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Sensitivity of inbred and selectively bred mice to ethanol
Alcohol, Vol. 4, pp. 5%62. Copyright©PergamonJournals Ltd., 1987. Printedin the U.S.A.
0741-8329/87$3.00 + .00
Sensitivity of Inbred and Selectively Bred Mice to Ethanol A N D R E W S M O L E N , 1 T O N I N. S M O L E N A N D J E N N I F E R L. VAN DE K A M P
Institute for Behavioral Genetics, and School of Pharmacy Molecular and Environmental Toxicology Program, University of Colorado Boulder, CO 80309-0447 R e c e i v e d 22 July 1986 SMOLEN, A., T. N. SMOLEN AND J. L. VAN DE KAMP. Sensitivity of inbred and selectively bred mice to ethanol. ALCOHOL 4(1) 57-62, 1987.--The Long-Sleep (LS) and Short-Sleep (SS) mice were bred for differences in sensitivity to ethanol as measured by duration of loss of the righting response (sleep time). The foundation population was a heterogeneous stock (HS) which was derived from a cross of eight inbred strains. Ethanol-induced sleep time and waking blood and brain ethanol levels were measured in the eight inbred strains, LS, SS and HS mice. The C3H and ISBI strains were quite resistant to ethanol as measured by sleep time, and only one, RIII, was very sensitive. Waking ethanol concentrations were similar for all of the inbreds, implying a narrow range of central nervous system sensitivity to ethanol. The HS mice had relatively short sleep times and blood ethanol levels equal to most of the inbreds. The LS mice were significantly more, and the SS mice significantly less sensitive to ethanol than any of the inbreds or HS mice. These studies suggest that the extremes of CNS sensitivities to ethanol manifested by the LS and SS mice cannot be traced to any of the inbred strains, and must have arisen through the selection process by changes in allelic frequencies of those genes conferring ethanol sensitivity and resistance. Long-Sleep mice Short-Sleep mice Inbred strains of mice Blood ethanol concentration Brain ethanol concentration Ethanol sleep time Acute tolerance
THE Long-Sleep (LS) and Short-Sleep (SS) mice were selectively bred for maximum differences in sensitivity to ethanol as measured by loss of the righting response, or sleep time [5]. The foundation population was a heterogeneous stock (HS) which was derived from a cross of eight inbred strains [6]. The LS and SS mice are currently in their 43rd generation, and differ so greatly in their sensitivity to ethanol that there is no overlap in their dose response curves for sleep time. The two lines began to diverge in a bidirectional fashion very early in the selection process, and this rapid response to selection was indicative of the effects of relatively few major genes acting in concert to determine the phenotypes. Dudek and Abbott have estimated the number of loci to be nine [3]. These animals have been widely studied in an effort to determine the factors responsible for their different sensitivity to ethanol. Many experimental approaches, including biochemical, behavioral and electrophysiological have been taken (for a review see [2]). Our studies have recently focused on the comparison of the LS and SS mice with the foundation population (HS) as well as the eight inbred strains which originally went into the production of the HS mice. The goal of these studies is to discover if a particular characteristic of the LS or SS mice can be traced back to one or two of the inbred strains from which they descended.
We used this approach in a previous study [I] in which we reported that sensitivity to ethanol as measured by the ED50 for loss of the righting response was similar for all of the inbred strains: between 2.30 and 2.75 g/kg. The HS were on the high end of the scale (2.8 g/kg); the LS were lower than any of the inbreds (2.07 g/kg) and the SS were higher than any of the inbreds (4.1 g/kg). We concluded that the difference in ED50 between the LS and SS could have diverged along with selection for duration of sleep time, but we have no direct way of knowing, since this measurement was not made during the selection process. In this paper we report on further studies comparing ethanol sensitivity of the LS, SS and HS mice with the eight inbred strains using sleep time as the measurement. In addition to ethanol-induced sleep time, we measured blood and brain ethanol concentrations at the time of regaining the righting response. We found that the inbred strains all awoke at similar blood and brain ethanol levels, even though there was a three-fold range of sleep times among them. The sleep times and waking ethanol levels of the HS mice were within the range established by the inbreds, the LS slept significantly longer and awoke with blood levels of ethanol significanlty lower than any of the inbreds, and the SS were significantly more resistant to ethanol than any of the inbreds or HS.
~Requests for reprints should be addressed to Dr. Andrew Smolen, Institute for Behavioral Genetics, Campus Box 447, University of Colorado, Boulder, CO 80309-0447.
57
58
S M O L E N , S M O L E N A N D VAN DE K A M P
200
E
Male
.a~
I
Female
150
E 1O0
50
oi A
K
B
3
C
D
I
R
H
L
S
A
K
B
3
C
D
I
R
H
L.
S
Strain
FIG. 1. Ethanol sleep times. The eight inbred strains, HS, LS and SS mice were injected with 3.8 g/kg ethanol in saline (20% w/v). Sleep time was measured as the time required to regain the fighting response after being placed on their backs in a V-shaped trough. Plotted values are the mean+SEM of 8 individuals (9 for C57BL). The abbreviations used are: A=A, K=AKR, B=BALB/cBY, 3=C3H, C=C57BL/6, D=DBA/2, I=ISBI, R=RIII, H=HS, L = L S and S=SS. *Indicates sex difference, p<0.05. **Indicates the SS mice did not lose the fighting response at this dose of ethanol.
TABLE 1 S L E E P TIMES AND W A K I N G BLOOD AND BRAIN
ETHANOL CONCENTRATIONS Waking Ethanol Concentration (mg/dl) Strain A AKR BALB C3H C57 DBA ISBI Rill HS LS SSt
Sleep Time (min) 76 ± 70-+ 84_ 36 66 ± 76 ± 38 ± 101 ± 44 _ 155 ± --
7 3 4 2 8 5 3 5 4 5*
Blood
Brain
Blood/Brain Ethanol
404 ± 6 378±5 386 ± 5 420 ± 7 383 ± 9 390 _ 7 373 ± 5 377 ± 8 418 ± 5 297 ± 7* 394 ± 7
435 ± 13 390± 8 385_+ 9 424 ± 14 390 ± 10 399 _+ 15 376 ± 7 389 ± 14 447 ± 15 309 ± 6* 422 ± 5
0.94 ± 0.02 0.98±0.02 1.01 ± 0.02 0.99 ± 0.03 0.98 ± 0.02 0.99 ± 0.03 0.99± 0.02 0.98 ± 0.03 0.95 ___0.03 0.96 ± 0.0l 0.94 ± 0.02
Tabled values are mean ± SEM of 16 mice per group (18 for C57BL). Mice were injected with 3.8 g/kg ethanol (20% w/v) in saline. After loss of the fighting response, they were placed on their backs in a V-shaped trough. At the time of awakening (able to right itself 3 times in 30 sec) a blood sample was taken from the retroorbital sinus and the brain was removed for ethanol analysis. Analysis of the inbred and HS comparisons are reported in Table 2. *Significantly different from all of the inbreds and HS mice, p<0.01. fSS mice did not lose the fighting response at this dose of ethanol. Blood and brain ethanol content was measured 35 min after injection.
