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Vasopressin and ethanol preference. II. Altered preference in two strains of diabetes insipidus rats and nephrogenic diabetes insipidus mice
Peptides, Vol. 6, pp. 677-683, 1985. ~ AnkhoInternationalInc. Printed in the U.S.A.
0196-9781/85$3.00 + .00
Vasopressin and Ethanol Preference. II. Altered Preference in Two Strains of Diabetes Insipidus Rats and Nephrogenic Diabetes Insipidus Mice J O H N C. C R A B B E 1 A N D H E N K R I G T E R 2
C N S P h a r m a c o l o g y D e p a r t m e n t , O r g a n o n , Oss, The N e t h e r l a n d s R e c e i v e d 20 F e b r u a r y 1985 CRABBE, J. C. AND H. RIGTER. Vasopressin and ethanol preference. H. Altered preference of two strains of diabetes insipidus rats and nephrogenic diabetes insipidus mice. PEPTIDES 6(4) 677-683, 1985.--In the first paper of this series, the influence of a single gene (di) for vasopressin deliciency on ethanol intake in rats was demonstrated. We studied preference for concentrations of ethanol between 2.2 and 10 percent versus tap water in Brattleboro rats homozygous for diabetes insipidus (di/di), heterozygous (di/+) or normal (+/+). The di/di rats, totally lacking in vasopressin, had greatly reduced preference scores for all concentrations of ethanol. Their intake of ethanol (g/day) was higher than heterozygotes or normals, but only when 2.2 percent ethanol was offered as a choice. Treatment with vasopressin or related peptides restored ethanol drinking to normal but also corrected water balance [13]. In the experiments reported here, Roman High Avoidance (RHA) rats of three genotypes (+/+, di/+, and di/di) were also tested for ethanol intake and preference with similar but not identical results. Thus, the effects of the di gene are independent of the genetic background on which it is placed to at least some extent. Chlorothiazide, a drug unrelated to vasopressin, also normalized ethanol drinking and corrected water balance in di/di rats. In nephrogenic diabetes insipidus mice, there was a strong negative correlation between severity of polydipsia and preference for ethanol. Thus, no paradigm tested was effective in dissociating polydipsia from reduced ethanol preference and increased ethanol intake. While these results cannot exclude a possible regulatory role for endogenous vasopressin in ethanol preference drinking, they more strongly suggest that reduced preference for ethanol and increased ethanol intake are epiphenomena secondary to a polydipsic state. Vasopressin Des-9-Glycinamide-[Arginines] vasopressin Ethanol preference drinking Diabetes insipidus Diurnal rhythm Pharmacogenetics Os mice Nephrogenic diabetes insipidus Chlorothiazide ence also restored water balance. These studies also revealed a surprising sensitivity to the antidiuretic effect of DGAVP in di/di Brattleboro rats, and they suggested that reduced ethanol preference was secondary to the polydipsia marking diabetes insipidus [13]. Exploration of the di/di deficit at the molecular level is just beginning. Schmale and Richter [14] have detected a single deletion in the sequence encoding neurophysin, but a recent report of low concentrations of vasopressin-like immunoreactivity in adrenals [11] suggests that a second gene may be involved. It was recently reported that an mRNA indistinguishable in size and similar in sequence to authentic vasopressin mRNA is present in very low levels in Brattleboro di/di hypothalamus [8]. The incomplete functional understanding of the di/di deficit was recently underscored by a report that postnatal administration of vasopressin to Brattleboro di/di rats had a long-lasting, marked effect on water balance without affecting gross measures of brain development [ 1].
IN the previous paper in this series, we reported that Brattleboro rats, homozygous for the di gene, display reduced preference for weak ethanol solutions, although they drink more ethanol on a gram basis than heterozygous or homozygous normal control rats when offered 2.2 percent ethanol versus water [13]. The effects of this gene have been characterized in the Brattleboro strain, and homozygous di/di Brattleboro rats virtually lack pituitary and brain vasopre.ssin [4,15] and have marked diabetes insipidus. Others have reported that Brattleboro rats are deficient in the development or maintenance of ethanol tolerance [12], but our results tended not to support deficiencies in tolerance as an explanation for the reduced ethanol preference ofdi/di rats. We demonstrated that preference for ethanol solutions could be increased by the administration of lysine vasopressin (LVP) or a fragment of vasopressin, des-9Glycinamide-[Arginines] vasopressin (DGAVP), which has reduced antidiuretic potency in normal rats. All treatments with LVP or DGAVP that successfully normalized ethanol prefer-
1Requests for reprints should be addressed to John C. Crabbe, Research Service (15 IP), Veterans Administration Medical Center, Portland, OR 97201. 2Present address: Gezondheidsraad, P.O. Box 95379, 2509 CJ The Hague, The Netherlands.
677
678
CRABBE A N D RIGTER
Research on the influence of single genes on behavior has demonstrated that a gene's effects may be highly dependent on the background genotype. F o r example, the db 2J gene leads to severe diabetes in C57BL/KsJ inbred mice, while it has greatly reduced effects when examined on the background of the relatively closely-related C57BL/6J mouse [6]. Such a difference in gene action dependent upon background genotype is presumably due to epistatic interactions; that is, the influence of one gene on the expression of another. In the experiments we report in this paper, we asked whether the diminished ethanol preference scores and enhanced ethanol intake in di/di rats were idiosyncratic to the Brattleboro genetic background or whether a similar influence of the di gene would be seen on RHA/N inbred rats. We also determined the effect of DGAVP on di/di rats of the RHA/N strain to see if they shared the enhanced sensitivity to antidiuretic effects of DGAVP seen in Brattleboro di/di rats, or whether we could successfully elevate ethanol preference at doses of D G A V P having no influence on water balance. We then ameliorated the diabetes insipidus with a compound unrelated to vasopressin (chlorothiazide) to see if this restored ethanol intake to normal. Finally, we examined mice with nephrogenic diabetes insipidus to see if polydipsia was inversely correlated with ethanol preference in animals without hypothalamic vasopressin insufficiency. In an attempt to promote clarity and to indicate how the results of one experiment led to the next, each experiment's Methods, Results, and Discussion are treated separately. EXPERIMENT 1: EFFECT OF THE DI GENE AND DES-9GLYCINAMIDE-[ARGININE a] VASOPRESSIN ON ETHANOL DRINKING BY ROMAN HIGH AVOIDANCE RATS
Dr. Carl Hansen of the National Institutes of Health has introduced the di gene, through a regimen of repeated backcrossing, onto the Roman High Avoidance/N (RHA) inbred rat strain. He kindly gave us some breeding stock of R H A di/di, RHA di/+, and R H A + / + . The animals studied were the product of 6 cycles of backcrossing. Animals born in our laboratory were tested for preference for three concentrations of ethanol solution. The results were similar but not identical to those obtained with Brattleboro rats. Method We selected 17 di/di and 20 + / + R H A rats for the experiment. The animals were 4-6 months old. We anesthetized the rats with ether and implanted an Alzet minipump (Model 2001; volume=200 /zl) with sterilized saline or 1 /zg/tzl DGAVP in the nape of the neck. The estimated rate of release of peptide was 780 ng/hr. The number of animals in the DGAVP-treated diMi and + / + groups was 10. There were 10 rats in the saline di/di and 7 rats in the saline + / + group. After surgery, the animals were housed one to a cage and offered a choice between tap water and ethanol. We measured ethanol preference for 6 days. The ethanol concentration was 2.2 percent on Days 1 and 2, 4.6 percent on Days 3 and 4, and 10 percent on Days 5 and 6. A more detailed treatment of the methods employed, calculations of preference ratios, statistical procedures, and related issues is given in the previous paper [13]. Results The groups of R H A rats differed in average daily fluid intake under each ethanol condition, H(2)~>30.6, p<0.001. Placebo-treated diabetes insipidus rats drank excessively.
