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hypnotic effects of alcohol
Pharmacology Biochemistry and Behavior,Vol. 53, No. 3, pp. 585-591, 1996 Copyright 0 1996 Elscvicr Science Inc. Printed in the USA. All rights reserved CNNl-30571%515.00 + .oo ELSEVIER
0091-3057(95)02055-1
Genetic Differences in Tolerance and Sensitization to the Sedative/Hypnotic Effects of Alcohol D. L. KURTZ,*$*
R. B. STEWART,*’
M. ZWEIFEL,
*2 T.-K.
LI*t
AND
J. C. FROEHLICH*t3
Departments of *Medicine, tBiochemistry/Molecular Biology, and $Physiology/Biophysics, Indiana UniversitySchool of Medicine, Indianapolis, IN 46202-5124 Received
30 December
1994; Revised 20 June 1995; Accepted
6 July 1995
KURT& D. L., R. B. STEWART, M. ZWEIFEL, T.-K. LI AND J. C. FROEHLICH. Genetic differences in toleronce and sensitization to the sedative/hypnotic effectsof alcohol. PHARMACOL BIOCHEM BEHAV 53(3) 585-591, 1996. -Initial sensitivity to alcohol and the development of alcohol tolerance were examined in rats of the selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines. All rats received two alcohol injections (3.0 g/kg b.wt., IP) separated by either 1 or 2 days. P rats were less sensitive to the behaviorally impairing effects of alcohol than were NP rats, as evidenced by a longer latency to lose righting reflex (RR) and a shorter time to recover RR following an initial alcohol injection. When 1 day separated the two alcohol injections. P rats recovered the RR more rapidly following a second injection compared to the first, indicating that the P rats developed tolerance to the sedative/hypnotic effects of alcohol. In contrast, the NP rats recovered the RR more slowly following the second injection compared to the first, indicating that the NP rats developed sensitization to alcohol. Tolerance in the P line and sensitization in the NP line disappeared when 2 days separated the two alcohol injections. Line differences in initial sensitivity and tolerance/sensitization to the behaviorally impairing effects of alcohol may contribute to the differences in alcohol consumption observed in the P and NP lines. Alcohol Alcohol sensitivity Alcohol-nonpreferring rats
Alcohol tolerance
Sensitization
REPEATED exposure to alcohol can result in decreased responsiveness or alcohol tolerance (16), which may be an important process that contributes to the development of alcoholism (35). The development of alcohol tolerance may serve to increase alcohol intake either by reducing the aversive effects of the drug that otherwise limit alcohol intake (2), or by reducing the reinforcing effects of the drug, resulting in increased intake to maintain the same level of drug effect (36). The degree of initial sensitivity to alcohol, in the absence of previous exposure to the drug, may also influence subsequent levels of alcohol intake. An association between high alcohol intake and the development of alcohol tolerance as well as high alcohol
intake and low initial sensitivity
to alcohol
Genetics
Alcohol-preferring
rats
amounts of a 10% (v/v) alcohol solution when given continuous access to the drug with water and food freely available (20,23). Rats of the P line show greater tolerance than do NP rats in experiments using a two-injection procedure in which alcohol-induced motor impairment in an escape/avoidance task is measured, and tolerance persists longer in P rats than in NP rats (10,28,39). When alcohol-induced hypothermia was used to index tolerance in P and NP rats using a two-injection procedure, the results depended on the temporal parameters of the alcohol injections (33). When the two alcohol injections were separated by 1 day, tolerance was observed in both lines of rats. When the two alcohol injections were separated by 2 days or 3 days, neither P nor NP rats showed tolerance; rather, NP rats exhibited sensitization to the hypothermic effect of alcohol. The present itivestigation extends this previous work by
have
been reported in various lines and strains of rodents (7,19,32, 37). The alcohol-preferring (P) and alcohol-nonpreferring (NP) lines of rats were selectively bred to drink high and low
’ R. B. Stewart is now in the Department of Psychology, Purdue School of Science, Indiana University/Purdue University at Indianapolis. 2 D. L. Kurtz and M. Zweifel are now in Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana. 3Requests for reprints should be addressed to J. C. Froehlich, Ph.D., Indiana University School of Medicine, Emerson Hall 421, 545 Barnhill Drive, Indianapolis, IN 46202-5124.
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examining the generality of the association between alcohol intake, initial sensitivity to alcohol, and the development of alcohol tolerance or sensitization. Time to lose and time to recover the righting reflex were measured in P and NP rats after each of two injections of a sedative/hypnotic dose of alcohol with the injections separated by either 1 or 2 days. EXPERIMENT
I
Responsiveness to the sedative/hypnotic effects of a high dose of alcohol was determined in P and NP rats. Initial sensitivity to alcohol and two-injection tolerance were measured. METHOD
Blood Sampling At the time of recovery of RR, a blood sample (0.3 ml) was collected from the cut tip of the tail and centrifuged. Plasma aliquots were stored at -20°C for later determination of BAC. BAC Determination A 1 : 1 solution of plasma and internal standard (isopropano1 solution) was injected in a volume of 1.O ~1 into a HewlettPackard 5730A gas chromatograph equipped with a flame ionization detector and 3380A integrator. The glass columns were packed with Poropak Q (100/120 mesh) and the oven temperature was 150%.
Subjects
Data Analysis
Male, alcohol-naive rats (341-492 g) were obtained from the 28th to the 31st generations of the alcohol-preferring (P) and -nonpreferring (NP) lines. They were housed in a quiet environment under controlled temperature and lighting conditions (lights on 0700 to 1900 h), with water and standard laboratory food pellets (Purina #5001) freely available. The rats were handled once daily for 4 days before the experiment began to reduce stress on the day of alcohol challenge.
Initial sensitivity. The P and NP lines were compared on time to loss of RR, time to regain RR, and BAC at recovery of RR following the first alcohol injection using unpaired Student’s t-tests. Tolerance and sensitization. Time to regain RR (time to loss of RR minus time of recovery of RR) following each of the two alcohol injections were compared. A difference score was obtained for each rat by subtracting the time to regain RR following the first injection from the time to regain RR following the second injection. The difference scores were analyzed using a two-way ANOVA with line (P or NP) and interval between exposures (1 or 2 days) as between-group factors. Student’s t-tests were used to determine if the mean difference score for each line at each interval was significantly less than zero (indicating tolerance) or greater than zero (indicating sensitization). A pooled estimate of variance within each line was used in calculating the t statistic. BAC. A difference score was obtained for each rat by subtracting the BAC at time of recovery of RR following the first injection from the BAC at time of recovery following the second injection. The data were analyzed in the manner described above for the time to regain RR.
