and D. A. Enger. Rebound hyperthermia follows ethanol-induced hypothermia in rats. Psychopharmaeology (Berlin) 91: 34-39, 1987. 7. Hilikivi, L., C. J. P. Eriksson, M. Sarviharju and J. D. Sinclair. Revitalization of the AA and ANA rat lines: Effects on some line characteristics. Alcohol 1: 71-77, 1984. 8. Kaivola, S., J. Parantainen, T. 0sterman and H. Timonen. n. Hangover headache and prostaglandins: Prophylactic treatment with tolfenamic acid. Cephalagia 3: 31-36, 1983. 9. Khan, M. A., K. Jensen and H. J. Krogh. Alcohol-induced hangover: A double blind comparison of pyritonol and placebo in preventing hangover symptoms. Q J Stud Alcohol 34: 11951201, 1973. 10. Mucha, R. F. and J. P. J. Pinel. Increased susceptibility to kindled seizures in rats following a single injection of alcohol. ,1 Stud Alcohol 40: 258--271, 1979. 11. Myrsten, A.-L., U. Rydberg, C.-M. Idestr6m and R. Lamble. Alcohol intoxication and hangover: Modification of hangover by chlormethiazole. Psychopharmacology (Berlin) 69: 117-125, 1980. 12. Parantainen, J. Prostaglandins in ethanol intolerance and hangover. Drug Alcohol Depend 11: 23%248, 1983.
13. Premack, D. Reversibility of the reinforcement relation. Scienee 136: 255-257, 1962. 14. Rydberg, U. Experimentally induced alcohol hangover. Excerpta Medica International Congress Series No. 407, Recent Advances in the Study of Alcoholism, 1977, pp. 32-40. 15. Sanders, B. Withdrawal-like signs induced by a single administration of ethanol in mice that differ in ethanol sensitivity. Psychopharmacology (Berlin)68: 10%113, 1980. 16. Scholtens, J., N. E. Van de Poll, H. B. M. Uylings, J. Van der Gugten and G. J. Boer. Effects of experimental suppression of active (REM) sleep during early development upon adult brain and behavior in the rat. Dev Brain Res 7: 277-286, 1983. 17. Siegel, S. Pavlovian conditioning and ethanol tolerance. Alcohol Alcohol Suppl 1: 25-36, 1987. 18. Sinclair, J. D., K. Gustafsson and J. Aalto. Hangover and the effects in rats on the day after a single ethanol injection. Acta Pharmaeol Toxicol (Copenh) 55: (Suppl I, Abstracts of the XV Annual Nordic Meeting on Biological Alcohol Research, Helsinki) No. 4, 1984. 19. Sinclair, J. D., K. Gustafsson and T. Taira. Aftereffects in rats of acute ethanol: Influence of administration route, tolerance, and the procedure for measuring body temperature. Alcohol Ah'ohol 21: (Abstracts of the Third Congress of the International Society for Biomedical Research on Alcoholism, Helsinki ) A44, 1986. 20. Sinclair, J. D. and T. Taira. Hangover hyperthermia in rats: Relation to tolerance and external stimuli. Psychopharmacology (Berlin) in press, 1987. 21. Skinner, B. F. The Behavior o f Organisms: An Experimental Analysis. Englewood Cliffs, N J: Century-Crofts, 1938. 22. Smith, C. M. and G. M. Barnes. Signs and symptoms of hangover: Prevalence and relationship to alcohol use in a general adult population. Drug Alcohol Depend 11: 249--269, 1983. 23. Ylikahri, R. Krapula ja kuntoilu (Hangover and exercize). Liikunta Tiede 24: 72-75, 1987. 24. Ylikahri, R. H., M. O. Huttunen, C. J. P. Eriksson and E. A. Nikkilh. Metabolic studies on the pathogenesis of hangover. Eur J Clin lm'est 4: 93-100, 1974.