Peptide treatment reduced fluid intake in diMi rats, although not to the level of + / + control rats. DGAVP also reduced fluid intake in + / + rats (Table 1). Ethanol concentration affected fluid intake in the placebo-treated, H(2)=6.7, p<0.05, but not in the peptide-treated + / + group (H=0.1). The placebo-treated rats drank more under the 4.6 percent condition than under either the 2.2 percent or 10 percent condition. A different pattern of resuRs was found for the di/di animals: ethanol concentration affected intake in the peptide-treated, H(2)= 18.7, p<0.001, but not in the placebo-treated group (H=3.8). The DGAVP-treated diMi rats drank less fluid with increasing ethanol concentration (Table 1). Groups differed in median ethanol preference score for each of the three ethanol concentrations (2.2 percent: H=28.1, p<0.001; 4.6 percent: H=20.6, p<0.001; 10 percent: H(2)=10.6, p<0.01 for each analysis). Under each ethanol condition, the diMi control rats had the lowest ethanol preference scores. DGAVP increased the ethanol preference score in the diabetes insipidus rats but not in the normal rats (Table 1, Fig. 1). There were also large variations among groups in the average amount of ethanol ingested (Table 1). Intake varied among groups under the 2.2 and 4.6 percent condition but not under the 10 percent condition (2.2 percent: H=29.6, p<0.001 ; 4.6 percent: H = 18.1, p<0.001 ; 10 percent: H(3)=5.8, p > 0 . 1 0 in each analysis). The placebo-treated diabetes insipidus rats ingested more ethanol, when offered 2.2 or 4.6 percent solutions, than any of the other groups. D G A V P reduced the intake of ethanol in diabetes insipidus rats, although, under the 2.2 percent condition, not quite to the level of normal rats. The concentration of the ethanol solution affected the amount of ethanol ingested in all groups, H(2)=9.8, p<0.05. Diabetes insipidus control rats decreased intake with increasing concentration. Rats from all other groups increased intake with increasing concentration (Table 1).
Discussion DGAVP largely corrected fluid intake in the diabetes insipidus R H A rats and also reduced drinking in the controls. The peptide enhanced the lowered ethanol preference scores of the di/di rats almost to the level of + / + rats. DGAVP also reduced the amount of ethanol ingested by di/di rats under the 2.2 and 4.6 percent conditions almost to the level of + / + rats. This pattern of results strongly resembles that found for Brattleboro rats [13]. There are a few exceptions. Ethanol preferences of the controls, and of the peptide-treated diabetes insipidus R H A rats, were not as markedly affected by the concentration of the ethanol solution as in Brattleboro rats. Thus, ethanol preference scores of the RHA rats remained relatively high when the concentration of ethanol was increased. The placebo-treated diabetes insipidus RHA rats also seemed to have a higher preference for ethanol than their Brattleboro counterparts. The placebo-treated di/di R H A not only ingested more grams o f ethanol than other R H A groups at the 2.2 percent condition (in agreement with the Brattleboro results) but also at the 4.6 percent condition. Finally, the R H A diMi rats appeared to have a slightly different pattern of sensitivity to D G A V P treatment. DGAVP seemed to be more potent in restoring ethanol preference (and g ethanol intake) to normal levels and tess potent in antidiuretic activity in the R H A di/di rats than in Brattleboro di/di rats. It would be tempting to conclude that the effects of DGAVP in this experiment successfully dissociated
ETHANOL PREFERENCE
AND GENETIC DIABETES INSIPIDUS
679
TABLE 1 THE EFFECT OF DES-9-GLYCINAMIDE-[ARGININE~]VASOPRESSIN (DGAVP) ON TOTAL FLUID INTAKE AND ETHANOL INTAKE IN RHA RATS Group
N
Days 1 and 2 (2.2% ethanol)
Days 3 and 4 (4.6% ethanol)
Days 5 and 6 (10% ethanol)
Average Daily Fluid Intake (ml)
di/di, di/di, +/+, +/+,
placebo DGAVP placebo DGAVP
10 10 7 10
230.9 52.8 31.7 24.8
± ± ± ±
8.1 2.5t¶ 1.0t 0.9t§
232.5 48.2 34.6 24.2
± ± ± ±
6.4 2.0t¶ 1.5t 0.7t¶
250.6 34.7 28.8 23.9
--- 3.6 ± 1.2t¶ ± 0.8t ± 0.7t§
Average Daily Ethanol Intake (g)
diMi, di/di. +/+, +/+,
placebo DGAVP placebo DGAVP
10 10 7 10
2.3 0.6 0.4 0.3
± 0.1 ± 0t¶ ± ± Ot
1.8 1.2 1.0 0.7
Ot
± ± ± ±
0.2 0.1 0.1" Ot~:
0.9 1.3 1.2 1.3
± ± ± ±
0.2 0.1 0.1 0.I
Median Ethanol Preference (Percent Daily Fluid Intake)
diMi. diMi. +/+, +/+,
placebo DGAVP placebo DGAVP
10 10 7 10
59 69* 73* 73*
22 69t~ 82t 84t
7 52t 50t 71t
Mean scores ± SE. di/di = diabetes insipidus; +/+ = normal. Significant difference from diMi placebo group, *p<0.05, tp<0.001. Significant differences from +/+ placebo group, ~p<0.05, §p<0.01, ¶p<0.001, by two-tailed randomization test. DGAVP was given 1.0 p,g//xl.
polydipsia from ethanol preference. We feel that the data suggest that there is a genetic difference in ethanol prefere n c e b e t w e e n B r a t t l e b o r o and R H A rats. H o w e v e r , insofar as the role of vasopressin is c o n c e r n e d , the pattern o f results for the two strains is m o r e similar than different. In o r d e r to dissociate the postulated central effect o f vasopressin on ethanol preference from its peripheral antidiuretic effect, which was less m a r k e d but nonetheless present in this experiment, we turned to a drug not related to vasopressin. EXPERIMENT 2: EFFECTS OF CHLOROTHIAZIDE ON ETHANOL PREFERENCE AND ETHANOL INTAKE We have thus far continued to find that genetic vasopressin d e f e c i e n c y leads to polydipsia, d e c r e a s e d ethanol prefere n c e , and increased absolute ethanol intake. The vasopressin deficiency e x e r t e d roughly the same influence on rats of two different genetic backgrounds, Brattleboro and R H A . A t t e m p t s to c o r r e c t the deficits with vasopressin or a related peptide were not c o m p l e t e l y successful in dissociating the polydipsia from either the m e a s u r e d absolute ethanol intake o r reduced ethanol preference. Since the increased absolute ethanol intake in di/di rats at low ethanol c o n c e n t r a t i o n s makes a taste or toxicitya v e r s i o n threshold that secondarily limits ethanol p r e f e r e n c e unlikely, we c o n s i d e r e d the possibility that the lowered ethanol p r e f e r e n c e scores in diabetes insipidus rats might be associated with their e x c e s s i v e drinking. If true, we would e x p e c t a negative correlation b e t w e e n r a t s ' p r e f e r e n c e scores and the total v o l u m e of fluid drunk. In an earlier e x p e r i m e n t ,
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FIG. 1. Lowered ethanol preferences scores in RHA diabetes insipidus rats and their reversal with Des-9-Glycinamide-[ArginineS] vasopressin. Median ethanol preference scores for di/di-placebo (x--x), di/di-DGAVP ( x - - - - x ) , +/+ placebo (I---O), and +/+ DGAVP (O-----O) rats. di/di=diabetes insipidus; +/+ = normal rats. *,***p<0.05 andp<0.001 for differences from the di/di-placebo group, by two-tailed randomization test. The difference between the +/+ group, on the one hand, and the diMi-DGAVP group, on the other was significant (p <0.05) at the 4.6 percent ethanol concentration.
680 we had tested Brattleboro di/di, d i / + and + / + normal rats for preference for 2.2 percent, 4.6 percent, or 10 percent ethanol versus water [13]. Table 2 shows that there was no significant inverse relationship for any of the groups between the two variables. There was one significant positive correlation coefficient (for the + / + rats under the 2.2 percent condition). However, the di/di groups showed consistent negative correlations between ethanol preference and total volume at each concentration studied. This suggests that such a correlation might be detectable if more animals were studied. In order to dissociate the influence of polydipsia versus vasopressin deficiency on ethanol intake and preference, we next attempted to correct the polydipsia using an agent, chlorothiazide, not related to vasooressin. We used 19 + / + and 11 di/di male R H A rats, varying in age from 6 to 12 months. The rats were offered a choice between tap water and 2.2 percent ethanol for 8 days. On days 5 and 6, we intraperitoneally injected the rats twice with chlorothiazide, 125 mg/kg in a mulgofen-saline suspension, or with placebo, at 0900 and 1600 hr. On Day 7, a single injection was given at 0900 hr. The + / + chlorothiazide group, + / + placebo group, diMi chlorothiazide group, and diMi placebo groups contained 9, 10, 6, and 5 rats, respectively. We did not treat the animals on Day 8.
CRABBE A N D RIGTER TABLE 2 SPEARMAN RANK CORRELATION COEFFICIENTS (r~)FOR THE ASSOCIATIONBETWEEN TOTAL FLUID INTAKE AND ETHANOL PREFERENCE SCORES IN BRATTLEBORO RATS Ethanol Concentration (%)
Group
N
diMi di/+
7
- O.36
2.2
7 7
+0.46 +0.89*
4.6
di/di di/+
+/+
7 7 7
-0.43 +0.29 -0.04
7
-0.30
10
di/di di/+
7
-0. I 1
+/+
7
-0.39
+/+
rs
The values used for the analysis were the median daily ethanol preference score for each rat and the average daily volume of fluid drunk by the animal. Data are from [13], Experiment 1. *p=0.02. diMi: diabetes insipidus Brattleboro rats; di/+: heterozygotes; +/+: normal rats.