METHOD
Animals Animals used in this study w e r e male and female mice, 60 to 80 days o f age. Mice were maintained on a 12 hour light cycle, and were allowed free access to food (Wayne Lab Blox) and water. The following inbred and selected lines w e r e used: A/lbg, A K R / J , Balb/cBylbg, C3H/Ibg, C57BL/6Ibg, DBA/2Ibg, ISBI/Crgl, R i l l s / J , H S , LS and SS.
The I B G substrains, H S , LS and SS were bred at the Institute for Behavioral Genetics. The " J " strains were obtained from J a c k s o n Laboratories, Bar Harbor, M E and the ISBI mice were obtained from the C a n c e r Research Genetics L a b o r a t o r y , Berkeley, CA. The Jackson L a b o r a t o r y and Crgl mice w e r e r e c e i v e d at approximately 6 weeks o f age and housed at I B G until tested.
Ethanol Solutions Ethanol solutions were injected intraperitoneally and were prepared as a 20% (w/v) solution in saline. Doses were varied by adjusting the v o l u m e injected, because of the marked c o n c e n t r a t i o n - d e p e n d e n t effects of ethanol on sleep time [4].
Sleep Time Determinations Sleep times were determined by injecting the mice with a 3.8 g/kg dose of ethanol. The animal was placed on its back in a V-shaped trough until it was able to right itself 3 times in a 30 sec period. Blood and brain samples were taken for ethanol m e a s u r e m e n t s at the time of awakening.
Measurement of Blood Ethanol Concentration A 10 /~1 venous blood sample was obtained from the retro-orbital sinus with a capillary pipet [9]. The a m o u n t of ethanol in the sample was m e a s u r e d using an enzymatic m e t h o d [11]. The 10/~1 blood sample was placed in a 6 x 5 0 mm test tube containing 200 p.l of ice-cold 0.55 M perchloric acid to precipitate the protein. The samples were centrifuged at 1500 x g for 10 rain at 4°C. Following centrifugation, 200 /~l of 0.60 M potassium hydroxide containing 50 m M acetic acid was added to adjust the p H of the samples to about 5 and precipitate the perchlorate anion (which may inhibit the alcohol d e h y d r o g e n a s e , A D H ) . The samples were centrifuged a second time (1500 x g, 10 rain) to pellet the resulting precipitate. The ethanol content was determined by measuring the amount of N A D H produced from N A D + (Boehringer Mannheim) in the p r e s e n c e of yeast A D H (Sigma). A 50 /zl aliquot of the resulting neutralized supernatant was added to 350/.d of 0.5 M Tris-HCl, p H 8.8 containing 2.30 m M N A D + (2.0 m M final) and 30 units/ml (8-10 units final) of A D H . Blanks without A D H were used routinely. This blank has
S E N S I T I V I T Y O F MICE TO E T H A N O L
59
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300
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200
100 c03
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A
K
B
3
C
D
I
R
H
L
S
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B
3
C
D
l
R
H
L
S
STRAIN
FIG. 2. Waking blood and brain ethanol concentrations. At the time of awakening blood and brain ethanol concentrations were measured. Each value is the mean_+SEM of 8 individuals (9 for C57BL). Abbreviations are given in the legend to Fig. 1. *Indicates a sex difference.
been found to work as well as a pyrazole-containing blank and was used for convenience. Samples and blanks were incubated at room temperature for 30 rain (this is an endpoint reaction, which is complete within 15 rain; temperature is not critical). The samples were read in Gilford 260 spectrophotometer at 340 nm zeroed against a water blank. Blood ethanol levels were calculated from a standard curve (0--600 mg%) which was analyzed simultaneously with the samples. The standards were prepared without blood from untreated mice, since the regression coefficients for standard curves run with and without blood have been found to be identical, and the results are therefore identical. All samples, blanks and standards were run in duplicate.
Measurement of Brain Ethanol Concentration After taking a 10 ~1 blood sample, the animals were killed by cervical dislocation, the brain removed, rinsed, blotted, weighed, homogenized in 4 ml ice-cold 0.55 M perchloric acid, and centrifuged (1500 x g, 10 rain) to precipitate the denatured proteins. Following centrifugation, a 200 /xl aliquot of each sample was neutralized by the addition of 200 /zl of 0.60 M potassium hydroxide containing 50 mM acetic acid. From this point on, the brain samples were assayed identically to the blood samples except that a 20/zl aliquot was taken.
Data Analysis Data were analyzed by linear regression analysis or analysis of variance as appropriate. Following a significant overall effect, (o<0.05) differences in individual group means were detected using Tukey's B-test. The inbred strain data reported in Table I were analyzed by one-way analysis of variance collapsed across sex, and these results are reported in Table 2 along with the comparisons between the HS mice and the inbreds. The HS, LS and SS data were
compared to the inbreds using separate analyses. The SS mice were not included in the sleep time analysis since they did not lose the righting response. RESULTS
Figure 1 shows the ethanol-induced sleep times in the eight inbred strains, HS, LS and SS mice. There were no sex differences in sleep time, and subsequent analyses were collapsed across sex (Tables 1 and 2). One-way analysis of variance revealed a significant strain difference in sleep time. The RIII mice had the longest sleep time, the C3H the shortest. HS were on the resistant end of the scale, and as expected, the LS slept significantly longer than any other group. SS mice did not lose the righting response at this dose o f ethanol. Figure 2 reports waking blood and brain ethanol concentrations in all 11 lines of mice. Blood and brain levels of ethanol in the SS, which did not lose the righting response at this dose, were determined after 35 min. This time was chosen since the least sensitive inbred, C3H, had a sleep time o f approximately 35 min. Sex differences were found only in BALB and RIII mice for blood, and for RIII mice for brain ethanol levels. When collapsed across sex (Tables 1 and 2), there was a significant effect of strain on both blood and brain ethanol levels at awakening. The ISBI mice had the lowest, and the C3H mice the highest waking ethanol levels. All of the strains had blood and brain ethanol concentrations higher than the LS at the time of awakening. F o r each individual, concentrations of ethanol in blood and brain at awakening were virtually identical, and there were no differences, either sex or strain, on the ratio of blood ethanol content to brain ethanol content. This indicates that for all genotypes, blood ethanol content accurately reflects brain ethanol content at the time of regaining the righting response. The relationship between waking blood ethanol concen-
60
SMOLEN, SMOLEN AND VAN DE KAMP
TABLE 2 RESULTS OF A N A L Y S I S OF VARIANCEOF DATA IN TABLE l
(~ 420
y=-O.27x ÷ 408
(~
r-=-0.32,
NS
C~
Anova for SLEEP TIME, F(7,122)=720.24, p<0.0001 Anova for WAKING BLOOD ETHANOL, F(7,122)=5.58, p<0.0001 Anova for WAKING BRAIN ETHANOL, F(7,122)=3.18, p<0.005 Anova for WAKING BLOOD/BRAIN ETHANOL, F(7,122)=0.98, NS
E o C
ru t-
400
Ld
_o rn
380
Post-Hoc Analysis of Sleep Time A
AKR BALB C3H 0.01 0.01 0.01
A AKR BALB C3H C57 DBA ISBI HS 0.01 0.01
C57
DBA
0.01
0.01
ISBI 0.01 0.01 0.01 0.01 0.01
0.01
0.01
0.01
Rill 0.01 0.01 0.05 0.01 0.01 0.01 0.01 0.01
Post-Hoc Analysis of Waking Blood Ethanol Concentrations A
AKR BALB
A AKR BALB C3H C57 DBA
C3H
C57
DBA
ISBI 0.05
RIII
0.01
0.01
0.01
0.01
0.01 0.01 0.01
0.05
l
~"
360
I
20
I
I
I
40 60 80 Sleep Time, min
I
100
FIG. 3. Correlation between sleep times and waking blood ethanol concentrations in eight inbred strains. Plotted values are taken from Table 1. Abbreviations are given in the legend to Fig. 1.