Procedure Alcohol-induced loss of righting reflex (RR) and recovery of RR were measured following each of two alcohol injections that were administered either 1 or 2 days apart. The group of rats given two alcohol injections separated by 1 day (day Oday 1) and the group given two alcohol injections separated by 2 days (day O-day 2) were tested at different times (4 months apart) and the two groups were similar in age (90 to 100 days old) at the time of tolerance testing. Alcohol was injected IP at a dose of 3.0 g alcohol/315 ml/kg b.wt. between 0800 and 1100 h. The concentration of the alcohol solution did not exceed 12% (v/v) to minimize concentrationinduced differences in alcohol absorption rates and tissue irritation at the site of injection (1,22). The apparatus used to measure RR was a V-shaped trough that was bent at a 90° angle and lined with sandpaper. The troughs were contained in a temperature-controlled chamber that also reduced sound disturbance. To determine loss of RR, each rat was placed on its back in the trough once every 30 s after the alcohol injection until it was unable to right itself within a 30-s interval. The time between the onset of the injection and the beginning of the 30-s interval during which the rat was unable to right itself was recorded as the time to loss of RR. The rat was then left undisturbed on its back until it turned over so that its forelegs were under its body and not visible from directly above. At this time righting trials were initiated. A righting trial was begun by placing the rat on its back. Righting was defined as the rat achieving the orientation in which at least three feet were under its body and not visible from above. Recovery of RR was defined as the ability of the rat to right itself in two consecutive 15-s trials. If the rat was unable to right itself in the first 15-s trial, it was left undisturbed until it turned over again, at which time righting trials were reinitiated. Tolerance was defined as a significantly faster recovery of the RR, at a higher blood alcohol concentration (BAC), following the second alcohol injection compared with the first. Sensitization was defined as significantly slower recovery of the RR, at a lower BAC, following the second alcohol injection compared with the first.
RESULTS
Initial Sensitivity The P rats took longer to lose RR (p < 0.05; Fig. 1) and recovered more quickly than the NP rats (p < 0.001; Fig. 1) following the first alcohol injection, which indicates that P rats were less sensitive to the behaviorally impairing effects of alcohol than were the NP rats. The mean BAC (+ SEM) at time of recovery was higher (p < 0.05) in the P rats (177 * 13 mg%) than in the NP rats (135 f 10 mg%). Tolerance and Sensitization P rats recovered the RR more rapidly following a second alcohol injection compared with the first, indicating that the P rats developed tolerance to the sedative/hypnotic effects of alcohol. By contrast, the NP rats recovered the RR more slowly following the second injection compared to the first, indicating that the NP rats developed sensitization to alcohol. The ANOVA of the difference scores of the time to regain RR indicated a significant effect of line, F(l, 34) = 25.2, p < 0.001, and an interaction of line with interval between alcohol exposures, F(l, 31) = 10.5, p < 0.01. The ANOVA of the BAC difference scores at time of recovery showed a significant effect of interval, F(1, 34) = 4.8, p < 0.05, line, F(1, 34) = 32.7, p < 0.001, and the interaction of line with interval, F(1,
GENETICS
OF ALCOHOL
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in rats of the P line in Experiment 1 could be attributed to the development of within-session tolerance.
FIGURE 1
METHOD 6.0
600.0
T
7.0
Subjects
500.0
6.0
5 400.0 5
g tz
5.0
8
4.0
300.0
G Y
3.0
P 200.0 s 3
i=
a
2.0
i
100.0
1.0
N=rn
N=10
N=20
N=18
FIG. 1. Mean ( f SEM) time to lose righting reflex (RR) and time to regain RR following a single alcohol injection (3.0 g/kg b.wt.). lp < 0.05; ***p < 0.001 significant difference between the P and NP lines.
34) = 11.8, p < 0.01. Therefore, t-tests were performed using the data for each line at each interval. When 1 day separated the two alcohol injections, the time to regain RR was shorter following the second injection compared with the first in the P line (p < O.Ol), indicating the presence of alcohol tolerance (Fig. 2, upper left panel). In these rats, there was a trend (p < 0.07, one tail) toward a higher BAC at recovery following the second alcohol injection compared with the first (Fig. 2, lower left panel). This would be expected due to the shorter time to regain RR, if alcohol metabolic rate is not changed. By contrast, in the NP line, time to regain RR following the second injection was longer compared with the first (p < 0.01) with a l-day interval between injections, indicating the presence of sensitization to alcohol (Fig. 2, upper left panel). For these rats, BAC was lower at recovery (p < 0.001, one tail) following the second injection compared with the first (Fig. 2, lower left panel). When 2 days separated the alcohol injections, neither tolerance nor sensitization was seen in either line (Fig. 2, upper right panel), although a nonsignificant trend toward sensitization was seen in the NP rats. For the P rats, BAC at recovery of RR was not different after each of the two injections when the injections were separated by 2 days. In the NP line, BAC was lower at recovery following the second injection compared with the first (p < 0.05) when the injections were separated by 2 days (Fig. 2, lower right panel). EXPERIMENT
2
In Experiment 1, rats of the P line took less time to recover RR following a single alcohol injection when compared to NP rats. Rats of the P line also showed faster recovery of RR following a second alcohol injection compared with the first (i.e., tolerance), while NP rats showed slower recovery after a second alcohol injection (sensitization). Experiment 2 was designed to determine whether tolerance to alcohol observed
Eight additional alcohol-naive adult male P rats (296-382 g) from the 33rd generation of selective breeding were tested. The rats were housed under the conditions described above in Experiment 1. Design and Procedure Tolerance testing. The rats were tested for two-injection tolerance as described in Experiment 1. Time to lose and recover RR were measured following each of the two alcohol injections that were separated by 1 day as described in Experiment 1. Blood sampling. Blood was collected via a chronic indwelling canulla to minimize behavioral disruption often associated with other blood collection procedures. A cannula was inserted into the right external jugular vein under Brevital anesthesia (50 mg/kg b.wt.) as described by Harms and Ojeda (13) 4 days prior to tolerance testing. Rats were handled daily for 4 days prior to testing to minimize stress on the day of alcohol challenge. We have previously determined that administration of 50 mg/kg Brevital 4 days prior to alcohol challenge does not alter the time to lose or recover RR relative to untreated controls (unpublished observation). Determination of BAC. A 0.3 ml blood sample was collected with heparinized saline replacement (200 units/ml) at the time of loss of RR and a second sample was obtained at the time of recovery of RR after each of two alcohol injections that were separated by 1 day. Blood was centrifuged and plasma aliquots were stored at - 20°C for later determination of BAC by gas chromatography as described in Experiment 1. Data Analysis Within-session BAC. BACs at time of loss and recovery of RR were compared following each of the two alcohol injections using paired Student’s t-tests. Within-session tolerance was defined as a higher BAC at time of recovery than at time of loss of RR. RESULTS
Table 1 shows that BAC at time of loss of RR did not differ from BAC at time of recovery of RR following the first or the second injection, indicating that within-session tolerance did not develop after either alcohol injection in P rats that exhibited two injection between-session tolerance. GENERAL
DISCUSSION
The results of the present study suggest that genetically determined differences in alcohol drinking behavior in rats of the P and NP lines are associated with differences in initial sensitivity to alcohol and with the development of alcohol tolerance or sensitization. Initial sensitivity to alcohol is generally measured by the magnitude of response to a single alcohol challenge in alcohol-naive subjects (16,35,39). The time required to lose a function during the ascending portion of the BAC curve (5,38,39) and/or the time required for the same function to be reinstated during the descending portion of the BAC curve (5,17,24) have both been used to index initial sensitivity. However, the time to lose a function may be a less
588
KURTZ ET AL.