0741-8329/87$3.00 + .00
Behavioral Changes in Rats on the Day After Acute Ethanol Intoxication ' J. D. S I N C L A I R A N D K. G U S T A F S S O N Research Laboratories o f the Finnish Ah:ohol Company (Alko Ltd), Box 350, SF-O0100 Helsinki, Finland R e c e i v e d 23 April 1987; A c c e p t e d 26 M a y 1987 SINCLAIR, J. D. AND K. GUSTAFSSON. Behavioral changes in rats on the day after acute ethanol intoxication. ALCOHOL 4(6) 503-507, 1987.--Activity in computer-monitored running wheels, behavior in open-field tests, body temperature measured by rectal probe, and vocalization were examined in rats after a single injection of 2.5 g/kg ethanol or saline. A reduction in running-wheel activity occurred during intoxication, followed by a significant increase in running 20--24 hr after the injection. Also 24 hr after ethanol, among rats kept in normal cages, there was an increase in body temperature and vocalization and a decrease in ambulation in a dark, quiet open field. Each of these 3 aftereffects was reduced or abolished in the rats with access to a running wheel. The results confirm and extend previous findings of aftereffects of acute intoxication in rats with a time course similar to hangover in humans. Hangover
Acute ethanol intoxication
Activity
Exercise
Open-field test
Temperature regulation
peripheral changes accompanying hangover, such as increased blood lactate and free fatty acids, the central nervous system changes related to the preceding intoxication are believed to be more important [24]. Therefore, the relevance of animal models to human hangover might be expected to be directly proportional to the relevance of the animal models of intoxication. Three aftereffects of acute intoxication in rats have previously been reported that fit Rydberg's definition of hangover and are apparently related to central mechanisms. The susceptibility to kindled seizures is increased [10], body temperature is increased [6, 18-20], and rats vocalize more when handled [19,20]. The present study was designed to search for other possible aftereffects of ethanol in rats. Ethanol intoxication in rats is accompanied by a monotonically dose-dependent decrease of activity in a running wheel [2] and hyperactivity is commonly found as a withdrawal symptom in various species [5], so the aftereffects on wheel running were examined. Open-field behavior was also measured because mice have been reported to show reduced activity in an open field as an aftereffect of a single ethanol injection [15]. This was with an open-field test made in a dark, quiet room, in contrast to the usual procedure in our laboratory of using high illumination and loud white noise during the test [7], so both conditions were included in the present study. Finally, many people say that jogging or other forms of exercise help to reduce hangover symptoms [23]; consequently, in order to see any effects of wheel running on the other aftereffects, control rats were included that were treated identically to the running-wheel animals but remained in their home cages.
THERE are several well-established animal models of withdrawal from chronic ethanol (cf. [5]), but to our knowledge none has been proposed for the aftereffects of acute intoxication, commonly referred to as hangover. If hangover is defined, not in terms of subjective reports [5], but rather as Rydberg [14] does, "as a state following the acute alcohol stage, appearing about the time when the blood alcohol concentration approaches zero, and continuing for several hours," then hangover should be as amenable to research with animal models as are withdrawal and acute ethanol intoxication. Certain aspects of intoxication, such as the influence of social factors and the subjective reports of euphoria, would at best be very difficult to study in animals. Nevertheless, intoxication is believed to be the result of ethanol's actions on the nervous system that are essentially the same in humans and laboratory animals. Consequently, there are a variety of animal models believed to be relevant for the study of intoxication [1]. Along the same lines, one cannot expect animal models of hangover to reflect all the components found in humans. For instance, the same social factors that affect intoxication in humans may also affect the subsequent hangover, and these would not be amenable to study with animal models. It should, however, be possible to develop animal models for the components of hangover that are caused by the aftereffects of ethanol on physiological mechanisms. The importance of such physiological factors, in contrast to social factors, is supported by the very high heritability estimate (0.62) for the number of hangovers that has been found in human twin studies (see [3]). Furthermore, although there are many
1portions of this material were presented at the XV Annual Nordic Meeting on Biological Alcohol Research, Helsinki, 1984 [18] and at the 3rd ISBRA Congress, Helsinki, 1986 [19].