equal concentrations as those found at the time of loss of righting. The magnitude of the effect varies with strain, but these results indicate that the potential to develop acute tolerance to ethanol was present in the foundation populations (inbreds and HS), but might have been selected out of the LS and SS during the course of the sleep time selection.
ISBI
0.01
HS
0.05
0.01
Post-Hoc Analysis of Waking Brain Ethanol Concentrations A A AKR BALB C3H C57 DBA
AKR BALB 0.05
C3H
C57
DBA
ISBI 0.01
Rill
0.01
0.01
0 . 0 5 0.05
0.01
0.01
ISBI
HS
0.01
The sleep time and waking blood and brain ethanol concentrations for the inbreds in Table 1 were collapsed across sex and analysed by one-way analysis of variance. Differences in group means were determined using Tukey's B-test. The HS were compared with the inbred strains using a separate analysis. Tabled values are p values for the significant strain comparisons. tration and sleep time is plotted in Fig. 3. There was a negative trend in the data indicating that animals which slept longer awoke at lower blood ethanol content, but the slope was not significantly different from zero. The potential for the development of acute tolerance is shown in Table 3. Blood ethanol at the time of loss of righting response (values taken from [1]) at the time of regaining the fighting response, and the difference in the two measures are shown for the inbreds, HS, LS and SS mice. In general, the mice all had higher blood ethanol content at the time of awakening than at the time of loss of the righting response, which may be a measure of the development of acute tolerance to ethanol. The only exceptions were the R i l l females and the LS and SS mice which awoke at lower or
DISCUSSION The results presented here show that there is a narrow, yet statistically significant, range of CNS sensitivities to ethanol among the eight inbred strains used in this study. The sleep response of the inbreds covers a wide spectrum, from the very resistant (C3H, 35 min) to very sensitive (RIII, 105 min). With the exception of the DBA mice, the albino strains (A, AKR, BALB, RIII) had significantly longer sleep times than all of the pigmented strains, which is in agreement with an earlier report [10]. None of the strains is as resistant to loss of fighting as the SS mice, nor as sensitive as the LS, although the difference at the sensitive end of the scale (RIII vs. LS) is not as great as on the resistant end (C3H vs. SS). In contrast to the 3-fold difference in sleep times among the inbreds, waking blood ethanol levels varied only 18% (359 to 424 mg%). The HS mice regained the fighting response at a time (44 min) and blood ethanol level (418 mg%) within the range established by the inbreds. Of the eight inbred strains, two (C3H and ISBI) are relatively resistant, one (Rill) is sensitive, and five are intermediate in their response to the soporific effect of ethanol. Yet when intercrossed, they established the relatively resistant HS stock. This suggests that more selection pressure should have been required to produce sensitivity than resistance to ethanol. The sleep times and waking ethanol concentrations for the LS and SS mice were far outside of the range established by the inbreds. The LS slept 50 rain longer and awoke at a blood ethanol concentration more than 100 rag% lower than the next most sensitive strain (RIII). The SS mice are so resistant to ethanol that they did not lose the righting response at this dose of ethanol (a dose of approximately 4.7 g/kg is required for a 35 rain sleep time, and waking blood ethanol levels are approximately 500 mg% for the SS).
S E N S I T I V I T Y O F MICE TO E T H A N O L
61
TABLE 3 BLOOD ETHANOL CONCENTRATIONSAT THE TIME OF LOSS AND REGAININGTHE RIGHTING RESPONSE
Sex
Blood Ethanol at Loss of Righting (mg/di)
Blood Ethanol at Awakening (mg/dl)
A
Male Female
374 372
405 401
31 * 29*
AKR
Male Female
383 372
387 369
4 3
BALB
Male Female
335 336
371 400
36* 64*
C3H
Male Female
368 350
424 417
56~ 67*
C57
Male Female
321 342
401 368
80* 26
DBA
Male Female
354 346
380 401
26* 55*
ISBI
Male Female
321 317
377 370
56* 53*
RII1
Male Female
358 395
394 359
36* -36
HS
Male Female
405 406
421 414
16* 8
LS
Male Female
327 313
268 243
-59 - 70
SS
Male Female
509 550
509 512
0 -38
Strain
Difference Awakening - Loss (mg/dl)
Blood ethanol at loss of righting are taken from [1]. Waking blood ethanol values for inbreds and HS are taken following 3.8 g/kg ethanol (Table 1). The values for LS are taken for a 2.5 g/kg dose. The values for SS are taken for a 5.0 g/kg dose. *Significantly higher at time of awakening than at time of loss of the righting response, p<0.05.