600, 500 400 300 200. looO-
P
NP
N=8
N=6
3 Day 0 I Day 1
NP N=12
7 I
N=12
Day0 Day 2
t T
P
NP
P
NP
N=8
N=6
N=12
N=12
FIG. 2. Upper panels: mean (+ SEM) time to regain RR following each of two alcohol injections (3.0 g/kg b.wt.), which were separated either by 1 day (upper left panel) or 2 days (upper right panel). Inserts depict the mean ( f SEM) difference scores (day 1 minus day 0 in the left panel and day 2 minus day 0 in the right panel). **p < 0.01 significant difference in time to recover RR following the second injection (day 1 or 2) compared with the first (day 0); i.e., difference scores significantly greater than or less than zero. Lower panels: mean ( f SEM) BAC at time of recovery of RR following each of two alcohol injections (3.0 g/kg b.wt.), which were separated either by 1 day (lower left panel) or 2 days (lower right panel). Inserts depict the mean ( f SEM) difference scores (day 1 minus day 0 in the left panel and day 2 minus day 0 in the right panel). ‘p < 0.07; *p < 0.05; ***p < 0.001 significant difference in BAC following the second injection (day 1 or 2) compared with the first (day 0); i.e., difference scores significantly greater than or less than zero.
equivocal measure of initial sensitivity than the time to recover a function, because the latter may also reflect the development of within-session or acute tolerance (27,39). Within-session tolerance, which can develop during the time course of the BAC curve following a single alcohol injection (36), is reflected by reinstatement of a function on the descending portion of the BAC curve at a higher BAC than that at which the function was lost on the ascending portion of the curve (27). The present study has the advantage of measuring both the time of loss and the time of recovery of RR in the same animals following a single alcohol injection. Rats of the P line were less sensitive than NP rats to the behaviorally-impairing effects of alcohol. P rats took longer
to lose RR, had a shorter recovery time, and recovered at a higher BAC following a single sedative/hypnotic dose of alco-
hol compared with rats of the NP line. These results are in agreement with previous reports that rats of the P line exhibit reduced sensitivity to alcohol relative to NP rats when sensitivity is indexed by degree of alcohol-induced hypnosis (21), motor impairment (23), and conditioned taste aversion (8). Differences in initial sensitivity to alcohol have also been reported in other rodent strains that differ in voluntary alcohol intake [e.g., C57BL and DBA mice; (37)]. In the present investigation (Experiment 2), we did not observe acute (within-session) tolerance in P rats, because BACs did not differ at time of loss and recovery of RR follow-
GENETICS
OF ALCOHOL
589
TOLERANCE
TABLE 1 MEAN (*SEM) TIME TO REGAIN RR AND BLOOD ALCOHOL CONCENTRATlON (BAC, mg’lo) AT TIME OF LOSS AND RECOVERY OF RR FOLLOWING EACH OF TWO ALCOHOL INJECTIONS (3.0 g/kg b.wt. IP) SEPARATED BY 1 DAY BAC Time to Regain RR (mw
Day 0 Day 1
220 f 24 151 f 25*
BAC at Loss of RR (meolo)
216 f 45 1% f 41
at Recovery of RR
(ms%‘o) 213 f 26 242 f 20
The data are from eight alcohol-preferring P rats. lp < 0.001, day 0 compared with day 1.
ing a single alcohol injection. In a previous investigation (39), acute tolerance to alcohol was investigated in P and NP rats using time to recover criterion performance on a jump-up escape task after two alcohol injections. In that study a 2.0 g/kg alcohol injection was administered initially. When escape performance recovered after the first alcohol injection, BAC was measured and a second alcohol injection (1 .O g/kg) was immediately administered to the same subjects. BAC was measured when escape performance recovered to criterion a second time. Because the second recovery of escape performance occurred at a higher BAC than the first, it was concluded that acute or within-session tolerance had developed in rats of the P line. It remains to be determined whether the processes underlying tolerance development are similar when tolerance is defined by a comparison of functional recovery following each of two alcohol injections given in succession (39) as opposed to the recovery of a function at a higher BAC than when the function was lost following a single alcohol injection as in the present study (27). Chronic alcohol tolerance is defined as a decrease in magnitude of the response to a second or subsequent alcohol exposure (36). One method for assessing chronic tolerance is the two-injection procedure in which functional tolerance is defined as a faster behavioral recovery at a higher BAC following a second alcohol injection compared to the first (10). In the present study, rats of the P line developed tolerance to the sedative/hypnotic effects of alcohol as indicated by faster behavioral recovery at a higher BAC following the second alcohol challenge compared to the first when 1 day separated the two injections. The tolerance retention time was short in the P line, and tolerance was no longer seen when 2 days separated the two alcohol injections. A short tolerance retention time has also been observed in rats of the P line when hypothermia was used to index tolerance (33), but tolerance retention was much more prolonged in P rats when an escapeavoidance task was used to index tolerance (10). Such variability suggests that the duration of tolerance. retention may depend on factors that are specific to the test used to measure tolerance. Other variables that may influence the duration of tolerance retention include the degree to which previous training is part of the task used to measure tolerance (18), and the degree of stress involved in the test used to measure tolerance (29). The importance of these variables has previously been discussed (33). The relationship between initial sensitivity to alcohol and the development of alcohol tolerance is of theoretical interest. It has been hypothesized that tolerance is an adaptive process
that occurs in response to drug-induced functional impairment, and that the greater the degree of functional impairment, the more rapid the development of tolerance (15). This concept was supported by the results of a study (9) that demonstrated that as the dose of alcohol is increased, and functional impairment is prolonged, recovery of function occurs at higher BACs, perhaps reflecting the development of withinsession tolerance. However, the results of the present study do not support this hypothesis because rats of the NP line, which exhibited a greater degree of functional impairment in response to a single alcohol challenge, did not develop tolerance. In fact, reduced functional impairment in rats of the P line was associated with the development of alcohol tolerance. It also has been suggested that alcohol tolerance may represent an extension of initial resistance to alcohol (36). Within this theoretical framework, tolerance is more likely to be seen in rats that are initially less sensitive (i.e., more resistant) to alcohol. The results of the present study tend to support this concept because rats of the P line that are initially less sensitive to alcohol develop alcohol tolerance more readily than the NP rats, which are initially more sensitive to alcohol. A similar association between low initial sensitivity to alcohol and the rapid development