503
504
S I N C L A I R AND G U S T A F S S O N METHOD 40
Animals
after saline
30
Male Long Evans rats (n= 31) of approximately 3 months of age were used. They were individually housed for 1 week before the experiment.
20 10 ...~-~va f t e r ®thanol
Equipment Four 36 cm running wheels with affixed living cages were monitored with a Commodore Vic 20 computer. The 83 cm diameter open field had 1830 luxes at floor level and 78 dB white noise in the light-noisy condition and only dim red light in the dark-quiet condition. Both the room containing the running wheels and the room with the suspended-wire home cages rooms were maintained at 21-23°C and illuminated from 6 p.m. to 6 a.m. by 4 overhead 40 watt fluorescent tubes. A TE 3 thermister (Ellab Instruments, Copenhagen) was used for temperature measurements. Water and standard powdered rat food (Astra-Ewos, Sweden) were available in both the home cages and the running wheels.
4
8 12 16 HOURS AFTER INJECTION
\~ 20
24
HOURS AFTER INJECTION 1
4
8
12
i~i~:l
.t
_> ,(
z
Procedure The general design was to measure the aftereffects of alcohol on running-wheel activity, body temperature, vocalization, and open-field behavior, plus the effect of the exercise in the running wheel on the other 3 measures. Each rat was tested twice, once after alcohol and once after saline, in a balanced order, and thus served as its own control. The rats were divided into two groups matched for body weight: one group (n = 16) was put into running wheels and the other (n= 15) remained in the home cages. After the first group had been in the running wheels for 24 hr, they were removed and had their temperature measured (6 cm anal insertion, recording after 40 sec). The number of audible vocalizations made during handling and temperature measurement were also recorded. Then they were injected with either 2.5 g/kg ethanol (as a warmed 12.5% w/v solution in saline, n = 8) or a similar volume of warmed saline (n = 8), and were returned to the running wheel. At the same time the home-cage rats also had their temperature and vocalization recorded and were injected with either ethanol (n=7) or saline (n=8). All of the rats had their temperature and vocalization again measured 24 hr after the injections, and then were tested in either the light-noisy or dark-quiet open field. They were then all placed in home cages, except when with temperature and vocalization was again recorded 48 hr after the injections. The procedure was repeated in exactly the same manner 13 days later except the animals previously given saline now had ethanol and the rats that previously had ethanol received saline. The experiment was run sequentially, with batches of 4 running-wheel rats and 4 home-cage rats started every 3 days, and with the other c o n d i t i o n . b a l a n c e d in each batch. The running-wheel results were combined into 6 segments of 4 hr each (except the first which was only 3 hr long because the data from the first hr were discarded).
Statistical Analyses The effects of ethanol on running-wheel activity were analyzed with matched-pair t-tests comparing each animal's activity after ethanol with its own activity at a comparable
W
O I/J O gl
10
0
2o
t
'
t
l
i
1 1 ii 3O
FIG. t. Effects of a 2.5 g/kg ethanol injection on activity in running wheels. The upper frame shows the mean number of revolutions/hr after ethanol and by the same rats after saline. The lower frame shows the mean (+_SE) of each rat's activity after ethanol minus its own activity after saline. **Significant difference between ethanol and saline results, p<0.01. time after saline. An analysis of variance was conducted on total ambulation, inner segment ambulation and defecation in the open field, and on the changes in body temperature and vocalization from the pre-injection measures. The significance of these changes themselves and the between cell comparisons of ambulation were analyzed with matched-pair t-tests. Since rather few animals vocalized but Some d i d so frequently, the vocalizations over all three measurements were ranked and statistical tests performed upon the ranks. RESULTS