Results
During the first 4 days, when the rats were not treated, the diabetes insipidus rats were polydipsic. During the period of treatment with chlorothiazide or placebo, the groups again differed in total fluid intake (p<0.001). Placebo treatment did not affect drinking. However, chlorothiazide reduced the average daily volume of fluid drunk by diabetes insipidus rats and increased the volume drunk by normal rats. During the " w a s h - o u t " day (Day 8) the drinking scores of the chlorothiazide-treated di/di and + / + rats tended to return to baseline levels (Table 3). Diabetes insipidus rats had lowered ethanol preference scores during Days 1-4. Ethanol preference scores also varied among groups during Days 5-7, at the time o f treatment with placebo or chlorothiazide (p<0.001). Chlorothiazide enhanced preference scores of di/di and + / + rats compared to their baseline (Days 1--4) scores. However, direct comparisons between placebo-treated and drug-treated di/di groups or + / + groups did not yield significant differences. Ethanol preference scores decreased within the di/di group when treatment with chlorothiazide was stopped (Day 8) (Table 3). Diabetes insipidus rats ingested more ethanol during Days 1-4 than + / + rats. Chlorothiazide reduced the amount of ethanol ingested by di/di rats and increased the amount ingested by + / + rats. When treatment was stopped scores of + / + rats tended to return to baseline levels (Table 3). Discussion
Chlorothiazide is a benzothiadiazide that exerts diuretic influences in normal individuals principally by increasing renal excretion of sodium and chloride and accompanying water. In diabetes insipidus, chlorothiazide has an established clinical use as an antidiuretic [5]. The antidiuretic effect is possibly due to increased reabsorption in the proximal tubule, in turn causing reduced sodium chloride and water delivery to the distal tubule [5]. Addition of chlorothiazide to the food of diMi Brattleboro rats leads to an approximate 50 percent reduction in their polyuria [3]. This effect is inde-
pendent of both increased sodium elimination and elevated plasma renin activity, and cannot yet be explained to satisfaction [2]. The effects of chlorothiazide in di/di R H A rats resembled those of vasopressin and DGAVP. In control animals, chlorothiazide tended to increase both ethanol preference and ethanol intake slightly. The relative normalization of drinking in vasopressin-deficient animals by administration of a compound unrelated to vasopressin offered the strongest evidence yet that the ethanol preference and gram intake abnormalities in diMi rats are secondary to the polydipsia. In a final experiment, we sought to determine the relationship between ethanol preference, ethanol intake, and polydipsia in an animal model with normal or high vasopressin levels, the nephrogenic diabetes insipidus mouse. EXPERIMENT 3: ETHANOL PREFERENCE AND INTAKE IN NEPHROGENIC DIABETICMICE Method
Mice with a single gene for oligosyndactyly (Os) have been reported to display diabetes insipidus [7,9]. Dr. Heinz Valtin kindly provided us with a breeding stock o f O s mice. The background strain was DI + / + non-severe [7]. DI + / + non-severe and DI O s / + mice were reared in our colonies in Portland, Oregon, and tested for ethanol preference in a two-bottle choice procedure in much the same w a y described above for rats. Mice were individually housed and offered ethanol at 2.2 percent (Days 1-7), 4.6 percent (Days 8--14), and 10 percent (Days 15--21) versus tap water. Three experiments were conducted. Since data were consistent from experiment to experiment, the results were combined 'or the analysis that follow. Results
Variances in the mouse drinking data were homogeneous, so parametric statistical analyses were employed. Daily av-
ETHANOL
PREFERENCE
AND GENETIC DIABETES INSIPIDUS
681
TABLE 3 THE EFFECT OF CHLOROTHIAZIDE ON DRINKING, 2.2 PERCENT ETHANOL PREFERENCE SCORES AND ETHANOL INTAKE IN DIABETES INSIPIDUS AND NORMAL RHA RATS
Group a
N
Average Daily Fluid Intake (ml)
Average Daily Ethanol Intake (g)
Median Daily Ethanol Preference (Percent Daily Fluid Intake)
Days 1--4b
di/di, di/di, +/+, +/+,
placebo CT placebo CT
5 6 10 9
243.1 224.3 32.7~ 36.0~
1.8 1.4 0.5 0.5
± 0.2 ± 0.2 ± 0¢
39.0 41.7 88.3~
± ,0~
88.8~:
± 0.2 ± 0.2*3 ± 0:~ -+ 0.15¶#
35.7 61.33 92.7~ 95.0:~§
± ± ± ±
38.0 41.0 96.5:~ 95.0:~
Days 5-7 b
diMi, diMi, +/+, +/+,
placebo CT placebo CT
5 6 10 9
216.4 76.6t§ 30.4¢ 47.5~§
1.4 0.6 0.5 0.8 Day 8"
di/di, di/di, +/+, +/+,
placebo CT placebo CT
5 6 10 9
231.5 118.0§ 32.6~: 38.2~§
1.3 0.7 0.5 0.6
0.3 0.2 0t 0.1t§
adi/di = diabetes insipidus; + / + = control; CT = chlorothiazide. hDays 1-4 = period prior to treatment; Days 5-7 = period of treatment wittt placebo or chlorothiazide; Day 8 = no treatment given. *, t, $p<0.05, p<0.01 and p<0.001, respectively for differences between a group and the corresponding scores of the diMi-placebo group for that same period (two-tailed randomization test). 3, ¶p<0.05 and p<0.001, respectively for changes within a group relative to the preceding period (Days 5--7 versus Days I-4; and Day 8 versus Days 5-7), by two-tailed sign test. #p<0.001 versus corresponding + / + placebo group, two-tailed randomization test.
TABLE 4 ETHANOL PREFERENCE AND INTAKE IN NEPHROGENIC DIABETES INSIPIDUS (Os/+) MICE Average Daily Ethanol Preference (Percent Daily Fluid Intake)
Group
N
Average Daily Fluid Intake
Average Daily Ethanol Intake (g/kg)
2.2
Os/+ +/+
22 27
33.4 ± 1.3 16.6 ± 1.1t
5.1 ± 0.8 3.4 ± 0.6*
27 ± 4 34 ± 7
4.6
Os/+ +/+
22 27
33.4 ± 1.9 17.1 ± 0.9t
6.6 ± 1.4 6.2 ± 1.3
23 ± 6 31 ± 6
10.0
Os/+ +/+
22 27
34.3 _+ 2.4 17.5 _+ 0.9
7.1 ± 1.5 5.6 ± 1.2
15 ± 5 15 ± 4
Ethanol Concentration
Os/+: nephrogenic diabetes insipidus group, DI Os/+ strain. + / + : homozygous DI + / + Non-Severe control strain. Means ± SE are given for the 7-day test period at each concentration. *p =0.06 versus Os/+ group by analysis of variance. tp<0.001 versus Os/+ group by analysis of variance.
e r a g e s for total v o l u m e d r u n k at e a c h c o n c e n t r a t i o n , e t h a n o l i n t a k e , a n d p r e f e r e n c e ratio are g i v e n in T a b l e 4. O s / + m i c e d r a n k a p p r o x i m a t e l y t w i c e the v o l u m e o f fluid o f c o n t r o l s in
e a c h w e e k o f the test, F = 4 8 . 9 , p < 0 . 0 0 1 . T h e polydipsic mice t e n d e d to h a v e r e d u c e d p r e f e r e n c e for e t h a n o l at the 2.2 a n d 4.6 p e r c e n t c o n c e n t r a t i o n s , b u t t h e s e d i f f e r e n c e s did n o t
682
CRABBE AND RIGTER was a clear negative correlation between total volume and preference in polydipsic animals. Since these animals have normal or even elevated vasopressin concentrations [9,10], the reduced preference could not easily be ascribed to a direct effect of vasopressin. This experiment strongly supported the hypothesis that polydipsia leads to a secondary reduction in preference for ethanol and an increased absolute intake of ethanol.