of alcohol tolerance has been reported by ourselves and others in rats and in mice (10,24,38). One of the novel findings of the present investigation is that rats of the NP line develop sensitization to the sedative/ hypnotic effects of a high dose of alcohol, as evidenced by a longer time to recover RR following the second alcohol challenge compared with the first. Sensitization to the motorstimulating effects of a low dose of alcohol has been observed following chronic alcohol administration in mice (4,25,26,30). Sensitization to low-dose alcohol effects may be analogous to neural sensitization, which is seen in response to excitatory stimuli (3,6,12). However, reports of sensitization to the sedative/hypnotic effects of high alcohol doses are relatively rare. We are aware of only one other report of sensitization to high alcohol doses in rodents (11). Thus, it is interesting that sensitization to a high dose of alcohol appears to be a relatively consistent finding in rats of the NP line. NP rats develop sensitization to both the sedative/hypnotic and the hypothermic effects of high dose alcohol as well as sensitization to alcohol-induced reductions in spontaneous motor activity that occur following moderate doses of alcohol (33,34). High alcohol drinking behavior is influenced by both environmental factors and neurobiological traits. A genetic propensity toward high alcohol drinking is most likely determined by multiple genes that regulate a number of predisposing neurobiological traits; hence, a number of alcohol-related traits may contribute to alcohol preference and high alcohol intake. Selectively bred rodent lines provide a powerful tool for identification of the alcohol-related traits that are associated with, and may be causally related to, the high aIcoho1 drinking phenotype. The results of the present study suggest that selective breeding for alcohol preference in P rats is associated with decreased initial sensitivity to alcohol and with the rapid development of alcohol tolerance. It would not be prudent to infer that a meaningful relationship exists among these variables based only upon the studies of the P and NP lines. However, low initial sensitivity to alcohol and the rapid development of alcohol tolerance arguably have been the most generalizable and robust traits that have been found to be associated with alcohol preference in rodents. Differences in either or both of these responses have been observed in the alcohol-preferring C57BL and alcohol-nonpreferring BALB and DBA mouse
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strains (14,37), in the HS/Ibg heterogeneous stock mice (7), in the selectively bred alcohol preferring AA and alcoholavoiding ANA rat lines (19), and in the inbred rat lines used to constitute the N/NIH heterogeneous stock rats (32). The appearance of an association between alcohol preference, and low initial sensitivity to alcohol, and/or the rapid development of alcohol tolerance in multiple lines and strains serves to minimize the possibility that the observed relationship is fortuitous. It is unlikely that any single trait, such as alcohol sensitivity or the rapid development of alcohol tolerance is sufficient for the expression of the alcohol drinking phenotype. Instead, the presence of certain environmental variables and alcoholrelated traits may serve to increase the probability of high alcohol drinking behavior, and a combination of these variables and traits may act additively or interact synergistically to produce high alcohol drinking behavior. Establishing trait causality and identifying the combination of alcohol-related traits and environmental factors which, together, will invariably produce high alcohol drinking behavior, is a formidable
task. However, the task begins with the identification of traits that are associated with high alcohol drinking behavior in multiple lines and strains. With regard to human drinking behavior, the results suggest that genetic differences among individuals in initial sensitivity to alcohol and/or the rapid development of alcohol tolerance may contribute to differences in susceptibility to alcohol abuse and dependence. This view is supported by the results of a study conducted with sons of alcoholics and nonalcoholics, which indicate that a lower intensity of response to the intoxicating effects of alcohol is predictive of a future propensity toward high alcohol intake (31). ACKNOWLEDGEMENTS
This work was in partial fulfillment of the requirements of D. L. Kurtz for the M.Sc. degree in Physiology and Biophysics at Indiana University School of Medicine. The authors wish to thank Dr. James Norton for statistical consultation. This research was supported by grants AA08312, AA08553, and AA07611 from the PHS.
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14. Kakihana, R.; Brown, D. R.; McClearn, G. E.; Tabershaw, I. R. Brain sensitivity to alcohol in inbred mouse strains. Science 154: 1574-1575; 1966. 15. Kalant, K. Comparative aspects of tolerance to, and dependence on, alcohol, barbiturates and opiates. Adv. Exp. Med. Biol. 85B: 169-186; 1977. 16. Kalant, H.; LeBlanc, A. E.; Gibbins, R. J. Tolerance to, and dependence on, some nonopiate psychotropic drugs. Pharmacol. Rev. 23:135-191; 1971. 17. Khanna, J. M.; Le, A. D.; LeBlanc, A. E.; Shah, G. Initial sensitivity vs. acquired tolerance to alcohol in rats selectively bred for alcohol sensitivity. Psychopharmacology (Berlin) 86:302-306; 1985. 18. Le. A. D.; Kalant, H.; Khanna, J. M. Interaction between desglycinamide’-[ARG’] vasopressin and serotonin on alcohol tolerance. Eur. J. Pharmacol. 80:337-345; 1982. 19. Le, A. D.; Kiianmaa, K. Characteristics of alcohol tolerance in alcohol drinking (AA) and alcohol avoiding (ANA) rats. Psychopharmacology (Berlin) 94:479-483; 1988. 20. Li, T.-K.; Lumeng, L.; Doolittle, D. P.; McBride, W. J.; Murphy, J. M.; Froehlich, J. C.; Morzorati, S. Behavioral and neurochemical associations of alcohol-seeking behavior. In: Kuriyama, K.; Takada, A.; Ishii, H., eds. Biomedical and social aspects of alcohol and alcoholism. Amsterdam: Excerpta Medica; 1988:435438. 21. Li, T.-K.; Lumeng, L.; McBride, W. J.; Waller, M. B. Indiana selection studies on alcohol-related behaviors. In: NIAAA research monograph No. 6, Development of animal models as pharmacoaenetic tools. Rockville. MD: U.S. Department of Health and Human Services; 1981:17-l-191. _ 22. Linakis, J. G.; Cunningham, C. L. Effects of concentration of ethanol injected intraperitonealiy on taste aversion, body temperature, and activity. Psychopharmacology (Berlin) 64:61-65; 1979. 23. Lumeng, L.; Hawkins, T.; Li, T.-K. New strains of rats with alcohol preference and nonpreference. In: Thurman, R.; Williamson, J.; Drott, H.; Chance, B., eds. Alcohol and aldehyde metabolizing systems, vol. 3. New York: Academic Press; 1977:537544. 24. Lumeng, L.; Waller, M. B.; McBride, W. J.; Li, T.-K. Different sensitivities to alcohol in alcohol-preferring and -nonpreferring rats. Pharmacol. Biochem. Behav. 16:125-130; 1982. 25. Masur, J.; Boerngen, R. The excitatory component of alcohol in mice: A chronic study. Pharmacol. Biochem. Behav. 13:777-780; 1980. 26. Masur, J.; Martins dos Santos, H. M. L. Response variability of ethanol-induced locomotor activation in mice. Psychopharmacology (Berlin) %:547-550; 1988.