Activity Ethanol significantly reduced activity during the first 12 hr (Fig. I). An increase in activity w a s then found during the last 8 hr, with the activity 20--24 hr after ethanol being significantly higher than after the saline injection, t(14)=2.95, p =0.0098. There were no significant differences in the hyperactivity results between the animals receiving ethanol first and saline first and there were no significant group differences on the activity on the days before the injections. The reduction in
HANGOVER IN RATS
505
TABLE 1 CHANGES IN BODYTEMPERATUREAFTER2.5 g,/kgETHANOLOR SALINE RELATIVETO PRE-INJECTION(°C, MEAN _+SE) 24 hr
48 hr
After Ethanol Home cage Ethanol first (n=7) Saline first (8) Running wheel Ethanol first (8) Saline first (8) All rats (31)
+0.90 _+ 0.20¢ +0.32 _+ 0.13" +0.59 _+ 0.13~t§#
+0.49 ± 0.18" +0.11 _~ 0.35 +0.29 _+ 0.20
+0.04 +0,30 +0.17 +0.37
-0.16 -0.11 -0.14 +0.07
_+ 0.09 _+ 0.17 _+ 0.10 _+ 0.09~t¶
_+ 0.12 + 0.24 _+ 0.13 _+ 0.12
After Saline Home cage Ethanol first (7) Saline first (8) Running wheel Ethanol first (8) Saline first (8) All rats (31)
+0.10 ± 0.23 -0.18 _+ 0.27 -0.05 _+ 0.18
+0.41 _+ 0.27 -0.21 _+ 0.18 +0.08 _+ 0.17
+0.22 -0.15 +0.04 -0.00
-0.03 -0.25 -0.14 -0.03
± 0.17 _+ 0.20 _+ 0.15 ± 0.11
_+ 0.22 ± 0.16 _+ 0.14 ± 0.11
*Significant increase over pre-injection p<0.05; ~p<0.01; ~p<0.001. §Significantly higher than increase in running wheel rats, p<0.01. ¶Significantly higher than 24 hr after saline, p<0,05; #p<0.01.
ethanol was first. The home-cage rats receiving ethanol first also had a significant increase at 48 hr over their preinjection temperatures, but showed a similar tendency after saline.
Open Field The aftereffect of ethanol on open-field performance differed in the home-cage rats and the running-wheel rats. The home-cage animals showed significantly less total ambulation and less activity in the inner segments of the dark-quite open field on the day after ethanol than on the day after saline (Table 2). This reduction was not seen, however, in the animals that had been running in the wheels prior to testing; the interaction between injection (ethanol vs. saline) and prior housing location was significant, F(1,27)---6.27, p =0.019. Activity in general was lower in the light-noisy open field than in the dark-quiet one, and no aftereffect of ethanol was seen under the light-noisy condition; the interaction between injection and open-field condition was significant (p =0.042). There were no significant results related to the order of injection and the only significant difference with defecation scores (not shown) was a higher value for the running-wheel rats (p =0.043), regardless of injection and other conditions.
Vocalization The changes in vocalization 24 hr after ethanol (Table 3) also differed according to the prior housing location. The home-cage rats vocalized significantly more than prior to the injection, the running-wheel rats tended (p =0.10) to vocalize less, and the difference between groups was significant. At 48 hr after ethanol, when the running-wheel rats had been back in their home cages for 24 hr, the difference between them and the home-cage group was no longer significant. There was, however, a significant increase over both the pre-injection vocalization and the vocalization 48 hr after saline in both the home-cage rats and in all animals combined. There also was a significant interaction between the order of injection, the substance injected, and time order of injection, the substance injected, and time (p =0.043). DISCUSSION
activity seen both 4--7 hr and 8--11 hr after ethanol was, however, significantly (p<0.05) greater in the rats receiving ethanol first.