70
r • -.75 df • 20 p < .001
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GENERAL DISCUSSION
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Average Doily Fluid Intake (ml) FIG. 2. Scatterplot for average daily fluid intake (ml) versus average daily ethanol preference ratio (percent daily fluid intake) for Os/+ nephrogenic diabetes insipidus mice offered a choice between 2.2 percent ethanol and water for 7 days.
reach significance (Table 4). Similarly, Os/+ mice tended to have increased ethanol intake at the lowest concentration (p=0.06) but not at higher concentrations. To assess the possibility that preference for ethanol was negatively correlated with total fluid intake, we correlated the average daily preference ratio versus average daily total fluid drunk for each group for each week. For the 27 control mice, these correlations were -0.13, -0.25, and -0.41, respectively, during weeks 1, 2 and 3. The first two were not significant, and a scatterplot revealed the third week's correlation to be an artifact of two mice with very high total fluid intake. For the 22 Os/+ nephrogenic diabetes insipidus mice, these correlations were -0.75, -0.87, and -0.91, respectively. All were statistically significant (p<0.001). The scatterplot for the data at 2.2 percent ethanol is shown in Fig. 2. Discussion
For mice with a kidney tubule defect leading to high fluid intake, the by-now expected pattern of increased ethanol intake at low concentrations, reduced preference for ethanol, and polydipsia, was clearly demonstrated. Both the polydipsia and the ethanol intake values were not as different from controls, which are themselves mildly diabetic, as those seen in vasopressin-deficient diabetes insipidus rats. While no significant relationship between ethanol preference and total fluid intake was detected in normal animals, there
In other studies, we found that Brattleboro rats homozygous for the di gene leading to diabetes insipidus had elevated gram ethanol intake but reduced ethanol preference when presented with a choice between weak ethanol solutions and water [13]. The current experiments confirmed both results in rats of a different genetic background (RHA/N) onto which the di gene had been introduced by backcrossing. RHA di/di rats showed a similar pattern of reduced ethanol preference to that seen in Brattleboro di/di rats. The di gene may have exerted a stronger influence on the RHA background: RHA di/di rats showed elevated grams-ethanol intake at both 2.2 percent and 4.6 percent concentrations, and they seemed to have a higher preference for 2.2 percent ethanol, than Brattleboro di/di rats. However, in one of our earlier experiments [13] Brattleboro diMi rats had higher preferences for 2.2 percent ethanol, resembling the RHA di/di rats, than in the other three experiments there reported (which were consistently lower). RHA di/di rats also seemed to be relatively more sensitive to DGAVP's effect on ethanol preference than to its effect on water balance, where as DGAVP in Brattleboro di/di rats had generally shown equipotency for both effects. These postulated differences between RHA and Brattleboro di/di rats have appeared consistently in several other unpublished experiments with each strain. However, since the magnitudes of the strain differences are no greater than the interexperiment variability within each strain, and since we have no direct contemporaneous comparisons between Brattleboro and RHA/N di/di rats, these generalizations should be viewed with caution. In sum, the effect of the di gene on ethanol preference and water balance was not identical in two genotypes of rats. It should be emphasized that the RHAJN di/di animals studied were the product of only 6 generations of backcrossing. Other Brattleboro genes closely linked to the di locus were undoubtedly still present in the RHA diMi animals we studied. Backcrossing has now proceeded to a sufficient number of generations that this problem may be considered negligible in future studies. While the pattern of responses we found was similar, the background genotypic differences may fruitfully be exploited in future research. For example, the RHA/N diMi rats would seem to be a better strain in which to study the effects of other vasopressin-related peptides in further attempts to dissociate polydipsia from abnormalities in ethanol intake. Clearly, our results caution against hastily generalizing about the "effects of a gene" without consideration of the background on which it is expressed, While we expect that RHA/N di/di rats will prove to have the same pattern of a single deletion in the sequence encoding the neurophysin for vasopressin [14] and detectable (but very low) levels of presumptive vasopressin mRNA in hypothalamus [8] reported for Brattleboro diMi rats, the regulation of gene activity could easily differ in the genotypes.
ETHANOL PREFERENCE AND GENETIC DIABETES INSIPIDUS In other studies [13], we found that treatment o f Brattleboro di/di rats with vasopressin or a related peptide was only effective in normalizing ethanol intake at doses that restored water balance. The current experiments showed that restoration o f water balance itself was sufficient to normalize ethanol intake. Chlorothiazide, a drug unrelated to vasopressin, reduced gram ethanol intake and elevated preference in R H A / N di/di rats. Furthermore, and most telling, mice with nephrogenic diabetes insipidus, who have normal or elevated vasopressin levels, also showed reduced preference for ethanol and increased absolute intake of ethanol. Within the diabetic groups of mice, a clear negative correlation between ethanol preference and total fluid intake strongly supports the hypothesis that the abnormalities of ethanol intake are secondary to the water balance disturbance. We must conclude that the mechanisms underlying the ethanol intake abnormalities of diabetes insipidus animals remain obscure. The most likely hypothesis is that a toxic threshold for ethanol is reached in diabetes insipidus animals which leads them to avoid ethanol solutions. In our earlier
683
paper [13] we discussed those aspects of our data not entirely consistent with this hypothesis. Although the elevated absolute intake of ethanol seen in di animals offered 2.2 percent ethanol versus water could be interpreted as indicating increased preference for ethanol, we do not feel that this is likely. The absolute intake is elevated only at 2.2 percent concentrations (also 4.6 percent for R H A / N di/di) and is reduced in all rats at 10 percent concentrations. Future studies will be needed to determine the nature o f the relationships among vasopressin, polydispsia, and ethanol intake.
ACKNOWLEDGEMENTS Part of the data were reported at the International Symposium on the Brattleboro Rat, Hanover, New Hampshire, 5-7 September 1981. The authors thank Yvonne Mous and Helen Hall for secretarial assistance, Chris Dortmans, Huub Rijk, Emmett R. Young, Brenda R. Tam and Ann Kosobud for help with the experiments, and an anonymous reviewer for helpful critical comments. These experiments were supported in part by Grant AA05828 from the NIAAA to JCC.
REFERENCES 1. Boer, G. J,, R. Kragten, J. Kruisbrink and D. F. Swaab. Vasopressin fails to restore postnatally the stunted brain development in the Brattleboro rat, but affects water metabolism permanently. Neurobehav Toxieol Teratol 6: 103-109, 1984. 2, DeGroat, C. The role of renin in the paradoxical antipolyuric effects of chlorothiazide in the homozygous Brattleboro rat. Ann N Y Acad Sci 394: 259-364, 1982. 3. DeGroat, C. and A. Tijssen. The effects of long-term oral treatment with chlorothiazide or furosemide on hereditary diabetes insipidus in rats. Arch lnt Pharmacodyn Ther 237: 75-87, 1979. 4. Dorsa, D. M. and L. C. Bottemiller. Age-related changes of vasopressin content of microdissected areas of rat brain. Brain Res 242: 151-156, 1982. 5. Gilman, A. G., L. S. Goodman and A. Gilman. The Pharmacological Basis of Therapeutics, sixth edition. New York: MacMillan Pub. Co., Inc., 1980, pp. 925-926. 6. Hummel, K. P., D. L. Coleman and P. W. Lane. The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BL/KsJ and C57BL/6J strains. Biochem Genet 7: 1-13, 1972. 7. Kettyle, W. M. and H. Valtin. Chemical and dimensional characterization of the renal countercurrent system in mice. Kidney lnt 1: 135-144, 1972. 8. Majzoub, J. A., A. Pappey, R. Burg and J. F. Habener. Vasopressin gene is expressed at low levels in the hypothalamus of the Brattleboro rat. Proc Natl Acad Sci USA, gl: 5296--5299, 1984.
9. Naik, D. Salt and water metabolism and neurohypophyseal vasopressor activity in mice with hereditary nephrngenic diabetes insipidus. Acta Endocrinol (Copenh) 69:434 4A,4, 1972. 10. Naik, D. and H. Kobayashi. Neurohypophysial hormones in the pars nervosa of the mouse with hereditary nephrogenic diabetes insipidus. Neuroendocrinology 7: 322-328, 1971. ! 1. Nussey, S. S., V. T. Y. Ang, J. S. Jenkins, H. S. Chowdrey and G. W. Bisset. Brattleboro rat adrenal contains vasopressin. Nature 310: 64-66, 1984. 12. Pittman, Q. J., J. Rogers and F. E. Bloom. Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol. Regul Pept4: 33-41, 1982. 13. Rigter, H. and J. C. Crabbe. Vasopressin and ethanol preference. I. Effects of vasopressin and the fragment DGAVP on altered ethanol preference in Brattleboro diabetes insipidus rats. Peptides 6: 669-676, 1985. 14. Schmale, H. and D. Richter. Single base deletion in the vasopressin gene is the cause of the diabetes insipidus in Brattleboro rats. Nature 308: 705-709, 1984. 15. Valtin, H. The discovery of the Brattleboro rat, recommended nomenclature, and the question of proper controls. Ann N Y Acad Sci 394: 1-9, 1982.