Spuhler and coworkers also measured sleep time responses in these eight inbred strains [12]. The rank ordering of the strains for sleep time response was different in their study and ours, but the four most sensitive, and the four most resistant strains were the same in each. These differences could be due to the dose (3.3 g/kg) and the concentration (30% w/v) they used. Others from our laboratory have reported that for a given dose of ethanol, sleep times increase with increasing concentration of ethanol [4]. This effect of concentration is minimal or absent at ethanol concentrations o f 20% (w/v) or less, which is the concentration we now use routinely. The sleep time response of the LS and SS mice is thought to be due to differential CNS sensitivity, and not to differ-
ences in ethanol pharmacokinetics. In previous studies we [11], and others [8] found that the small metabolism differences in these mice affected the shape of the dose response curve for ethanol elimination, and not so much the overall rate. At the dose used in this study, 3.8 g/kg (IP) the LS and SS differ in metabolism by 10--15% (SS faster), which is not great enough to explain the marked behavioral differences in these two lines of mice. We have not measured ethanol elimination rates in the eight inbred strains. Acute tolerance has been described as the development of tolerance during a single exposure to the agent in question [7]. Tabakoff and Ritzman [13] reported that C57BL mice had higher brain ethanol levels at the time of regaining, than at the time of loss of the righting response, but that D B A mice had similar levels at both times. They suggested that the C57 mice had the potential to develop acute tolerance, but the DBA mice did not. In a subsequent paper [14] this group showed that neither the LS nor the SS mice could develop acute tolerance. Our data (Table 3) largely support this contention, even though our C57/DBA data do not agree totally with their previous study. Among the inbreds, the A K R strain shows the least evidence of development of acute tolerance. We found no evidence for acute tolerance in the LS or SS mice. These data imply that the potential for acute tolerance was lost during the selection for the LS and SS lines. Our studies of the LS and SS mice have recently focused on comparing their responses to alcohol with the HS and inbred mice which were the progenitors of these selected lines. These studies require the assumption that by sampling inbred strains now, we can draw inferences to what these animals were like at the beginning of the selection study some years ago. This assumption is based on the stability of inbred strains over generations, and while this may be difficult to prove, we believe it to be generally true. Still, with the caution that genetic drift could account for some of our findings, this approach may be useful in understanding the events which occurred early in a study such as the LS/SS selection. In summary, the sleep time responses of the eight inbred strains spaned a wide range of values. These sleep time differences were apparently not due to intrinsic CNS sensitivity differences among the strains since they all awoke at approximately the same blood and brain levels of ethanol regardless of their sleep times. Figure 3 shows that there was a trend for animals with the longest sleep times to awake with lower ethanol levels than those animals with short sleep times, but this relationship was not statistically significant. In contrast to the inbreds, the extended sleep time of the LS mice and the brief sleep time of the SS mice are associated with very low, and very high waking ethanol levels, respectively. These results are clearly indicative of marked CNS sensitivity differences of these mice to ethanol. Since the differences in sensitivity to ethanol among the inbred strains examined were small, the extreme CNS sensitivities of the LS and SS mice cannot be traced back to one inbred more than any other. The CNS sensitivity difference in the LS and SS must have arisen exclusively via the selection process by changes in allelic frequencies of those genes conferring ethanol sensitivity and resistance.
62
S M O L E N , S M O L E N A N D VAN DE K A M P ACKNOWLEDGEMENTS This work was supported in part by grants from the National Institute of Neurological and Communicative Disorders and Stroke, NS 20748 (A.S.), the National Institute of Child Health and Human Development, HD 21709 (A.S.) and the National Institute on Alcohol Abuse and Alcoholism, AA 06487 and AA 06527 (T.N.S). Preparation of this manuscript was facilitated by a grant from the Biomedical Research Support Grant Program, RR-07013-20 awarded to the University of Colorado, Boulder, CO. The authors wish to thank Dr. Richard A, Deitrich of the University of Colorado Alcohol Research Center for providing some of the AKR, RIII and ISBI mice for these studies.
REFERENCES 1. Baker, R. L., T. N. Smolen, A. Smolen and R. A, Deitrich. Relationship between acute ethanol related responses in long sleep and short sleep mice. Alcoholism: Clin Exp Res, in press. 2. Collins, A. C. A review of research using Short-Sleep and Long-Sleep mice. In: The Development of Animal Models as Pharmacogenetic Tools, edited by G. E. McClearn, R. A. Deitrich and V. G. Erwin. DHHS Publication No. (ADM 81-1133). Washington, DC: U.S. Government Printing Office, 1981, pp. 161-170. 3. Dudek, B. C. and M. E. Abbott. A biometricat genetic analysis of ethanol response in selectively bred long-sleep and shortsleep mice. Behav Genet 14: 1-19, 1984. 4. Gilliam, D. M. and A. C. Collins. Concentration-dependent effects of ethanol in long-sleep and short-sleep mice. Ah'oholism: Clin Exp Res 7: 337-342, 1983. 5. McClearn, G. E. and R. Kakihana. Selective breeding for ethanol sensitivity: SS and LS mice. In: The Development of Animal Models as Pharmacogenetic Tools, edited by G. E. McClearn, R. A. Deitrich and V. G. Erwin. DHHS Publication No. (ADM 81-1133). Washington, DC: U.S. Government Printing Office, 1981, pp. 147-159. 6. McClearn, G. E., J. R. Wilson and W. Meridith. In: Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype. edited by G. Lindzey and D. D. Thiessen. New York: Appleton-Century-Crofts, 1970, pp. 3-22.
7. Mellanby, E. Medical Research Council (Great Britain), Special Report Series No. 31, London, 1919. 8. Phillips, T. J., D. M. Gilliam and B, C. Dudek. An evaluation of the role of ethanol clearance rate in the differential response of long-sleep and short-sleep mice to ethanol. Ah.ohol 1: 373-378, 1984. 9. Riley, V. Adaptation of orbital bleeding technic to rapid serial blood studies. Proc Soc Exp Biol Med 104: 751-754, 1960. 10. Rush, W. A. and R. A. King. Effect of the albino gene on sleep time in mice. Behav Genet 6:116, 1976. 11. Smolen, A., M. Marks, T. N. Smolen and A. C. Collins. Dose and route of administration alter the relative elimination of ethanol by long-sleep and short-sleep mice. Alcoholism: Clin Exp Res 10: 198-204, 1986. 12. Spuhler, K., B. Hoffer, N. Weiner and M. Palmer. Evidence for genetic correlation of hypnotic effects and cerebellar Purkinje neuron depression in response to ethanol in mice." Pharmacol Biochem Behav 17: 569-578, 1982. 13. Tabakoff, B. and R. F. Ritzman. Acute tolerance in inbred and selected lines of mice. Drug Alcohol Depend 4: 87-90, 1979. 14. Tabakoff, B., R. F. Ritzman, T. S. Raju and R. A. Deitrich. Characterization of acute and chronic tolerance in mice selected for inherent differences in sensitivity to ethanol. Alcoholism: Clin I£xp Res 4: 70-73, 1980.