Bo@ Temperature The temperature, for all 31 rats combined, was significantly higher on the day after ethanol than just prior to the injection: the increase was also significantly different from what they showed 24 hr after saline (Table 1). The mean temperatures (+SE) with ethanol were 37.20+0.10 ° (preinjection), 37.57__0.06 ° (24 hr), and 37.26_+0.08 ° (48 hr); the corresponding temperatures with saline were 37.10_+0.09 °, 37.10_+0.10 ° and 37.07_+0.08 °. The hyperthermia 24 hr after ethanol was significantly larger in the home-cage rats than in the running-wheel animals (F=8.94, p =0.0059). The injection order (ethanol vs. saline first) did not have a direct significant effect on the hyperthermia, but it did interact with location (F=7.97, p=0.0089): the home-cage rats showed more hyperthermia on the day after ethanol when ethanol was the first injection but the running-wheel rats had less hyperthermia when
Several aftereffects of acute ethanol were observed: hyperactivity in the running wheel, hyperthermia, under certain conditions a decrease in open-field activity, and an increase in vocalization. The activity records from the running wheel (Fig. 1) have a strong resemblance to the time course for body temperature changes observed previously when measuring by rectal probe [20]. Both running and temperature were reduced during intoxication; the decrease disappeared at about the time the ethanol would be expected to have been eliminated [10]; and thereafter a change opposite to that produced by ethanol was seen, reaching maximum significance 20--24 hr after alcohol. The hyperthermia now seen 24 hr after ethanol among the home-cage animals was similar in magnitude to that previously observed [20]. Their increase in vocalization at 24 hr is also in agreement with the earlier study, but the significant increase at 48 hr was not seen previously; the only major procedural differences are that temperature was measured every 4 hr in the previous study and the open-field test was not given. The 48 hr increase could be related to the hyper-
506
H A N G O V E R IN R A T S
TABLE 2 AMBULATION IN THE OPEN FIELD 24 HR AFTER AN ETHANOL OR SALINE INJECTION (NUMBER OF SEGMENTS ENTERED. MEAN ~ SE fN))
TABLE 3 CHANGES IN VOCALIZATION AFTER 2~5g~kgETHANOL OR SAHNE RELATIVE TO PRE-INJECTION (MEAN +:_SE) 24 hr
48 hr
Open-Field Condition Injection
Dark-Quiet
Light-Noisy
After Etham~l Home cage Ethanol first (n:-71 Saline first (8)
Total Activity in All Segments Home cage Ethanol Saline
17.4 ___3.6*(7) 28.0 -+ 2.1 (7)
16.9 ± 3.7 (8) 16.0 ± 3.0 (8)
Running wheel Ethanol Saline
24.1 ± 3.2 (8) 22.2 ± 4.2 (8)
20.6 ± 4.6 (8) 16.8 ± 4.1 (8)
Running wheel Ethanol first (8) Saline first (8) All rats
Activity in Inner Segments Home cage Ethanol Saline
3.4 ± 2.5*(7) 6.9 ± 1.7 (7)
2.9 ± 1.6 (8) 2.1 ± 1.5 (8)
Running wheel Ethanol Saline
5.0 + 1.7 (8) 5.5 + 1.7 (8)
5.8 ± 2.4 (8) 3.4 ± 1.6 (8)
*Significantly less than after saline, p<0.05. thermia on the s e c o n d day after ethanol r e p o r t e d by Gallaher and E n g e r [6]; this late aftereffect on t e m p e r a t u r e had not been seen in our p r e v i o u s study but s o m e indication for it was found n o w a m o n g the rats receiving ethanol as their first injection. T h e d e c r e a s e in open-field activity 24 hr after ethanol is in a g r e e m e n t with the results with mice [15] which were also obtained with a dark-quiet o p e n field. Since suppression of open-field ambulation, and particularly o f exploration of the inner segments, is supposedly a mark o f emotional reactions [16], the result could be due to a greater reaction on the day after ethanol to stress or fear elicited by the open-field situation. The light-noisy o p e n field may h a v e b e e n so stressful that it suppressed activity in all animals regardless of their prior injection. E a c h of the three effects seen 24 hr after ethanol in the h o m e - c a g e rats was significantly r e d u c e d in the runningw h e e l rats. T h e r e are various possible explanations that will have to be e x a m i n e d in future studies, but one possibility