0196-9781/85$3.00 + .00
Vasopressin and Ethanol Preference. II. Altered Preference in Two Strains of Diabetes Insipidus Rats and Nephrogenic Diabetes Insipidus Mice J O H N C. C R A B B E 1 A N D H E N K R I G T E R 2
C N S P h a r m a c o l o g y D e p a r t m e n t , O r g a n o n , Oss, The N e t h e r l a n d s R e c e i v e d 20 F e b r u a r y 1985 CRABBE, J. C. AND H. RIGTER. Vasopressin and ethanol preference. H. Altered preference of two strains of diabetes insipidus rats and nephrogenic diabetes insipidus mice. PEPTIDES 6(4) 677-683, 1985.--In the first paper of this series, the influence of a single gene (di) for vasopressin deliciency on ethanol intake in rats was demonstrated. We studied preference for concentrations of ethanol between 2.2 and 10 percent versus tap water in Brattleboro rats homozygous for diabetes insipidus (di/di), heterozygous (di/+) or normal (+/+). The di/di rats, totally lacking in vasopressin, had greatly reduced preference scores for all concentrations of ethanol. Their intake of ethanol (g/day) was higher than heterozygotes or normals, but only when 2.2 percent ethanol was offered as a choice. Treatment with vasopressin or related peptides restored ethanol drinking to normal but also corrected water balance [13]. In the experiments reported here, Roman High Avoidance (RHA) rats of three genotypes (+/+, di/+, and di/di) were also tested for ethanol intake and preference with similar but not identical results. Thus, the effects of the di gene are independent of the genetic background on which it is placed to at least some extent. Chlorothiazide, a drug unrelated to vasopressin, also normalized ethanol drinking and corrected water balance in di/di rats. In nephrogenic diabetes insipidus mice, there was a strong negative correlation between severity of polydipsia and preference for ethanol. Thus, no paradigm tested was effective in dissociating polydipsia from reduced ethanol preference and increased ethanol intake. While these results cannot exclude a possible regulatory role for endogenous vasopressin in ethanol preference drinking, they more strongly suggest that reduced preference for ethanol and increased ethanol intake are epiphenomena secondary to a polydipsic state. Vasopressin Des-9-Glycinamide-[Arginines] vasopressin Ethanol preference drinking Diabetes insipidus Diurnal rhythm Pharmacogenetics Os mice Nephrogenic diabetes insipidus Chlorothiazide ence also restored water balance. These studies also revealed a surprising sensitivity to the antidiuretic effect of DGAVP in di/di Brattleboro rats, and they suggested that reduced ethanol preference was secondary to the polydipsia marking diabetes insipidus [13]. Exploration of the di/di deficit at the molecular level is just beginning. Schmale and Richter [14] have detected a single deletion in the sequence encoding neurophysin, but a recent report of low concentrations of vasopressin-like immunoreactivity in adrenals [11] suggests that a second gene may be involved. It was recently reported that an mRNA indistinguishable in size and similar in sequence to authentic vasopressin mRNA is present in very low levels in Brattleboro di/di hypothalamus [8]. The incomplete functional understanding of the di/di deficit was recently underscored by a report that postnatal administration of vasopressin to Brattleboro di/di rats had a long-lasting, marked effect on water balance without affecting gross measures of brain development [ 1].
IN the previous paper in this series, we reported that Brattleboro rats, homozygous for the di gene, display reduced preference for weak ethanol solutions, although they drink more ethanol on a gram basis than heterozygous or homozygous normal control rats when offered 2.2 percent ethanol versus water [13]. The effects of this gene have been characterized in the Brattleboro strain, and homozygous di/di Brattleboro rats virtually lack pituitary and brain vasopre.ssin [4,15] and have marked diabetes insipidus. Others have reported that Brattleboro rats are deficient in the development or maintenance of ethanol tolerance [12], but our results tended not to support deficiencies in tolerance as an explanation for the reduced ethanol preference ofdi/di rats. We demonstrated that preference for ethanol solutions could be increased by the administration of lysine vasopressin (LVP) or a fragment of vasopressin, des-9Glycinamide-[Arginines] vasopressin (DGAVP), which has reduced antidiuretic potency in normal rats. All treatments with LVP or DGAVP that successfully normalized ethanol prefer-
1Requests for reprints should be addressed to John C. Crabbe, Research Service (15 IP), Veterans Administration Medical Center, Portland, OR 97201. 2Present address: Gezondheidsraad, P.O. Box 95379, 2509 CJ The Hague, The Netherlands.
677
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CRABBE A N D RIGTER
Research on the influence of single genes on behavior has demonstrated that a gene's effects may be highly dependent on the background genotype. F o r example, the db 2J gene leads to severe diabetes in C57BL/KsJ inbred mice, while it has greatly reduced effects when examined on the background of the relatively closely-related C57BL/6J mouse [6]. Such a difference in gene action dependent upon background genotype is presumably due to epistatic interactions; that is, the influence of one gene on the expression of another. In the experiments we report in this paper, we asked whether the diminished ethanol preference scores and enhanced ethanol intake in di/di rats were idiosyncratic to the Brattleboro genetic background or whether a similar influence of the di gene would be seen on RHA/N inbred rats. We also determined the effect of DGAVP on di/di rats of the RHA/N strain to see if they shared the enhanced sensitivity to antidiuretic effects of DGAVP seen in Brattleboro di/di rats, or whether we could successfully elevate ethanol preference at doses of D G A V P having no influence on water balance. We then ameliorated the diabetes insipidus with a compound unrelated to vasopressin (chlorothiazide) to see if this restored ethanol intake to normal. Finally, we examined mice with nephrogenic diabetes insipidus to see if polydipsia was inversely correlated with ethanol preference in animals without hypothalamic vasopressin insufficiency. In an attempt to promote clarity and to indicate how the results of one experiment led to the next, each experiment's Methods, Results, and Discussion are treated separately. EXPERIMENT 1: EFFECT OF THE DI GENE AND DES-9GLYCINAMIDE-[ARGININE a] VASOPRESSIN ON ETHANOL DRINKING BY ROMAN HIGH AVOIDANCE RATS
Dr. Carl Hansen of the National Institutes of Health has introduced the di gene, through a regimen of repeated backcrossing, onto the Roman High Avoidance/N (RHA) inbred rat strain. He kindly gave us some breeding stock of R H A di/di, RHA di/+, and R H A + / + . The animals studied were the product of 6 cycles of backcrossing. Animals born in our laboratory were tested for preference for three concentrations of ethanol solution. The results were similar but not identical to those obtained with Brattleboro rats. Method We selected 17 di/di and 20 + / + R H A rats for the experiment. The animals were 4-6 months old. We anesthetized the rats with ether and implanted an Alzet minipump (Model 2001; volume=200 /zl) with sterilized saline or 1 /zg/tzl DGAVP in the nape of the neck. The estimated rate of release of peptide was 780 ng/hr. The number of animals in the DGAVP-treated diMi and + / + groups was 10. There were 10 rats in the saline di/di and 7 rats in the saline + / + group. After surgery, the animals were housed one to a cage and offered a choice between tap water and ethanol. We measured ethanol preference for 6 days. The ethanol concentration was 2.2 percent on Days 1 and 2, 4.6 percent on Days 3 and 4, and 10 percent on Days 5 and 6. A more detailed treatment of the methods employed, calculations of preference ratios, statistical procedures, and related issues is given in the previous paper [13]. Results The groups of R H A rats differed in average daily fluid intake under each ethanol condition, H(2)~>30.6, p<0.001. Placebo-treated diabetes insipidus rats drank excessively.
Peptide treatment reduced fluid intake in diMi rats, although not to the level of + / + control rats. DGAVP also reduced fluid intake in + / + rats (Table 1). Ethanol concentration affected fluid intake in the placebo-treated, H(2)=6.7, p<0.05, but not in the peptide-treated + / + group (H=0.1). The placebo-treated rats drank more under the 4.6 percent condition than under either the 2.2 percent or 10 percent condition. A different pattern of resuRs was found for the di/di animals: ethanol concentration affected intake in the peptide-treated, H(2)= 18.7, p<0.001, but not in the placebo-treated group (H=3.8). The DGAVP-treated diMi rats drank less fluid with increasing ethanol concentration (Table 1). Groups differed in median ethanol preference score for each of the three ethanol concentrations (2.2 percent: H=28.1, p<0.001; 4.6 percent: H=20.6, p<0.001; 10 percent: H(2)=10.6, p<0.01 for each analysis). Under each ethanol condition, the diMi control rats had the lowest ethanol preference scores. DGAVP increased the ethanol preference score in the diabetes insipidus rats but not in the normal rats (Table 1, Fig. 1). There were also large variations among groups in the average amount of ethanol ingested (Table 1). Intake varied among groups under the 2.2 and 4.6 percent condition but not under the 10 percent condition (2.2 percent: H=29.6, p<0.001 ; 4.6 percent: H = 18.1, p<0.001 ; 10 percent: H(3)=5.8, p > 0 . 1 0 in each analysis). The placebo-treated diabetes insipidus rats ingested more ethanol, when offered 2.2 or 4.6 percent solutions, than any of the other groups. D G A V P reduced the intake of ethanol in diabetes insipidus rats, although, under the 2.2 percent condition, not quite to the level of normal rats. The concentration of the ethanol solution affected the amount of ethanol ingested in all groups, H(2)=9.8, p<0.05. Diabetes insipidus control rats decreased intake with increasing concentration. Rats from all other groups increased intake with increasing concentration (Table 1).