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27. Mellanby, E. Alcohol: Its absorption into and disappearance from the blood under different conditions. Special Report Series No. 31. London: Medical Research Committee; 1919. 28. Murphy, J. M.; Gatto, G. J.; McBride, W. J.; Lumeng, L.; Li, T.-K. Persistence of tolerance in the P line of alcohol-preferring rats does not require performance while intoxicated. Alcohol 7: 367-369; 1990. 29. Peris, J.; Cunningham, C. L. Stress enhances the development of tolerance to the hypnotic effects of ethanol. Alcohol. Drug. Res. 7:187-193; 1987. 30. Phillips, T. J.; Burkart-Kasch, S.; Crabbe, J. C. Locomotor activity response to chronic alcohol treatment in selectively bred FAST and SLOW mice. Alcohol Alcohol. Suppl. 1:109-113; 1991. 31. Schuckit, M. A.; Low level of response to alcohol as a predictor of future alcoholism. Am. J. Psychiatry 151(2):184-189; 1994. 32. Spuhler, K.; Deitrich, R. A. Correlative analysis of ethanol-related phenotypes in rat inbred strains. Alcoholism 8:480-484, 1984. 33. Stewart, R. B.; Kurtz, D. L.; Zweifel, M. J.; Li, T.-K.; Froehlich, J. C. Differences in the hypothermic response to ethanol in rats
34.
35. 36. 37. 38.
39.
selectively bred for oral ethanol preference and nonpreference. Psychopharmacology (Berlin) 106:169-174; 1992. Stewart, R. B.; Li, T.-K.; McBride, W. J.; Lumeng, L.; Murphy, J. M. Chronic tolerance in ethanol-preferring P rats and enhanced responsiveness in ethanol-nonpreferring NP rats to the effects of alcohol on spontaneous motor activity (SMA). Alcohol.: Chn. Exp. Res. 15:317; 1991. Tabakoff, B.; Cornell, N.; Hoffman, P. L. Alcohol tolerance. Ann. Emerg. Med. 15:1005-1012; 1986. Tabakoff, B.; Melchior, C. L.; Hoffman, P. L. Commentary on alcohol tolerance. Alcohol.: Clin. Exp. Res. 6:252-259; 1982. Tabakoff, B.; Ritzman, R. F. Acute tolerance in inbred and selected lines of mice. Drug Alcohol. Depend. 4:87-w 1979. Tabakoff, B.; Ritzman, R. F.; Raju, T. S.; Deitrich, R. A. Characterization of acute and chronic tolerance in mice selected for inherent differences in sensitivity to ethanol. Alcohol.: Clin. Exp. Res. 4:70-73; 1980. Waller, M. B.; McBride, W. J.; Lumeng, L.; Li, T.-K. Initial sensitivity and acute tolerance to alcohol in the P and NP lines of rats. Pharmacol. B&hem. Behav. 19:683-686; 1983.
0091-3057(95)02055-1
Genetic Differences in Tolerance and Sensitization to the Sedative/Hypnotic Effects of Alcohol D. L. KURTZ,*$*
R. B. STEWART,*’
M. ZWEIFEL,
*2 T.-K.
LI*t
AND
J. C. FROEHLICH*t3
Departments of *Medicine, tBiochemistry/Molecular Biology, and $Physiology/Biophysics, Indiana UniversitySchool of Medicine, Indianapolis, IN 46202-5124 Received
30 December
1994; Revised 20 June 1995; Accepted
6 July 1995
KURT& D. L., R. B. STEWART, M. ZWEIFEL, T.-K. LI AND J. C. FROEHLICH. Genetic differences in toleronce and sensitization to the sedative/hypnotic effectsof alcohol. PHARMACOL BIOCHEM BEHAV 53(3) 585-591, 1996. -Initial sensitivity to alcohol and the development of alcohol tolerance were examined in rats of the selectively bred alcohol-preferring (P) and -nonpreferring (NP) lines. All rats received two alcohol injections (3.0 g/kg b.wt., IP) separated by either 1 or 2 days. P rats were less sensitive to the behaviorally impairing effects of alcohol than were NP rats, as evidenced by a longer latency to lose righting reflex (RR) and a shorter time to recover RR following an initial alcohol injection. When 1 day separated the two alcohol injections. P rats recovered the RR more rapidly following a second injection compared to the first, indicating that the P rats developed tolerance to the sedative/hypnotic effects of alcohol. In contrast, the NP rats recovered the RR more slowly following the second injection compared to the first, indicating that the NP rats developed sensitization to alcohol. Tolerance in the P line and sensitization in the NP line disappeared when 2 days separated the two alcohol injections. Line differences in initial sensitivity and tolerance/sensitization to the behaviorally impairing effects of alcohol may contribute to the differences in alcohol consumption observed in the P and NP lines. Alcohol Alcohol sensitivity Alcohol-nonpreferring rats
Alcohol tolerance
Sensitization
REPEATED exposure to alcohol can result in decreased responsiveness or alcohol tolerance (16), which may be an important process that contributes to the development of alcoholism (35). The development of alcohol tolerance may serve to increase alcohol intake either by reducing the aversive effects of the drug that otherwise limit alcohol intake (2), or by reducing the reinforcing effects of the drug, resulting in increased intake to maintain the same level of drug effect (36). The degree of initial sensitivity to alcohol, in the absence of previous exposure to the drug, may also influence subsequent levels of alcohol intake. An association between high alcohol intake and the development of alcohol tolerance as well as high alcohol
intake and low initial sensitivity
to alcohol
Genetics
Alcohol-preferring
rats
amounts of a 10% (v/v) alcohol solution when given continuous access to the drug with water and food freely available (20,23). Rats of the P line show greater tolerance than do NP rats in experiments using a two-injection procedure in which alcohol-induced motor impairment in an escape/avoidance task is measured, and tolerance persists longer in P rats than in NP rats (10,28,39). When alcohol-induced hypothermia was used to index tolerance in P and NP rats using a two-injection procedure, the results depended on the temporal parameters of the alcohol injections (33). When the two alcohol injections were separated by 1 day, tolerance was observed in both lines of rats. When the two alcohol injections were separated by 2 days or 3 days, neither P nor NP rats showed tolerance; rather, NP rats exhibited sensitization to the hypothermic effect of alcohol. The present itivestigation extends this previous work by
have
been reported in various lines and strains of rodents (7,19,32, 37). The alcohol-preferring (P) and alcohol-nonpreferring (NP) lines of rats were selectively bred to drink high and low
’ R. B. Stewart is now in the Department of Psychology, Purdue School of Science, Indiana University/Purdue University at Indianapolis. 2 D. L. Kurtz and M. Zweifel are now in Lilly Research Laboratories, Eli Lilly and Co., Indianapolis, Indiana. 3Requests for reprints should be addressed to J. C. Froehlich, Ph.D., Indiana University School of Medicine, Emerson Hall 421, 545 Barnhill Drive, Indianapolis, IN 46202-5124.