is that the exercise m a d e by the running-wheel rats r e d u c e d the o t h e r aftereffects o f ethanol. This suggests the importance o f having a controlled study in h u m a n s to see if vigorous exercise really does reduce hangover. O n e can e v e n speculate that a reduction in unpleasant aftereffects reinforced the running and thus caused the increase in this activity. T h e r e are, h o w e v e r , several o t h e r h y p o t h e s e s that might a c c o u n t for the increase in running. It could be (1) a direct aftereffect of p r e v i o u s ethanol, (2) an aftereffect o f the p r e v i o u s hypoactivity [13,21 (p. 364)], (3) a result of an alteration in normal circadian r h y t h m s [6], (4) a result of increased sensitivity or reactivity to stimulation on the day after ethanol, or (5) a c o m p e n s a t o r y reaction, opposite to the drug effect (i.e., hypoactivity), elicited by stimuli that had p r e v i o u s l y b e e n present w h e n e t h a n o l was administered or during the early phases of intoxication [17]. Essentially these h y p o t h e s e s w e r e evaluated previously
Home cage Ethanol first (7) Saline first (8) Running wheel Ethanol first (8) Saline first 181 All rats
! 7 , ~.!~ 0.6 : 0.6 1.1 ~ 0.5* 1.6 '_ 1,9 -0.4 ~: 0.4 1.0 :_~ 1.0 0 c 0.6 After Saline
1.3 r- 0.4 2.2 ± 2.2 1.8 +-. 1.2+ 2.6 -0.3 1.2 ~.5
:~ 1.2 ± 0.3 ± 0.7 ± 075
0~1 :-. 0.7 0,3 ~- 0.3 0.2 .t 0.3
0,9 _- 0.9 (1.4 ~c 0.4 --0.2 ± 0.5
0~3 ~ 0.5 0. ! :!: 0 t 0.2 0.2 0.2 - {).2
0. I 0 0.1 - 0.1
+ 0.3 :~ 0 = 0.1 - 0.2
*Significant increase over pre-injection, p =0.41, and significantly higher than change in runnmg wheel rats, p =0.043. tSignificant increase over pre-injection, p=0.004, and significantly larger increase than 48 hr after saline. # =0.022. ;Significant increase over pre-injection, p=0.001, and significantly larger increase than 48 hr after saline, p =0.012. with regards to r e b o u n d h y p e r t h e r m i a and its changes with tolerance and with isolation [20], and the e v i d e n c e s e e m e d to favor the last two. T h e r e is yet little basis for evaluating t h e m with regards to the increase in running, but the finding that the rats w e r e not also hyperactive in the open field may h a v e some relevance. This difference could h a v e b e e n caused by the running wheel but not the open field being c o n d u c i v e to the type o f vigorous exercise n e e d e d to reduce unpleasant aftereffects or by either of the last 2 hypotheses. W h e e l running differs f r o m open-field activity in that it p r o d u c e s a large a m o u n t o f stimulation and this stimulation apparently promotes further running (cf. [4]). An increased sensitivity or reactivity to stimulation might, therefore, produce m o r e wheel running, but it would increase the stress or fear reaction in the o p e n field and d e c r e a s e ambulation there. Alternatively, it can be noted that the increased wheel running occurred in the same situation present during the early phases of intoxication and nearly the s a m e situation as ethanol administration (same room. s a m e lighting conditions). The o p e n field, h o w e v e r , was a n o v e l situation which could not h a v e b e e n associated with the p r e v i o u s ethanol administration. Thus c o m p e n s a t o r y reactions, including hyperactivity, could h a v e been elicited in the running w h e e l but not in the o p e n field. Despite the high cost to society and the discomfort to the individual caused by h a n g o v e r [22], there has b e e n relative!y little research on the problem. O n e purpose of the present line of r e s e a r c h is, therefore, the d e v e l o p m e n t of an animal
SINCLAIR AND GUSTAFSSON
507