Discussion DGAVP largely corrected fluid intake in the diabetes insipidus R H A rats and also reduced drinking in the controls. The peptide enhanced the lowered ethanol preference scores of the di/di rats almost to the level of + / + rats. DGAVP also reduced the amount of ethanol ingested by di/di rats under the 2.2 and 4.6 percent conditions almost to the level of + / + rats. This pattern of results strongly resembles that found for Brattleboro rats [13]. There are a few exceptions. Ethanol preferences of the controls, and of the peptide-treated diabetes insipidus R H A rats, were not as markedly affected by the concentration of the ethanol solution as in Brattleboro rats. Thus, ethanol preference scores of the RHA rats remained relatively high when the concentration of ethanol was increased. The placebo-treated diabetes insipidus RHA rats also seemed to have a higher preference for ethanol than their Brattleboro counterparts. The placebo-treated di/di R H A not only ingested more grams o f ethanol than other R H A groups at the 2.2 percent condition (in agreement with the Brattleboro results) but also at the 4.6 percent condition. Finally, the R H A diMi rats appeared to have a slightly different pattern of sensitivity to D G A V P treatment. DGAVP seemed to be more potent in restoring ethanol preference (and g ethanol intake) to normal levels and tess potent in antidiuretic activity in the R H A di/di rats than in Brattleboro di/di rats. It would be tempting to conclude that the effects of DGAVP in this experiment successfully dissociated
ETHANOL PREFERENCE
AND GENETIC DIABETES INSIPIDUS
679
TABLE 1 THE EFFECT OF DES-9-GLYCINAMIDE-[ARGININE~]VASOPRESSIN (DGAVP) ON TOTAL FLUID INTAKE AND ETHANOL INTAKE IN RHA RATS Group
N
Days 1 and 2 (2.2% ethanol)
Days 3 and 4 (4.6% ethanol)
Days 5 and 6 (10% ethanol)
Average Daily Fluid Intake (ml)
di/di, di/di, +/+, +/+,
placebo DGAVP placebo DGAVP
10 10 7 10
230.9 52.8 31.7 24.8
± ± ± ±
8.1 2.5t¶ 1.0t 0.9t§
232.5 48.2 34.6 24.2
± ± ± ±
6.4 2.0t¶ 1.5t 0.7t¶
250.6 34.7 28.8 23.9
--- 3.6 ± 1.2t¶ ± 0.8t ± 0.7t§
Average Daily Ethanol Intake (g)
diMi, di/di. +/+, +/+,
placebo DGAVP placebo DGAVP
10 10 7 10
2.3 0.6 0.4 0.3
± 0.1 ± 0t¶ ± ± Ot
1.8 1.2 1.0 0.7
Ot
± ± ± ±
0.2 0.1 0.1" Ot~:
0.9 1.3 1.2 1.3
± ± ± ±
0.2 0.1 0.1 0.I
Median Ethanol Preference (Percent Daily Fluid Intake)
diMi. diMi. +/+, +/+,
placebo DGAVP placebo DGAVP
10 10 7 10
59 69* 73* 73*
22 69t~ 82t 84t
7 52t 50t 71t
Mean scores ± SE. di/di = diabetes insipidus; +/+ = normal. Significant difference from diMi placebo group, *p<0.05, tp<0.001. Significant differences from +/+ placebo group, ~p<0.05, §p<0.01, ¶p<0.001, by two-tailed randomization test. DGAVP was given 1.0 p,g//xl.
polydipsia from ethanol preference. We feel that the data suggest that there is a genetic difference in ethanol prefere n c e b e t w e e n B r a t t l e b o r o and R H A rats. H o w e v e r , insofar as the role of vasopressin is c o n c e r n e d , the pattern o f results for the two strains is m o r e similar than different. In o r d e r to dissociate the postulated central effect o f vasopressin on ethanol preference from its peripheral antidiuretic effect, which was less m a r k e d but nonetheless present in this experiment, we turned to a drug not related to vasopressin. EXPERIMENT 2: EFFECTS OF CHLOROTHIAZIDE ON ETHANOL PREFERENCE AND ETHANOL INTAKE We have thus far continued to find that genetic vasopressin d e f e c i e n c y leads to polydipsia, d e c r e a s e d ethanol prefere n c e , and increased absolute ethanol intake. The vasopressin deficiency e x e r t e d roughly the same influence on rats of two different genetic backgrounds, Brattleboro and R H A . A t t e m p t s to c o r r e c t the deficits with vasopressin or a related peptide were not c o m p l e t e l y successful in dissociating the polydipsia from either the m e a s u r e d absolute ethanol intake o r reduced ethanol preference. Since the increased absolute ethanol intake in di/di rats at low ethanol c o n c e n t r a t i o n s makes a taste or toxicitya v e r s i o n threshold that secondarily limits ethanol p r e f e r e n c e unlikely, we c o n s i d e r e d the possibility that the lowered ethanol p r e f e r e n c e scores in diabetes insipidus rats might be associated with their e x c e s s i v e drinking. If true, we would e x p e c t a negative correlation b e t w e e n r a t s ' p r e f e r e n c e scores and the total v o l u m e of fluid drunk. In an earlier e x p e r i m e n t ,
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FIG. 1. Lowered ethanol preferences scores in RHA diabetes insipidus rats and their reversal with Des-9-Glycinamide-[ArginineS] vasopressin. Median ethanol preference scores for di/di-placebo (x--x), di/di-DGAVP ( x - - - - x ) , +/+ placebo (I---O), and +/+ DGAVP (O-----O) rats. di/di=diabetes insipidus; +/+ = normal rats. *,***p<0.05 andp<0.001 for differences from the di/di-placebo group, by two-tailed randomization test. The difference between the +/+ group, on the one hand, and the diMi-DGAVP group, on the other was significant (p <0.05) at the 4.6 percent ethanol concentration.
680 we had tested Brattleboro di/di, d i / + and + / + normal rats for preference for 2.2 percent, 4.6 percent, or 10 percent ethanol versus water [13]. Table 2 shows that there was no significant inverse relationship for any of the groups between the two variables. There was one significant positive correlation coefficient (for the + / + rats under the 2.2 percent condition). However, the di/di groups showed consistent negative correlations between ethanol preference and total volume at each concentration studied. This suggests that such a correlation might be detectable if more animals were studied. In order to dissociate the influence of polydipsia versus vasopressin deficiency on ethanol intake and preference, we next attempted to correct the polydipsia using an agent, chlorothiazide, not related to vasooressin. We used 19 + / + and 11 di/di male R H A rats, varying in age from 6 to 12 months. The rats were offered a choice between tap water and 2.2 percent ethanol for 8 days. On days 5 and 6, we intraperitoneally injected the rats twice with chlorothiazide, 125 mg/kg in a mulgofen-saline suspension, or with placebo, at 0900 and 1600 hr. On Day 7, a single injection was given at 0900 hr. The + / + chlorothiazide group, + / + placebo group, diMi chlorothiazide group, and diMi placebo groups contained 9, 10, 6, and 5 rats, respectively. We did not treat the animals on Day 8.
CRABBE A N D RIGTER TABLE 2 SPEARMAN RANK CORRELATION COEFFICIENTS (r~)FOR THE ASSOCIATIONBETWEEN TOTAL FLUID INTAKE AND ETHANOL PREFERENCE SCORES IN BRATTLEBORO RATS Ethanol Concentration (%)
Group
N
diMi di/+
7
- O.36
2.2
7 7
+0.46 +0.89*
4.6
di/di di/+
+/+
7 7 7
-0.43 +0.29 -0.04
7
-0.30
10
di/di di/+
7
-0. I 1
+/+
7
-0.39
+/+
rs
The values used for the analysis were the median daily ethanol preference score for each rat and the average daily volume of fluid drunk by the animal. Data are from [13], Experiment 1. *p=0.02. diMi: diabetes insipidus Brattleboro rats; di/+: heterozygotes; +/+: normal rats.