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examining the generality of the association between alcohol intake, initial sensitivity to alcohol, and the development of alcohol tolerance or sensitization. Time to lose and time to recover the righting reflex were measured in P and NP rats after each of two injections of a sedative/hypnotic dose of alcohol with the injections separated by either 1 or 2 days. EXPERIMENT
I
Responsiveness to the sedative/hypnotic effects of a high dose of alcohol was determined in P and NP rats. Initial sensitivity to alcohol and two-injection tolerance were measured. METHOD
Blood Sampling At the time of recovery of RR, a blood sample (0.3 ml) was collected from the cut tip of the tail and centrifuged. Plasma aliquots were stored at -20°C for later determination of BAC. BAC Determination A 1 : 1 solution of plasma and internal standard (isopropano1 solution) was injected in a volume of 1.O ~1 into a HewlettPackard 5730A gas chromatograph equipped with a flame ionization detector and 3380A integrator. The glass columns were packed with Poropak Q (100/120 mesh) and the oven temperature was 150%.
Subjects
Data Analysis
Male, alcohol-naive rats (341-492 g) were obtained from the 28th to the 31st generations of the alcohol-preferring (P) and -nonpreferring (NP) lines. They were housed in a quiet environment under controlled temperature and lighting conditions (lights on 0700 to 1900 h), with water and standard laboratory food pellets (Purina #5001) freely available. The rats were handled once daily for 4 days before the experiment began to reduce stress on the day of alcohol challenge.
Initial sensitivity. The P and NP lines were compared on time to loss of RR, time to regain RR, and BAC at recovery of RR following the first alcohol injection using unpaired Student’s t-tests. Tolerance and sensitization. Time to regain RR (time to loss of RR minus time of recovery of RR) following each of the two alcohol injections were compared. A difference score was obtained for each rat by subtracting the time to regain RR following the first injection from the time to regain RR following the second injection. The difference scores were analyzed using a two-way ANOVA with line (P or NP) and interval between exposures (1 or 2 days) as between-group factors. Student’s t-tests were used to determine if the mean difference score for each line at each interval was significantly less than zero (indicating tolerance) or greater than zero (indicating sensitization). A pooled estimate of variance within each line was used in calculating the t statistic. BAC. A difference score was obtained for each rat by subtracting the BAC at time of recovery of RR following the first injection from the BAC at time of recovery following the second injection. The data were analyzed in the manner described above for the time to regain RR.
Procedure Alcohol-induced loss of righting reflex (RR) and recovery of RR were measured following each of two alcohol injections that were administered either 1 or 2 days apart. The group of rats given two alcohol injections separated by 1 day (day Oday 1) and the group given two alcohol injections separated by 2 days (day O-day 2) were tested at different times (4 months apart) and the two groups were similar in age (90 to 100 days old) at the time of tolerance testing. Alcohol was injected IP at a dose of 3.0 g alcohol/315 ml/kg b.wt. between 0800 and 1100 h. The concentration of the alcohol solution did not exceed 12% (v/v) to minimize concentrationinduced differences in alcohol absorption rates and tissue irritation at the site of injection (1,22). The apparatus used to measure RR was a V-shaped trough that was bent at a 90° angle and lined with sandpaper. The troughs were contained in a temperature-controlled chamber that also reduced sound disturbance. To determine loss of RR, each rat was placed on its back in the trough once every 30 s after the alcohol injection until it was unable to right itself within a 30-s interval. The time between the onset of the injection and the beginning of the 30-s interval during which the rat was unable to right itself was recorded as the time to loss of RR. The rat was then left undisturbed on its back until it turned over so that its forelegs were under its body and not visible from directly above. At this time righting trials were initiated. A righting trial was begun by placing the rat on its back. Righting was defined as the rat achieving the orientation in which at least three feet were under its body and not visible from above. Recovery of RR was defined as the ability of the rat to right itself in two consecutive 15-s trials. If the rat was unable to right itself in the first 15-s trial, it was left undisturbed until it turned over again, at which time righting trials were reinitiated. Tolerance was defined as a significantly faster recovery of the RR, at a higher blood alcohol concentration (BAC), following the second alcohol injection compared with the first. Sensitization was defined as significantly slower recovery of the RR, at a lower BAC, following the second alcohol injection compared with the first.
RESULTS
Initial Sensitivity The P rats took longer to lose RR (p < 0.05; Fig. 1) and recovered more quickly than the NP rats (p < 0.001; Fig. 1) following the first alcohol injection, which indicates that P rats were less sensitive to the behaviorally impairing effects of alcohol than were the NP rats. The mean BAC (+ SEM) at time of recovery was higher (p < 0.05) in the P rats (177 * 13 mg%) than in the NP rats (135 f 10 mg%). Tolerance and Sensitization P rats recovered the RR more rapidly following a second alcohol injection compared with the first, indicating that the P rats developed tolerance to the sedative/hypnotic effects of alcohol. By contrast, the NP rats recovered the RR more slowly following the second injection compared to the first, indicating that the NP rats developed sensitization to alcohol. The ANOVA of the difference scores of the time to regain RR indicated a significant effect of line, F(l, 34) = 25.2, p < 0.001, and an interaction of line with interval between alcohol exposures, F(l, 31) = 10.5, p < 0.01. The ANOVA of the BAC difference scores at time of recovery showed a significant effect of interval, F(1, 34) = 4.8, p < 0.05, line, F(1, 34) = 32.7, p < 0.001, and the interaction of line with interval, F(1,
GENETICS
OF ALCOHOL
587
TOLERANCE
in rats of the P line in Experiment 1 could be attributed to the development of within-session tolerance.
FIGURE 1
METHOD 6.0
600.0
T
7.0
Subjects
500.0
6.0
5 400.0 5
g tz
5.0
8
4.0
300.0
G Y
3.0
P 200.0 s 3
i=
a
2.0
i
100.0
1.0
N=rn
N=10
N=20
N=18
FIG. 1. Mean ( f SEM) time to lose righting reflex (RR) and time to regain RR following a single alcohol injection (3.0 g/kg b.wt.). lp < 0.05; ***p < 0.001 significant difference between the P and NP lines.