model that might be useful in hangover research. Perhaps one could study h a n g o v e r with the existing animal models of withdrawal, if h a n g o v e r is, in fact, merely a w e a k form of withdrawal [5], but this relationship has yet to be firmly established. Indeed, one apparent difference has already been noted. Physical d e p e n d e n c e and the severity of withdrawal generally increase in parallel with the d e v e l o p m e n t of tolerance, but h a n g o v e r severity in humans is decreased by tolerance (see [20]). F u r t h e r m o r e , e v e n if h a n g o v e r and withdrawal do differ only quantitatively, there may well be effective h a n g o v e r treatments that would do little to reduce the more severe withdrawal s y m p t o m s , and a specific animal model of h a n g o v e r might be useful in finding these treatments. As discussed elsewhere [20], the face validity of the known aftereffects o f acute intoxication in rats for human h a n g o v e r is rather good. The predictive validity of the animal model, which is of more practical importance, can be partially tested by seeing if the aftereffects in rats are decreased by factors or treatments reported to lessen h a n g o v e r in humans. So far the only evidence of this sort is that tolerance
reduces not only human h a n g o v e r but also rebound hyperthermia in rats [20], and perhaps the present finding that exercise apparently reduced hyperthermia, vocalization, and the open-field aftereffects. Other treatments that might be tested include low doses of ethanol, aspirin [12] or the prophylactic use of tolfenamic acid [8], pyritinol [9], and chlormethiazole [11]. (The validity of the testing is, of course, limited by the reliability of the data from humans establishing that these treatments are effective.) Increasing the n u m b e r of aftereffects known to o c c u r in rats greatly improves the possibility for testing the relevance. F o r instance, a finding that aspirin or tolfenamic acid reduced the rebound hyperthermia would not be very meaningful. Testing w h e t h e r they counteracted a wide variety of aftereffects could, h o w e v e r , help to show w h e t h e r the rat model has relevance to human hangover. ACKNOWLEDGEMENT We wish to thank Dr. Jouni Aalto for developing the system for computer monitoring of the running wheels.
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13. Premack, D. Reversibility of the reinforcement relation. Scienee 136: 255-257, 1962. 14. Rydberg, U. Experimentally induced alcohol hangover. Excerpta Medica International Congress Series No. 407, Recent Advances in the Study of Alcoholism, 1977, pp. 32-40. 15. Sanders, B. Withdrawal-like signs induced by a single administration of ethanol in mice that differ in ethanol sensitivity. Psychopharmacology (Berlin)68: 10%113, 1980. 16. Scholtens, J., N. E. Van de Poll, H. B. M. Uylings, J. Van der Gugten and G. J. Boer. Effects of experimental suppression of active (REM) sleep during early development upon adult brain and behavior in the rat. Dev Brain Res 7: 277-286, 1983. 17. Siegel, S. Pavlovian conditioning and ethanol tolerance. Alcohol Alcohol Suppl 1: 25-36, 1987. 18. Sinclair, J. D., K. Gustafsson and J. Aalto. Hangover and the effects in rats on the day after a single ethanol injection. Acta Pharmaeol Toxicol (Copenh) 55: (Suppl I, Abstracts of the XV Annual Nordic Meeting on Biological Alcohol Research, Helsinki) No. 4, 1984. 19. Sinclair, J. D., K. Gustafsson and T. Taira. Aftereffects in rats of acute ethanol: Influence of administration route, tolerance, and the procedure for measuring body temperature. Alcohol Ah'ohol 21: (Abstracts of the Third Congress of the International Society for Biomedical Research on Alcoholism, Helsinki ) A44, 1986. 20. Sinclair, J. D. and T. Taira. Hangover hyperthermia in rats: Relation to tolerance and external stimuli. Psychopharmacology (Berlin) in press, 1987. 21. Skinner, B. F. The Behavior o f Organisms: An Experimental Analysis. Englewood Cliffs, N J: Century-Crofts, 1938. 22. Smith, C. M. and G. M. Barnes. Signs and symptoms of hangover: Prevalence and relationship to alcohol use in a general adult population. Drug Alcohol Depend 11: 249--269, 1983. 23. Ylikahri, R. Krapula ja kuntoilu (Hangover and exercize). Liikunta Tiede 24: 72-75, 1987. 24. Ylikahri, R. H., M. O. Huttunen, C. J. P. Eriksson and E. A. Nikkilh. Metabolic studies on the pathogenesis of hangover. Eur J Clin lm'est 4: 93-100, 1974.