Results
During the first 4 days, when the rats were not treated, the diabetes insipidus rats were polydipsic. During the period of treatment with chlorothiazide or placebo, the groups again differed in total fluid intake (p<0.001). Placebo treatment did not affect drinking. However, chlorothiazide reduced the average daily volume of fluid drunk by diabetes insipidus rats and increased the volume drunk by normal rats. During the " w a s h - o u t " day (Day 8) the drinking scores of the chlorothiazide-treated di/di and + / + rats tended to return to baseline levels (Table 3). Diabetes insipidus rats had lowered ethanol preference scores during Days 1-4. Ethanol preference scores also varied among groups during Days 5-7, at the time o f treatment with placebo or chlorothiazide (p<0.001). Chlorothiazide enhanced preference scores of di/di and + / + rats compared to their baseline (Days 1--4) scores. However, direct comparisons between placebo-treated and drug-treated di/di groups or + / + groups did not yield significant differences. Ethanol preference scores decreased within the di/di group when treatment with chlorothiazide was stopped (Day 8) (Table 3). Diabetes insipidus rats ingested more ethanol during Days 1-4 than + / + rats. Chlorothiazide reduced the amount of ethanol ingested by di/di rats and increased the amount ingested by + / + rats. When treatment was stopped scores of + / + rats tended to return to baseline levels (Table 3). Discussion
Chlorothiazide is a benzothiadiazide that exerts diuretic influences in normal individuals principally by increasing renal excretion of sodium and chloride and accompanying water. In diabetes insipidus, chlorothiazide has an established clinical use as an antidiuretic [5]. The antidiuretic effect is possibly due to increased reabsorption in the proximal tubule, in turn causing reduced sodium chloride and water delivery to the distal tubule [5]. Addition of chlorothiazide to the food of diMi Brattleboro rats leads to an approximate 50 percent reduction in their polyuria [3]. This effect is inde-
pendent of both increased sodium elimination and elevated plasma renin activity, and cannot yet be explained to satisfaction [2]. The effects of chlorothiazide in di/di R H A rats resembled those of vasopressin and DGAVP. In control animals, chlorothiazide tended to increase both ethanol preference and ethanol intake slightly. The relative normalization of drinking in vasopressin-deficient animals by administration of a compound unrelated to vasopressin offered the strongest evidence yet that the ethanol preference and gram intake abnormalities in diMi rats are secondary to the polydipsia. In a final experiment, we sought to determine the relationship between ethanol preference, ethanol intake, and polydipsia in an animal model with normal or high vasopressin levels, the nephrogenic diabetes insipidus mouse. EXPERIMENT 3: ETHANOL PREFERENCE AND INTAKE IN NEPHROGENIC DIABETICMICE Method
Mice with a single gene for oligosyndactyly (Os) have been reported to display diabetes insipidus [7,9]. Dr. Heinz Valtin kindly provided us with a breeding stock o f O s mice. The background strain was DI + / + non-severe [7]. DI + / + non-severe and DI O s / + mice were reared in our colonies in Portland, Oregon, and tested for ethanol preference in a two-bottle choice procedure in much the same w a y described above for rats. Mice were individually housed and offered ethanol at 2.2 percent (Days 1-7), 4.6 percent (Days 8--14), and 10 percent (Days 15--21) versus tap water. Three experiments were conducted. Since data were consistent from experiment to experiment, the results were combined 'or the analysis that follow. Results
Variances in the mouse drinking data were homogeneous, so parametric statistical analyses were employed. Daily av-
ETHANOL
PREFERENCE
AND GENETIC DIABETES INSIPIDUS
681
TABLE 3 THE EFFECT OF CHLOROTHIAZIDE ON DRINKING, 2.2 PERCENT ETHANOL PREFERENCE SCORES AND ETHANOL INTAKE IN DIABETES INSIPIDUS AND NORMAL RHA RATS
Group a
N
Average Daily Fluid Intake (ml)
Average Daily Ethanol Intake (g)
Median Daily Ethanol Preference (Percent Daily Fluid Intake)
Days 1--4b
di/di, di/di, +/+, +/+,
placebo CT placebo CT
5 6 10 9
243.1 224.3 32.7~ 36.0~
1.8 1.4 0.5 0.5
± 0.2 ± 0.2 ± 0¢
39.0 41.7 88.3~
± ,0~
88.8~:
± 0.2 ± 0.2*3 ± 0:~ -+ 0.15¶#
35.7 61.33 92.7~ 95.0:~§
± ± ± ±
38.0 41.0 96.5:~ 95.0:~
Days 5-7 b
diMi, diMi, +/+, +/+,
placebo CT placebo CT
5 6 10 9
216.4 76.6t§ 30.4¢ 47.5~§
1.4 0.6 0.5 0.8 Day 8"
di/di, di/di, +/+, +/+,
placebo CT placebo CT
5 6 10 9
231.5 118.0§ 32.6~: 38.2~§
1.3 0.7 0.5 0.6
0.3 0.2 0t 0.1t§
adi/di = diabetes insipidus; + / + = control; CT = chlorothiazide. hDays 1-4 = period prior to treatment; Days 5-7 = period of treatment wittt placebo or chlorothiazide; Day 8 = no treatment given. *, t, $p<0.05, p<0.01 and p<0.001, respectively for differences between a group and the corresponding scores of the diMi-placebo group for that same period (two-tailed randomization test). 3, ¶p<0.05 and p<0.001, respectively for changes within a group relative to the preceding period (Days 5--7 versus Days I-4; and Day 8 versus Days 5-7), by two-tailed sign test. #p<0.001 versus corresponding + / + placebo group, two-tailed randomization test.
TABLE 4 ETHANOL PREFERENCE AND INTAKE IN NEPHROGENIC DIABETES INSIPIDUS (Os/+) MICE Average Daily Ethanol Preference (Percent Daily Fluid Intake)
Group
N
Average Daily Fluid Intake
Average Daily Ethanol Intake (g/kg)
2.2
Os/+ +/+
22 27
33.4 ± 1.3 16.6 ± 1.1t
5.1 ± 0.8 3.4 ± 0.6*
27 ± 4 34 ± 7
4.6
Os/+ +/+
22 27
33.4 ± 1.9 17.1 ± 0.9t
6.6 ± 1.4 6.2 ± 1.3
23 ± 6 31 ± 6
10.0
Os/+ +/+
22 27
34.3 _+ 2.4 17.5 _+ 0.9
7.1 ± 1.5 5.6 ± 1.2
15 ± 5 15 ± 4
Ethanol Concentration
Os/+: nephrogenic diabetes insipidus group, DI Os/+ strain. + / + : homozygous DI + / + Non-Severe control strain. Means ± SE are given for the 7-day test period at each concentration. *p =0.06 versus Os/+ group by analysis of variance. tp<0.001 versus Os/+ group by analysis of variance.
e r a g e s for total v o l u m e d r u n k at e a c h c o n c e n t r a t i o n , e t h a n o l i n t a k e , a n d p r e f e r e n c e ratio are g i v e n in T a b l e 4. O s / + m i c e d r a n k a p p r o x i m a t e l y t w i c e the v o l u m e o f fluid o f c o n t r o l s in
e a c h w e e k o f the test, F = 4 8 . 9 , p < 0 . 0 0 1 . T h e polydipsic mice t e n d e d to h a v e r e d u c e d p r e f e r e n c e for e t h a n o l at the 2.2 a n d 4.6 p e r c e n t c o n c e n t r a t i o n s , b u t t h e s e d i f f e r e n c e s did n o t
682
CRABBE AND RIGTER was a clear negative correlation between total volume and preference in polydipsic animals. Since these animals have normal or even elevated vasopressin concentrations [9,10], the reduced preference could not easily be ascribed to a direct effect of vasopressin. This experiment strongly supported the hypothesis that polydipsia leads to a secondary reduction in preference for ethanol and an increased absolute intake of ethanol.