34) = 11.8, p < 0.01. Therefore, t-tests were performed using the data for each line at each interval. When 1 day separated the two alcohol injections, the time to regain RR was shorter following the second injection compared with the first in the P line (p < O.Ol), indicating the presence of alcohol tolerance (Fig. 2, upper left panel). In these rats, there was a trend (p < 0.07, one tail) toward a higher BAC at recovery following the second alcohol injection compared with the first (Fig. 2, lower left panel). This would be expected due to the shorter time to regain RR, if alcohol metabolic rate is not changed. By contrast, in the NP line, time to regain RR following the second injection was longer compared with the first (p < 0.01) with a l-day interval between injections, indicating the presence of sensitization to alcohol (Fig. 2, upper left panel). For these rats, BAC was lower at recovery (p < 0.001, one tail) following the second injection compared with the first (Fig. 2, lower left panel). When 2 days separated the alcohol injections, neither tolerance nor sensitization was seen in either line (Fig. 2, upper right panel), although a nonsignificant trend toward sensitization was seen in the NP rats. For the P rats, BAC at recovery of RR was not different after each of the two injections when the injections were separated by 2 days. In the NP line, BAC was lower at recovery following the second injection compared with the first (p < 0.05) when the injections were separated by 2 days (Fig. 2, lower right panel). EXPERIMENT
2
In Experiment 1, rats of the P line took less time to recover RR following a single alcohol injection when compared to NP rats. Rats of the P line also showed faster recovery of RR following a second alcohol injection compared with the first (i.e., tolerance), while NP rats showed slower recovery after a second alcohol injection (sensitization). Experiment 2 was designed to determine whether tolerance to alcohol observed
Eight additional alcohol-naive adult male P rats (296-382 g) from the 33rd generation of selective breeding were tested. The rats were housed under the conditions described above in Experiment 1. Design and Procedure Tolerance testing. The rats were tested for two-injection tolerance as described in Experiment 1. Time to lose and recover RR were measured following each of the two alcohol injections that were separated by 1 day as described in Experiment 1. Blood sampling. Blood was collected via a chronic indwelling canulla to minimize behavioral disruption often associated with other blood collection procedures. A cannula was inserted into the right external jugular vein under Brevital anesthesia (50 mg/kg b.wt.) as described by Harms and Ojeda (13) 4 days prior to tolerance testing. Rats were handled daily for 4 days prior to testing to minimize stress on the day of alcohol challenge. We have previously determined that administration of 50 mg/kg Brevital 4 days prior to alcohol challenge does not alter the time to lose or recover RR relative to untreated controls (unpublished observation). Determination of BAC. A 0.3 ml blood sample was collected with heparinized saline replacement (200 units/ml) at the time of loss of RR and a second sample was obtained at the time of recovery of RR after each of two alcohol injections that were separated by 1 day. Blood was centrifuged and plasma aliquots were stored at - 20°C for later determination of BAC by gas chromatography as described in Experiment 1. Data Analysis Within-session BAC. BACs at time of loss and recovery of RR were compared following each of the two alcohol injections using paired Student’s t-tests. Within-session tolerance was defined as a higher BAC at time of recovery than at time of loss of RR. RESULTS
Table 1 shows that BAC at time of loss of RR did not differ from BAC at time of recovery of RR following the first or the second injection, indicating that within-session tolerance did not develop after either alcohol injection in P rats that exhibited two injection between-session tolerance. GENERAL
DISCUSSION
The results of the present study suggest that genetically determined differences in alcohol drinking behavior in rats of the P and NP lines are associated with differences in initial sensitivity to alcohol and with the development of alcohol tolerance or sensitization. Initial sensitivity to alcohol is generally measured by the magnitude of response to a single alcohol challenge in alcohol-naive subjects (16,35,39). The time required to lose a function during the ascending portion of the BAC curve (5,38,39) and/or the time required for the same function to be reinstated during the descending portion of the BAC curve (5,17,24) have both been used to index initial sensitivity. However, the time to lose a function may be a less
588
KURTZ ET AL.
600, 500 400 300 200. looO-
P
NP
N=8
N=6
3 Day 0 I Day 1
NP N=12
7 I
N=12
Day0 Day 2
t T
P
NP
P
NP
N=8
N=6
N=12
N=12
FIG. 2. Upper panels: mean (+ SEM) time to regain RR following each of two alcohol injections (3.0 g/kg b.wt.), which were separated either by 1 day (upper left panel) or 2 days (upper right panel). Inserts depict the mean ( f SEM) difference scores (day 1 minus day 0 in the left panel and day 2 minus day 0 in the right panel). **p < 0.01 significant difference in time to recover RR following the second injection (day 1 or 2) compared with the first (day 0); i.e., difference scores significantly greater than or less than zero. Lower panels: mean ( f SEM) BAC at time of recovery of RR following each of two alcohol injections (3.0 g/kg b.wt.), which were separated either by 1 day (lower left panel) or 2 days (lower right panel). Inserts depict the mean ( f SEM) difference scores (day 1 minus day 0 in the left panel and day 2 minus day 0 in the right panel). ‘p < 0.07; *p < 0.05; ***p < 0.001 significant difference in BAC following the second injection (day 1 or 2) compared with the first (day 0); i.e., difference scores significantly greater than or less than zero.
equivocal measure of initial sensitivity than the time to recover a function, because the latter may also reflect the development of within-session or acute tolerance (27,39). Within-session tolerance, which can develop during the time course of the BAC curve following a single alcohol injection (36), is reflected by reinstatement of a function on the descending portion of the BAC curve at a higher BAC than that at which the function was lost on the ascending portion of the curve (27). The present study has the advantage of measuring both the time of loss and the time of recovery of RR in the same animals following a single alcohol injection. Rats of the P line were less sensitive than NP rats to the behaviorally-impairing effects of alcohol. P rats took longer
to lose RR, had a shorter recovery time, and recovered at a higher BAC following a single sedative/hypnotic dose of alco-
hol compared with rats of the NP line. These results are in agreement with previous reports that rats of the P line exhibit reduced sensitivity to alcohol relative to NP rats when sensitivity is indexed by degree of alcohol-induced hypnosis (21), motor impairment (23), and conditioned taste aversion (8). Differences in initial sensitivity to alcohol have also been reported in other rodent strains that differ in voluntary alcohol intake [e.g., C57BL and DBA mice; (37)]. In the present investigation (Experiment 2), we did not observe acute (within-session) tolerance in P rats, because BACs did not differ at time of loss and recovery of RR follow-
GENETICS
OF ALCOHOL
589
TOLERANCE
TABLE 1 MEAN (*SEM) TIME TO REGAIN RR AND BLOOD ALCOHOL CONCENTRATlON (BAC, mg’lo) AT TIME OF LOSS AND RECOVERY OF RR FOLLOWING EACH OF TWO ALCOHOL INJECTIONS (3.0 g/kg b.wt. IP) SEPARATED BY 1 DAY BAC Time to Regain RR (mw
Day 0 Day 1
220 f 24 151 f 25*
BAC at Loss of RR (meolo)
216 f 45 1% f 41
at Recovery of RR
(ms%‘o) 213 f 26 242 f 20
The data are from eight alcohol-preferring P rats. lp < 0.001, day 0 compared with day 1.