0741-8329/87$3.00 + .00
Sensitivity of Inbred and Selectively Bred Mice to Ethanol A N D R E W S M O L E N , 1 T O N I N. S M O L E N A N D J E N N I F E R L. VAN DE K A M P
Institute for Behavioral Genetics, and School of Pharmacy Molecular and Environmental Toxicology Program, University of Colorado Boulder, CO 80309-0447 R e c e i v e d 22 July 1986 SMOLEN, A., T. N. SMOLEN AND J. L. VAN DE KAMP. Sensitivity of inbred and selectively bred mice to ethanol. ALCOHOL 4(1) 57-62, 1987.--The Long-Sleep (LS) and Short-Sleep (SS) mice were bred for differences in sensitivity to ethanol as measured by duration of loss of the righting response (sleep time). The foundation population was a heterogeneous stock (HS) which was derived from a cross of eight inbred strains. Ethanol-induced sleep time and waking blood and brain ethanol levels were measured in the eight inbred strains, LS, SS and HS mice. The C3H and ISBI strains were quite resistant to ethanol as measured by sleep time, and only one, RIII, was very sensitive. Waking ethanol concentrations were similar for all of the inbreds, implying a narrow range of central nervous system sensitivity to ethanol. The HS mice had relatively short sleep times and blood ethanol levels equal to most of the inbreds. The LS mice were significantly more, and the SS mice significantly less sensitive to ethanol than any of the inbreds or HS mice. These studies suggest that the extremes of CNS sensitivities to ethanol manifested by the LS and SS mice cannot be traced to any of the inbred strains, and must have arisen through the selection process by changes in allelic frequencies of those genes conferring ethanol sensitivity and resistance. Long-Sleep mice Short-Sleep mice Inbred strains of mice Blood ethanol concentration Brain ethanol concentration Ethanol sleep time Acute tolerance
THE Long-Sleep (LS) and Short-Sleep (SS) mice were selectively bred for maximum differences in sensitivity to ethanol as measured by loss of the righting response, or sleep time [5]. The foundation population was a heterogeneous stock (HS) which was derived from a cross of eight inbred strains [6]. The LS and SS mice are currently in their 43rd generation, and differ so greatly in their sensitivity to ethanol that there is no overlap in their dose response curves for sleep time. The two lines began to diverge in a bidirectional fashion very early in the selection process, and this rapid response to selection was indicative of the effects of relatively few major genes acting in concert to determine the phenotypes. Dudek and Abbott have estimated the number of loci to be nine [3]. These animals have been widely studied in an effort to determine the factors responsible for their different sensitivity to ethanol. Many experimental approaches, including biochemical, behavioral and electrophysiological have been taken (for a review see [2]). Our studies have recently focused on the comparison of the LS and SS mice with the foundation population (HS) as well as the eight inbred strains which originally went into the production of the HS mice. The goal of these studies is to discover if a particular characteristic of the LS or SS mice can be traced back to one or two of the inbred strains from which they descended.
We used this approach in a previous study [I] in which we reported that sensitivity to ethanol as measured by the ED50 for loss of the righting response was similar for all of the inbred strains: between 2.30 and 2.75 g/kg. The HS were on the high end of the scale (2.8 g/kg); the LS were lower than any of the inbreds (2.07 g/kg) and the SS were higher than any of the inbreds (4.1 g/kg). We concluded that the difference in ED50 between the LS and SS could have diverged along with selection for duration of sleep time, but we have no direct way of knowing, since this measurement was not made during the selection process. In this paper we report on further studies comparing ethanol sensitivity of the LS, SS and HS mice with the eight inbred strains using sleep time as the measurement. In addition to ethanol-induced sleep time, we measured blood and brain ethanol concentrations at the time of regaining the righting response. We found that the inbred strains all awoke at similar blood and brain ethanol levels, even though there was a three-fold range of sleep times among them. The sleep times and waking ethanol levels of the HS mice were within the range established by the inbreds, the LS slept significantly longer and awoke with blood levels of ethanol significanlty lower than any of the inbreds, and the SS were significantly more resistant to ethanol than any of the inbreds or HS.
~Requests for reprints should be addressed to Dr. Andrew Smolen, Institute for Behavioral Genetics, Campus Box 447, University of Colorado, Boulder, CO 80309-0447.
57
58
S M O L E N , S M O L E N A N D VAN DE K A M P
200
E
Male
.a~
I
Female
150
E 1O0
50
oi A
K
B
3
C
D
I
R
H
L
S
A
K
B
3
C
D
I
R
H
L.
S
Strain
FIG. 1. Ethanol sleep times. The eight inbred strains, HS, LS and SS mice were injected with 3.8 g/kg ethanol in saline (20% w/v). Sleep time was measured as the time required to regain the fighting response after being placed on their backs in a V-shaped trough. Plotted values are the mean+SEM of 8 individuals (9 for C57BL). The abbreviations used are: A=A, K=AKR, B=BALB/cBY, 3=C3H, C=C57BL/6, D=DBA/2, I=ISBI, R=RIII, H=HS, L = L S and S=SS. *Indicates sex difference, p<0.05. **Indicates the SS mice did not lose the fighting response at this dose of ethanol.
TABLE 1 S L E E P TIMES AND W A K I N G BLOOD AND BRAIN
ETHANOL CONCENTRATIONS Waking Ethanol Concentration (mg/dl) Strain A AKR BALB C3H C57 DBA ISBI Rill HS LS SSt
Sleep Time (min) 76 ± 70-+ 84_ 36 66 ± 76 ± 38 ± 101 ± 44 _ 155 ± --
7 3 4 2 8 5 3 5 4 5*
Blood
Brain
Blood/Brain Ethanol
404 ± 6 378±5 386 ± 5 420 ± 7 383 ± 9 390 _ 7 373 ± 5 377 ± 8 418 ± 5 297 ± 7* 394 ± 7
435 ± 13 390± 8 385_+ 9 424 ± 14 390 ± 10 399 _+ 15 376 ± 7 389 ± 14 447 ± 15 309 ± 6* 422 ± 5
0.94 ± 0.02 0.98±0.02 1.01 ± 0.02 0.99 ± 0.03 0.98 ± 0.02 0.99 ± 0.03 0.99± 0.02 0.98 ± 0.03 0.95 ___0.03 0.96 ± 0.0l 0.94 ± 0.02
Tabled values are mean ± SEM of 16 mice per group (18 for C57BL). Mice were injected with 3.8 g/kg ethanol (20% w/v) in saline. After loss of the fighting response, they were placed on their backs in a V-shaped trough. At the time of awakening (able to right itself 3 times in 30 sec) a blood sample was taken from the retroorbital sinus and the brain was removed for ethanol analysis. Analysis of the inbred and HS comparisons are reported in Table 2. *Significantly different from all of the inbreds and HS mice, p<0.01. fSS mice did not lose the fighting response at this dose of ethanol. Blood and brain ethanol content was measured 35 min after injection.