70
r • -.75 df • 20 p < .001
60
50 C
GENERAL DISCUSSION
~
40
ot O ¢-
.~
o ~
2O
I0 •
• •
~4 2'6
IO
1
32
36
• I
I
40 42
l
46
Average Doily Fluid Intake (ml) FIG. 2. Scatterplot for average daily fluid intake (ml) versus average daily ethanol preference ratio (percent daily fluid intake) for Os/+ nephrogenic diabetes insipidus mice offered a choice between 2.2 percent ethanol and water for 7 days.
reach significance (Table 4). Similarly, Os/+ mice tended to have increased ethanol intake at the lowest concentration (p=0.06) but not at higher concentrations. To assess the possibility that preference for ethanol was negatively correlated with total fluid intake, we correlated the average daily preference ratio versus average daily total fluid drunk for each group for each week. For the 27 control mice, these correlations were -0.13, -0.25, and -0.41, respectively, during weeks 1, 2 and 3. The first two were not significant, and a scatterplot revealed the third week's correlation to be an artifact of two mice with very high total fluid intake. For the 22 Os/+ nephrogenic diabetes insipidus mice, these correlations were -0.75, -0.87, and -0.91, respectively. All were statistically significant (p<0.001). The scatterplot for the data at 2.2 percent ethanol is shown in Fig. 2. Discussion
For mice with a kidney tubule defect leading to high fluid intake, the by-now expected pattern of increased ethanol intake at low concentrations, reduced preference for ethanol, and polydipsia, was clearly demonstrated. Both the polydipsia and the ethanol intake values were not as different from controls, which are themselves mildly diabetic, as those seen in vasopressin-deficient diabetes insipidus rats. While no significant relationship between ethanol preference and total fluid intake was detected in normal animals, there
In other studies, we found that Brattleboro rats homozygous for the di gene leading to diabetes insipidus had elevated gram ethanol intake but reduced ethanol preference when presented with a choice between weak ethanol solutions and water [13]. The current experiments confirmed both results in rats of a different genetic background (RHA/N) onto which the di gene had been introduced by backcrossing. RHA di/di rats showed a similar pattern of reduced ethanol preference to that seen in Brattleboro di/di rats. The di gene may have exerted a stronger influence on the RHA background: RHA di/di rats showed elevated grams-ethanol intake at both 2.2 percent and 4.6 percent concentrations, and they seemed to have a higher preference for 2.2 percent ethanol, than Brattleboro di/di rats. However, in one of our earlier experiments [13] Brattleboro diMi rats had higher preferences for 2.2 percent ethanol, resembling the RHA di/di rats, than in the other three experiments there reported (which were consistently lower). RHA di/di rats also seemed to be relatively more sensitive to DGAVP's effect on ethanol preference than to its effect on water balance, where as DGAVP in Brattleboro di/di rats had generally shown equipotency for both effects. These postulated differences between RHA and Brattleboro di/di rats have appeared consistently in several other unpublished experiments with each strain. However, since the magnitudes of the strain differences are no greater than the interexperiment variability within each strain, and since we have no direct contemporaneous comparisons between Brattleboro and RHA/N di/di rats, these generalizations should be viewed with caution. In sum, the effect of the di gene on ethanol preference and water balance was not identical in two genotypes of rats. It should be emphasized that the RHAJN di/di animals studied were the product of only 6 generations of backcrossing. Other Brattleboro genes closely linked to the di locus were undoubtedly still present in the RHA diMi animals we studied. Backcrossing has now proceeded to a sufficient number of generations that this problem may be considered negligible in future studies. While the pattern of responses we found was similar, the background genotypic differences may fruitfully be exploited in future research. For example, the RHA/N diMi rats would seem to be a better strain in which to study the effects of other vasopressin-related peptides in further attempts to dissociate polydipsia from abnormalities in ethanol intake. Clearly, our results caution against hastily generalizing about the "effects of a gene" without consideration of the background on which it is expressed, While we expect that RHA/N di/di rats will prove to have the same pattern of a single deletion in the sequence encoding the neurophysin for vasopressin [14] and detectable (but very low) levels of presumptive vasopressin mRNA in hypothalamus [8] reported for Brattleboro diMi rats, the regulation of gene activity could easily differ in the genotypes.
ETHANOL PREFERENCE AND GENETIC DIABETES INSIPIDUS In other studies [13], we found that treatment o f Brattleboro di/di rats with vasopressin or a related peptide was only effective in normalizing ethanol intake at doses that restored water balance. The current experiments showed that restoration o f water balance itself was sufficient to normalize ethanol intake. Chlorothiazide, a drug unrelated to vasopressin, reduced gram ethanol intake and elevated preference in R H A / N di/di rats. Furthermore, and most telling, mice with nephrogenic diabetes insipidus, who have normal or elevated vasopressin levels, also showed reduced preference for ethanol and increased absolute intake of ethanol. Within the diabetic groups of mice, a clear negative correlation between ethanol preference and total fluid intake strongly supports the hypothesis that the abnormalities of ethanol intake are secondary to the water balance disturbance. We must conclude that the mechanisms underlying the ethanol intake abnormalities of diabetes insipidus animals remain obscure. The most likely hypothesis is that a toxic threshold for ethanol is reached in diabetes insipidus animals which leads them to avoid ethanol solutions. In our earlier
683
paper [13] we discussed those aspects of our data not entirely consistent with this hypothesis. Although the elevated absolute intake of ethanol seen in di animals offered 2.2 percent ethanol versus water could be interpreted as indicating increased preference for ethanol, we do not feel that this is likely. The absolute intake is elevated only at 2.2 percent concentrations (also 4.6 percent for R H A / N di/di) and is reduced in all rats at 10 percent concentrations. Future studies will be needed to determine the nature o f the relationships among vasopressin, polydispsia, and ethanol intake.
ACKNOWLEDGEMENTS Part of the data were reported at the International Symposium on the Brattleboro Rat, Hanover, New Hampshire, 5-7 September 1981. The authors thank Yvonne Mous and Helen Hall for secretarial assistance, Chris Dortmans, Huub Rijk, Emmett R. Young, Brenda R. Tam and Ann Kosobud for help with the experiments, and an anonymous reviewer for helpful critical comments. These experiments were supported in part by Grant AA05828 from the NIAAA to JCC.
REFERENCES 1. Boer, G. J,, R. Kragten, J. Kruisbrink and D. F. Swaab. Vasopressin fails to restore postnatally the stunted brain development in the Brattleboro rat, but affects water metabolism permanently. Neurobehav Toxieol Teratol 6: 103-109, 1984. 2, DeGroat, C. The role of renin in the paradoxical antipolyuric effects of chlorothiazide in the homozygous Brattleboro rat. Ann N Y Acad Sci 394: 259-364, 1982. 3. DeGroat, C. and A. Tijssen. The effects of long-term oral treatment with chlorothiazide or furosemide on hereditary diabetes insipidus in rats. Arch lnt Pharmacodyn Ther 237: 75-87, 1979. 4. Dorsa, D. M. and L. C. Bottemiller. Age-related changes of vasopressin content of microdissected areas of rat brain. Brain Res 242: 151-156, 1982. 5. Gilman, A. G., L. S. Goodman and A. Gilman. The Pharmacological Basis of Therapeutics, sixth edition. New York: MacMillan Pub. Co., Inc., 1980, pp. 925-926. 6. Hummel, K. P., D. L. Coleman and P. W. Lane. The influence of genetic background on expression of mutations at the diabetes locus in the mouse. I. C57BL/KsJ and C57BL/6J strains. Biochem Genet 7: 1-13, 1972. 7. Kettyle, W. M. and H. Valtin. Chemical and dimensional characterization of the renal countercurrent system in mice. Kidney lnt 1: 135-144, 1972. 8. Majzoub, J. A., A. Pappey, R. Burg and J. F. Habener. Vasopressin gene is expressed at low levels in the hypothalamus of the Brattleboro rat. Proc Natl Acad Sci USA, gl: 5296--5299, 1984.
9. Naik, D. Salt and water metabolism and neurohypophyseal vasopressor activity in mice with hereditary nephrngenic diabetes insipidus. Acta Endocrinol (Copenh) 69:434 4A,4, 1972. 10. Naik, D. and H. Kobayashi. Neurohypophysial hormones in the pars nervosa of the mouse with hereditary nephrogenic diabetes insipidus. Neuroendocrinology 7: 322-328, 1971. ! 1. Nussey, S. S., V. T. Y. Ang, J. S. Jenkins, H. S. Chowdrey and G. W. Bisset. Brattleboro rat adrenal contains vasopressin. Nature 310: 64-66, 1984. 12. Pittman, Q. J., J. Rogers and F. E. Bloom. Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol. Regul Pept4: 33-41, 1982. 13. Rigter, H. and J. C. Crabbe. Vasopressin and ethanol preference. I. Effects of vasopressin and the fragment DGAVP on altered ethanol preference in Brattleboro diabetes insipidus rats. Peptides 6: 669-676, 1985. 14. Schmale, H. and D. Richter. Single base deletion in the vasopressin gene is the cause of the diabetes insipidus in Brattleboro rats. Nature 308: 705-709, 1984. 15. Valtin, H. The discovery of the Brattleboro rat, recommended nomenclature, and the question of proper controls. Ann N Y Acad Sci 394: 1-9, 1982.