ing a single alcohol injection. In a previous investigation (39), acute tolerance to alcohol was investigated in P and NP rats using time to recover criterion performance on a jump-up escape task after two alcohol injections. In that study a 2.0 g/kg alcohol injection was administered initially. When escape performance recovered after the first alcohol injection, BAC was measured and a second alcohol injection (1 .O g/kg) was immediately administered to the same subjects. BAC was measured when escape performance recovered to criterion a second time. Because the second recovery of escape performance occurred at a higher BAC than the first, it was concluded that acute or within-session tolerance had developed in rats of the P line. It remains to be determined whether the processes underlying tolerance development are similar when tolerance is defined by a comparison of functional recovery following each of two alcohol injections given in succession (39) as opposed to the recovery of a function at a higher BAC than when the function was lost following a single alcohol injection as in the present study (27). Chronic alcohol tolerance is defined as a decrease in magnitude of the response to a second or subsequent alcohol exposure (36). One method for assessing chronic tolerance is the two-injection procedure in which functional tolerance is defined as a faster behavioral recovery at a higher BAC following a second alcohol injection compared to the first (10). In the present study, rats of the P line developed tolerance to the sedative/hypnotic effects of alcohol as indicated by faster behavioral recovery at a higher BAC following the second alcohol challenge compared to the first when 1 day separated the two injections. The tolerance retention time was short in the P line, and tolerance was no longer seen when 2 days separated the two alcohol injections. A short tolerance retention time has also been observed in rats of the P line when hypothermia was used to index tolerance (33), but tolerance retention was much more prolonged in P rats when an escapeavoidance task was used to index tolerance (10). Such variability suggests that the duration of tolerance. retention may depend on factors that are specific to the test used to measure tolerance. Other variables that may influence the duration of tolerance retention include the degree to which previous training is part of the task used to measure tolerance (18), and the degree of stress involved in the test used to measure tolerance (29). The importance of these variables has previously been discussed (33). The relationship between initial sensitivity to alcohol and the development of alcohol tolerance is of theoretical interest. It has been hypothesized that tolerance is an adaptive process
that occurs in response to drug-induced functional impairment, and that the greater the degree of functional impairment, the more rapid the development of tolerance (15). This concept was supported by the results of a study (9) that demonstrated that as the dose of alcohol is increased, and functional impairment is prolonged, recovery of function occurs at higher BACs, perhaps reflecting the development of withinsession tolerance. However, the results of the present study do not support this hypothesis because rats of the NP line, which exhibited a greater degree of functional impairment in response to a single alcohol challenge, did not develop tolerance. In fact, reduced functional impairment in rats of the P line was associated with the development of alcohol tolerance. It also has been suggested that alcohol tolerance may represent an extension of initial resistance to alcohol (36). Within this theoretical framework, tolerance is more likely to be seen in rats that are initially less sensitive (i.e., more resistant) to alcohol. The results of the present study tend to support this concept because rats of the P line that are initially less sensitive to alcohol develop alcohol tolerance more readily than the NP rats, which are initially more sensitive to alcohol. A similar association between low initial sensitivity to alcohol and the rapid development of alcohol tolerance has been reported by ourselves and others in rats and in mice (10,24,38). One of the novel findings of the present investigation is that rats of the NP line develop sensitization to the sedative/ hypnotic effects of a high dose of alcohol, as evidenced by a longer time to recover RR following the second alcohol challenge compared with the first. Sensitization to the motorstimulating effects of a low dose of alcohol has been observed following chronic alcohol administration in mice (4,25,26,30). Sensitization to low-dose alcohol effects may be analogous to neural sensitization, which is seen in response to excitatory stimuli (3,6,12). However, reports of sensitization to the sedative/hypnotic effects of high alcohol doses are relatively rare. We are aware of only one other report of sensitization to high alcohol doses in rodents (11). Thus, it is interesting that sensitization to a high dose of alcohol appears to be a relatively consistent finding in rats of the NP line. NP rats develop sensitization to both the sedative/hypnotic and the hypothermic effects of high dose alcohol as well as sensitization to alcohol-induced reductions in spontaneous motor activity that occur following moderate doses of alcohol (33,34). High alcohol drinking behavior is influenced by both environmental factors and neurobiological traits. A genetic propensity toward high alcohol drinking is most likely determined by multiple genes that regulate a number of predisposing neurobiological traits; hence, a number of alcohol-related traits may contribute to alcohol preference and high alcohol intake. Selectively bred rodent lines provide a powerful tool for identification of the alcohol-related traits that are associated with, and may be causally related to, the high aIcoho1 drinking phenotype. The results of the present study suggest that selective breeding for alcohol preference in P rats is associated with decreased initial sensitivity to alcohol and with the rapid development of alcohol tolerance. It would not be prudent to infer that a meaningful relationship exists among these variables based only upon the studies of the P and NP lines. However, low initial sensitivity to alcohol and the rapid development of alcohol tolerance arguably have been the most generalizable and robust traits that have been found to be associated with alcohol preference in rodents. Differences in either or both of these responses have been observed in the alcohol-preferring C57BL and alcohol-nonpreferring BALB and DBA mouse
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strains (14,37), in the HS/Ibg heterogeneous stock mice (7), in the selectively bred alcohol preferring AA and alcoholavoiding ANA rat lines (19), and in the inbred rat lines used to constitute the N/NIH heterogeneous stock rats (32). The appearance of an association between alcohol preference, and low initial sensitivity to alcohol, and/or the rapid development of alcohol tolerance in multiple lines and strains serves to minimize the possibility that the observed relationship is fortuitous. It is unlikely that any single trait, such as alcohol sensitivity or the rapid development of alcohol tolerance is sufficient for the expression of the alcohol drinking phenotype. Instead, the presence of certain environmental variables and alcoholrelated traits may serve to increase the probability of high alcohol drinking behavior, and a combination of these variables and traits may act additively or interact synergistically to produce high alcohol drinking behavior. Establishing trait causality and identifying the combination of alcohol-related traits and environmental factors which, together, will invariably produce high alcohol drinking behavior, is a formidable
task. However, the task begins with the identification of traits that are associated with high alcohol drinking behavior in multiple lines and strains. With regard to human drinking behavior, the results suggest that genetic differences among individuals in initial sensitivity to alcohol and/or the rapid development of alcohol tolerance may contribute to differences in susceptibility to alcohol abuse and dependence. This view is supported by the results of a study conducted with sons of alcoholics and nonalcoholics, which indicate that a lower intensity of response to the intoxicating effects of alcohol is predictive of a future propensity toward high alcohol intake (31). ACKNOWLEDGEMENTS
This work was in partial fulfillment of the requirements of D. L. Kurtz for the M.Sc. degree in Physiology and Biophysics at Indiana University School of Medicine. The authors wish to thank Dr. James Norton for statistical consultation. This research was supported by grants AA08312, AA08553, and AA07611 from the PHS.
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