METHOD
Animals Animals used in this study w e r e male and female mice, 60 to 80 days o f age. Mice were maintained on a 12 hour light cycle, and were allowed free access to food (Wayne Lab Blox) and water. The following inbred and selected lines w e r e used: A/lbg, A K R / J , Balb/cBylbg, C3H/Ibg, C57BL/6Ibg, DBA/2Ibg, ISBI/Crgl, R i l l s / J , H S , LS and SS.
The I B G substrains, H S , LS and SS were bred at the Institute for Behavioral Genetics. The " J " strains were obtained from J a c k s o n Laboratories, Bar Harbor, M E and the ISBI mice were obtained from the C a n c e r Research Genetics L a b o r a t o r y , Berkeley, CA. The Jackson L a b o r a t o r y and Crgl mice w e r e r e c e i v e d at approximately 6 weeks o f age and housed at I B G until tested.
Ethanol Solutions Ethanol solutions were injected intraperitoneally and were prepared as a 20% (w/v) solution in saline. Doses were varied by adjusting the v o l u m e injected, because of the marked c o n c e n t r a t i o n - d e p e n d e n t effects of ethanol on sleep time [4].
Sleep Time Determinations Sleep times were determined by injecting the mice with a 3.8 g/kg dose of ethanol. The animal was placed on its back in a V-shaped trough until it was able to right itself 3 times in a 30 sec period. Blood and brain samples were taken for ethanol m e a s u r e m e n t s at the time of awakening.
Measurement of Blood Ethanol Concentration A 10 /~1 venous blood sample was obtained from the retro-orbital sinus with a capillary pipet [9]. The a m o u n t of ethanol in the sample was m e a s u r e d using an enzymatic m e t h o d [11]. The 10/~1 blood sample was placed in a 6 x 5 0 mm test tube containing 200 p.l of ice-cold 0.55 M perchloric acid to precipitate the protein. The samples were centrifuged at 1500 x g for 10 rain at 4°C. Following centrifugation, 200 /~l of 0.60 M potassium hydroxide containing 50 m M acetic acid was added to adjust the p H of the samples to about 5 and precipitate the perchlorate anion (which may inhibit the alcohol d e h y d r o g e n a s e , A D H ) . The samples were centrifuged a second time (1500 x g, 10 rain) to pellet the resulting precipitate. The ethanol content was determined by measuring the amount of N A D H produced from N A D + (Boehringer Mannheim) in the p r e s e n c e of yeast A D H (Sigma). A 50 /zl aliquot of the resulting neutralized supernatant was added to 350/.d of 0.5 M Tris-HCl, p H 8.8 containing 2.30 m M N A D + (2.0 m M final) and 30 units/ml (8-10 units final) of A D H . Blanks without A D H were used routinely. This blank has
S E N S I T I V I T Y O F MICE TO E T H A N O L
59
400
300
E C" O
200
100 c03
0 U
22 ....,
t~ c
A
K
B
3
C
D
I
R
H
L
S
A
K
B
3
C
D
l
R
H
L
S
STRAIN
FIG. 2. Waking blood and brain ethanol concentrations. At the time of awakening blood and brain ethanol concentrations were measured. Each value is the mean_+SEM of 8 individuals (9 for C57BL). Abbreviations are given in the legend to Fig. 1. *Indicates a sex difference.
been found to work as well as a pyrazole-containing blank and was used for convenience. Samples and blanks were incubated at room temperature for 30 rain (this is an endpoint reaction, which is complete within 15 rain; temperature is not critical). The samples were read in Gilford 260 spectrophotometer at 340 nm zeroed against a water blank. Blood ethanol levels were calculated from a standard curve (0--600 mg%) which was analyzed simultaneously with the samples. The standards were prepared without blood from untreated mice, since the regression coefficients for standard curves run with and without blood have been found to be identical, and the results are therefore identical. All samples, blanks and standards were run in duplicate.
Measurement of Brain Ethanol Concentration After taking a 10 ~1 blood sample, the animals were killed by cervical dislocation, the brain removed, rinsed, blotted, weighed, homogenized in 4 ml ice-cold 0.55 M perchloric acid, and centrifuged (1500 x g, 10 rain) to precipitate the denatured proteins. Following centrifugation, a 200 /xl aliquot of each sample was neutralized by the addition of 200 /zl of 0.60 M potassium hydroxide containing 50 mM acetic acid. From this point on, the brain samples were assayed identically to the blood samples except that a 20/zl aliquot was taken.
Data Analysis Data were analyzed by linear regression analysis or analysis of variance as appropriate. Following a significant overall effect, (o<0.05) differences in individual group means were detected using Tukey's B-test. The inbred strain data reported in Table I were analyzed by one-way analysis of variance collapsed across sex, and these results are reported in Table 2 along with the comparisons between the HS mice and the inbreds. The HS, LS and SS data were
compared to the inbreds using separate analyses. The SS mice were not included in the sleep time analysis since they did not lose the righting response. RESULTS
Figure 1 shows the ethanol-induced sleep times in the eight inbred strains, HS, LS and SS mice. There were no sex differences in sleep time, and subsequent analyses were collapsed across sex (Tables 1 and 2). One-way analysis of variance revealed a significant strain difference in sleep time. The RIII mice had the longest sleep time, the C3H the shortest. HS were on the resistant end of the scale, and as expected, the LS slept significantly longer than any other group. SS mice did not lose the righting response at this dose o f ethanol. Figure 2 reports waking blood and brain ethanol concentrations in all 11 lines of mice. Blood and brain levels of ethanol in the SS, which did not lose the righting response at this dose, were determined after 35 min. This time was chosen since the least sensitive inbred, C3H, had a sleep time o f approximately 35 min. Sex differences were found only in BALB and RIII mice for blood, and for RIII mice for brain ethanol levels. When collapsed across sex (Tables 1 and 2), there was a significant effect of strain on both blood and brain ethanol levels at awakening. The ISBI mice had the lowest, and the C3H mice the highest waking ethanol levels. All of the strains had blood and brain ethanol concentrations higher than the LS at the time of awakening. F o r each individual, concentrations of ethanol in blood and brain at awakening were virtually identical, and there were no differences, either sex or strain, on the ratio of blood ethanol content to brain ethanol content. This indicates that for all genotypes, blood ethanol content accurately reflects brain ethanol content at the time of regaining the righting response. The relationship between waking blood ethanol concen-
60
SMOLEN, SMOLEN AND VAN DE KAMP
TABLE 2 RESULTS OF A N A L Y S I S OF VARIANCEOF DATA IN TABLE l
(~ 420
y=-O.27x ÷ 408
(~
r-=-0.32,
NS
C~
Anova for SLEEP TIME, F(7,122)=720.24, p<0.0001 Anova for WAKING BLOOD ETHANOL, F(7,122)=5.58, p<0.0001 Anova for WAKING BRAIN ETHANOL, F(7,122)=3.18, p<0.005 Anova for WAKING BLOOD/BRAIN ETHANOL, F(7,122)=0.98, NS
E o C
ru t-
400
Ld
_o rn
380
Post-Hoc Analysis of Sleep Time A
AKR BALB C3H 0.01 0.01 0.01
A AKR BALB C3H C57 DBA ISBI HS 0.01 0.01
C57
DBA
0.01
0.01
ISBI 0.01 0.01 0.01 0.01 0.01
0.01
0.01
0.01
Rill 0.01 0.01 0.05 0.01 0.01 0.01 0.01 0.01
Post-Hoc Analysis of Waking Blood Ethanol Concentrations A
AKR BALB
A AKR BALB C3H C57 DBA
C3H
C57
DBA
ISBI 0.05
RIII
0.01
0.01
0.01
0.01
0.01 0.01 0.01
0.05
l
~"
360
I
20
I
I
I
40 60 80 Sleep Time, min
I
100
FIG. 3. Correlation between sleep times and waking blood ethanol concentrations in eight inbred strains. Plotted values are taken from Table 1. Abbreviations are given in the legend to Fig. 1.
equal concentrations as those found at the time of loss of righting. The magnitude of the effect varies with strain, but these results indicate that the potential to develop acute tolerance to ethanol was present in the foundation populations (inbreds and HS), but might have been selected out of the LS and SS during the course of the sleep time selection.
ISBI
0.01
HS
0.05
0.01
Post-Hoc Analysis of Waking Brain Ethanol Concentrations A A AKR BALB C3H C57 DBA
AKR BALB 0.05
C3H
C57
DBA
ISBI 0.01
Rill
0.01
0.01
0 . 0 5 0.05
0.01
0.01
ISBI
HS
0.01
The sleep time and waking blood and brain ethanol concentrations for the inbreds in Table 1 were collapsed across sex and analysed by one-way analysis of variance. Differences in group means were determined using Tukey's B-test. The HS were compared with the inbred strains using a separate analysis. Tabled values are p values for the significant strain comparisons. tration and sleep time is plotted in Fig. 3. There was a negative trend in the data indicating that animals which slept longer awoke at lower blood ethanol content, but the slope was not significantly different from zero. The potential for the development of acute tolerance is shown in Table 3. Blood ethanol at the time of loss of righting response (values taken from [1]) at the time of regaining the fighting response, and the difference in the two measures are shown for the inbreds, HS, LS and SS mice. In general, the mice all had higher blood ethanol content at the time of awakening than at the time of loss of the righting response, which may be a measure of the development of acute tolerance to ethanol. The only exceptions were the R i l l females and the LS and SS mice which awoke at lower or
DISCUSSION The results presented here show that there is a narrow, yet statistically significant, range of CNS sensitivities to ethanol among the eight inbred strains used in this study. The sleep response of the inbreds covers a wide spectrum, from the very resistant (C3H, 35 min) to very sensitive (RIII, 105 min). With the exception of the DBA mice, the albino strains (A, AKR, BALB, RIII) had significantly longer sleep times than all of the pigmented strains, which is in agreement with an earlier report [10]. None of the strains is as resistant to loss of fighting as the SS mice, nor as sensitive as the LS, although the difference at the sensitive end of the scale (RIII vs. LS) is not as great as on the resistant end (C3H vs. SS). In contrast to the 3-fold difference in sleep times among the inbreds, waking blood ethanol levels varied only 18% (359 to 424 mg%). The HS mice regained the fighting response at a time (44 min) and blood ethanol level (418 mg%) within the range established by the inbreds. Of the eight inbred strains, two (C3H and ISBI) are relatively resistant, one (Rill) is sensitive, and five are intermediate in their response to the soporific effect of ethanol. Yet when intercrossed, they established the relatively resistant HS stock. This suggests that more selection pressure should have been required to produce sensitivity than resistance to ethanol. The sleep times and waking ethanol concentrations for the LS and SS mice were far outside of the range established by the inbreds. The LS slept 50 rain longer and awoke at a blood ethanol concentration more than 100 rag% lower than the next most sensitive strain (RIII). The SS mice are so resistant to ethanol that they did not lose the righting response at this dose of ethanol (a dose of approximately 4.7 g/kg is required for a 35 rain sleep time, and waking blood ethanol levels are approximately 500 mg% for the SS).
S E N S I T I V I T Y O F MICE TO E T H A N O L
61
TABLE 3 BLOOD ETHANOL CONCENTRATIONSAT THE TIME OF LOSS AND REGAININGTHE RIGHTING RESPONSE
Sex
Blood Ethanol at Loss of Righting (mg/di)
Blood Ethanol at Awakening (mg/dl)
A
Male Female
374 372
405 401
31 * 29*
AKR
Male Female
383 372
387 369
4 3
BALB
Male Female
335 336
371 400
36* 64*
C3H
Male Female
368 350
424 417
56~ 67*
C57
Male Female
321 342
401 368
80* 26
DBA
Male Female
354 346
380 401
26* 55*
ISBI
Male Female
321 317
377 370
56* 53*
RII1
Male Female
358 395
394 359
36* -36
HS
Male Female
405 406
421 414
16* 8
LS
Male Female
327 313
268 243
-59 - 70
SS
Male Female
509 550
509 512
0 -38
Strain
Difference Awakening - Loss (mg/dl)
Blood ethanol at loss of righting are taken from [1]. Waking blood ethanol values for inbreds and HS are taken following 3.8 g/kg ethanol (Table 1). The values for LS are taken for a 2.5 g/kg dose. The values for SS are taken for a 5.0 g/kg dose. *Significantly higher at time of awakening than at time of loss of the righting response, p<0.05.
Spuhler and coworkers also measured sleep time responses in these eight inbred strains [12]. The rank ordering of the strains for sleep time response was different in their study and ours, but the four most sensitive, and the four most resistant strains were the same in each. These differences could be due to the dose (3.3 g/kg) and the concentration (30% w/v) they used. Others from our laboratory have reported that for a given dose of ethanol, sleep times increase with increasing concentration of ethanol [4]. This effect of concentration is minimal or absent at ethanol concentrations o f 20% (w/v) or less, which is the concentration we now use routinely. The sleep time response of the LS and SS mice is thought to be due to differential CNS sensitivity, and not to differ-
ences in ethanol pharmacokinetics. In previous studies we [11], and others [8] found that the small metabolism differences in these mice affected the shape of the dose response curve for ethanol elimination, and not so much the overall rate. At the dose used in this study, 3.8 g/kg (IP) the LS and SS differ in metabolism by 10--15% (SS faster), which is not great enough to explain the marked behavioral differences in these two lines of mice. We have not measured ethanol elimination rates in the eight inbred strains. Acute tolerance has been described as the development of tolerance during a single exposure to the agent in question [7]. Tabakoff and Ritzman [13] reported that C57BL mice had higher brain ethanol levels at the time of regaining, than at the time of loss of the righting response, but that D B A mice had similar levels at both times. They suggested that the C57 mice had the potential to develop acute tolerance, but the DBA mice did not. In a subsequent paper [14] this group showed that neither the LS nor the SS mice could develop acute tolerance. Our data (Table 3) largely support this contention, even though our C57/DBA data do not agree totally with their previous study. Among the inbreds, the A K R strain shows the least evidence of development of acute tolerance. We found no evidence for acute tolerance in the LS or SS mice. These data imply that the potential for acute tolerance was lost during the selection for the LS and SS lines. Our studies of the LS and SS mice have recently focused on comparing their responses to alcohol with the HS and inbred mice which were the progenitors of these selected lines. These studies require the assumption that by sampling inbred strains now, we can draw inferences to what these animals were like at the beginning of the selection study some years ago. This assumption is based on the stability of inbred strains over generations, and while this may be difficult to prove, we believe it to be generally true. Still, with the caution that genetic drift could account for some of our findings, this approach may be useful in understanding the events which occurred early in a study such as the LS/SS selection. In summary, the sleep time responses of the eight inbred strains spaned a wide range of values. These sleep time differences were apparently not due to intrinsic CNS sensitivity differences among the strains since they all awoke at approximately the same blood and brain levels of ethanol regardless of their sleep times. Figure 3 shows that there was a trend for animals with the longest sleep times to awake with lower ethanol levels than those animals with short sleep times, but this relationship was not statistically significant. In contrast to the inbreds, the extended sleep time of the LS mice and the brief sleep time of the SS mice are associated with very low, and very high waking ethanol levels, respectively. These results are clearly indicative of marked CNS sensitivity differences of these mice to ethanol. Since the differences in sensitivity to ethanol among the inbred strains examined were small, the extreme CNS sensitivities of the LS and SS mice cannot be traced back to one inbred more than any other. The CNS sensitivity difference in the LS and SS must have arisen exclusively via the selection process by changes in allelic frequencies of those genes conferring ethanol sensitivity and resistance.
62
S M O L E N , S M O L E N A N D VAN DE K A M P ACKNOWLEDGEMENTS This work was supported in part by grants from the National Institute of Neurological and Communicative Disorders and Stroke, NS 20748 (A.S.), the National Institute of Child Health and Human Development, HD 21709 (A.S.) and the National Institute on Alcohol Abuse and Alcoholism, AA 06487 and AA 06527 (T.N.S). Preparation of this manuscript was facilitated by a grant from the Biomedical Research Support Grant Program, RR-07013-20 awarded to the University of Colorado, Boulder, CO. The authors wish to thank Dr. Richard A, Deitrich of the University of Colorado Alcohol Research Center for providing some of the AKR, RIII and ISBI mice for these studies.
REFERENCES 1. Baker, R. L., T. N. Smolen, A. Smolen and R. A, Deitrich. Relationship between acute ethanol related responses in long sleep and short sleep mice. Alcoholism: Clin Exp Res, in press. 2. Collins, A. C. A review of research using Short-Sleep and Long-Sleep mice. In: The Development of Animal Models as Pharmacogenetic Tools, edited by G. E. McClearn, R. A. Deitrich and V. G. Erwin. DHHS Publication No. (ADM 81-1133). Washington, DC: U.S. Government Printing Office, 1981, pp. 161-170. 3. Dudek, B. C. and M. E. Abbott. A biometricat genetic analysis of ethanol response in selectively bred long-sleep and shortsleep mice. Behav Genet 14: 1-19, 1984. 4. Gilliam, D. M. and A. C. Collins. Concentration-dependent effects of ethanol in long-sleep and short-sleep mice. Ah'oholism: Clin Exp Res 7: 337-342, 1983. 5. McClearn, G. E. and R. Kakihana. Selective breeding for ethanol sensitivity: SS and LS mice. In: The Development of Animal Models as Pharmacogenetic Tools, edited by G. E. McClearn, R. A. Deitrich and V. G. Erwin. DHHS Publication No. (ADM 81-1133). Washington, DC: U.S. Government Printing Office, 1981, pp. 147-159. 6. McClearn, G. E., J. R. Wilson and W. Meridith. In: Contributions to Behavior-Genetic Analysis: The Mouse as a Prototype. edited by G. Lindzey and D. D. Thiessen. New York: Appleton-Century-Crofts, 1970, pp. 3-22.
7. Mellanby, E. Medical Research Council (Great Britain), Special Report Series No. 31, London, 1919. 8. Phillips, T. J., D. M. Gilliam and B, C. Dudek. An evaluation of the role of ethanol clearance rate in the differential response of long-sleep and short-sleep mice to ethanol. Ah.ohol 1: 373-378, 1984. 9. Riley, V. Adaptation of orbital bleeding technic to rapid serial blood studies. Proc Soc Exp Biol Med 104: 751-754, 1960. 10. Rush, W. A. and R. A. King. Effect of the albino gene on sleep time in mice. Behav Genet 6:116, 1976. 11. Smolen, A., M. Marks, T. N. Smolen and A. C. Collins. Dose and route of administration alter the relative elimination of ethanol by long-sleep and short-sleep mice. Alcoholism: Clin Exp Res 10: 198-204, 1986. 12. Spuhler, K., B. Hoffer, N. Weiner and M. Palmer. Evidence for genetic correlation of hypnotic effects and cerebellar Purkinje neuron depression in response to ethanol in mice." Pharmacol Biochem Behav 17: 569-578, 1982. 13. Tabakoff, B. and R. F. Ritzman. Acute tolerance in inbred and selected lines of mice. Drug Alcohol Depend 4: 87-90, 1979. 14. Tabakoff, B., R. F. Ritzman, T. S. Raju and R. A. Deitrich. Characterization of acute and chronic tolerance in mice selected for inherent differences in sensitivity to ethanol. Alcoholism: Clin I£xp Res 4: 70-73, 1980.