Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol

4 (1982) 33-41 Elsevier Biomedical Press

Regulatory Peptides,

33

Arginine vasopressin deficient Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol Quentin J. Pittman *, Joseph Rogers and Floyd E. Bloom Alcohol Research Center, The Salk Institute, P.O. Box 85800, San Diego, CA 92138, U.S.A.

(Received l February 1982; accepted for publication 9 March 1982)

Summary We have tested the hypothesis that animals with reduced levels of arginine vasopressin (AVP) would show reduced tolerance to ethanol. Brattleboro rats either heterozygous or homozygous for the diabetes insipidus (DI) trait and normal Sprague-Dawley rats were exposed to ethanol vapor for 21 days. Two days later, tolerance was evaluated by monitoring body temperature reductions after intraperitoneal injection of 2 g / k g (20% w / v ) ethanol. Under the same conditions of chronic ethanol exposure, Sprague-Dawley rats, but not Brattleboro rats, displayed tolerance to the hypothermic effects of intraperitoneal ethanol. This phenomenon did not appear to be related to differences in ethanol metabolism or blood alcohol levels in Brattleboro rats. These data support a possible role for AVP in the development or maintenance of tolerance. vasopressin; ethanol; tolerance; Brattleboro rat; hypothermia

Introduction Repeated exposure to ethan01 leads to tolerance to its physiological and behavioral effects. Recent studies indicate that neurohypophysial peptides can influence both the acquisition of ethanol drinking behavior and the development of tolerance to ethanol. For example, rats forced to drink ethanol as part of their liquid diet

University of Calgary, Dept. Pharmacology and Therapeutics, 3330 Hospital Drive N.W., Calgary, Alberta, T2N 4Nl, Canada. Correspondence to: Dr. J. Rogers, The Salk Institute, P.O. Box 85800, San Diego, CA 92138, U.S.A. * Present address:

0167-0115/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

34 accept higher concentrations of ethanol if they are concurrently treated with certain vasopressin analogs [4,11 ], whereas hypophysectomized rats accept almost no ethanol. Further studies also indicate that administration of arginine vasopressin (AVP) or related analogs during chronic exposure of mice to ethanol enhances the development of tolerance to ethanol's hypothermic and sedative effects and exacerbates the severity of withdrawal [5,6,13-15]. These observations raise the possibility that endogenous AVP may play a role in the development of tolerance to ethanol. To test this hypothesis, tolerance to ethanol was evaluated in Brattleboro rats, which in the homozygous state ( D I / D I ) lack AVP and in the heterozygous state ( D I / + ) exhibit reduced stores of AVP [22]. We report here that Brattleboro rats fail to develop tolerance to the hypothermic effects of ethanol despite prolonged chronic exposure.

Materials and Methods

Effects of rat strain and ethanol injection on plasma blood alcohol levels and tissue levels of immunoreactive (IR) A VP and IR ~-endorphin Two strains of naive male rat, both of which were 45 days-old on the day of the experiment, were employed. The first strain, Sprague-Dawley (Charles River, Wilmington, Delaware), consisted of 16 rats with a mean weight of 313 ± 6 g. The second strain, Brattleboro (Blue Spruce Farms, Altamont, New York), consisted of 32 rats with a mean weight of 1 8 5 ± 5 g . The latter group included 16 rats homozygous for the diabetes insipidus (DI) trait ( D I / D I ) and 16 rats heterozygous for the DI trait ( D I / + ) . Rats within each strain were randomly assigned to one of four groups given either 0.15 M saline, or 0.50, 1.25, or 2.00 g / k g ethanol. The injection volume for saline control rats was equivalent to the volume for a 1.25 g / k g ethanol injection. Ethanol was formulated as a 20% ( w / v ) solution in 0.15 M saline and injected intraperitoneally. Tail vein blood samples were collected 10, 30, 60 and 120 min after ethanol administration. Blood alcohol levels were assayed using the Sigma N A D - A D H test kit No. 331-10. 2 h after ethanol administration rats were decapitated, thebrain rapidly removed, and the hypothalamus, pituitary, and rest of brain dissected and stored on dry ice. These tissues were subsequently assayed for immunoreactive (IR) AVP and /3-endorphin in order to verify AVP content of mutant rats ~nd to provide baseline levels of /3-endorphin, another peptide implicated in memory acquisition [3]. The assays employed the methods of Iversen et al. [7] and Rossier et al. [18], respectively. Statistical analyses were by means of standard two-way analysis of variance tests (ANOVAs), with ethanol dose (0.00, 0.50, 1.25 or 2.0 g/kg) as the first factor and rat strain (Sprague-Dawley versus Brattleboro) as the second factor. Hypothermic effects of ethanol injection after chronic exposure and withdrawal Additional groups of naive male Brattleboro (Blue Spruce Farms, Altamont, New York) (n = 20) and Sprague-Dawley (Charles River Farms, Wilmingion, Delaware) (n = 28) rats were employed. All animals were 100-120 days-old on the day of the experiment. Sprague-Dawley rats weighed an average of 420 - 13 g. Brattleboro rats

35 averaged 351 --- 15 g body weight. Within each strain, rats were randomly assigned to either a chronic ethanol treatment or control condition. For chronic treatment, the ethanol inhalation paradigm of Rogers et al. [17] was used to provide uniform, constant high blood alcohol levels for a period of 21 days. Briefly, rats were put in sealed chambers continuously infused with ethanol vapor. The ambient room temperature was maintained at 23°C during chronic ethanol inhalation. For the first 4 days, a low ethanol vapor concentration (20 mg/liter) was employed to accustom rats to breathing ethanol-laden air. Ethanol vapor concentration was then raised to 24 mg/liter. We have previously shown [17] that 20 mg/liter ethanol vapor typically results in blood alcohol levels less than 100 mg%, whereas 24 mg/liter produces blood alcohol levels of 150 mg% or more. Blood alcohol levels were checked every other day by tail vein blood sample. When a rat's blood alcohol level dropped below 160 mg%, it was moved to a chamber 2 mg/liter higher in ethanol vapor concentration. Rats with blood alcohol levels above 220 mg% were moved to chambers 2 mg/liter lower in concentration. By this means, equivalent exposure was ensured among all treated rats regardless of body weight or any potential differences in ability to metabolize ethanol. Control animals were also housed in sealed chambers and tail vein blood sampled. After 21 days of ethanol inhalation, rats were withdrawn from ethanol for 48 h, then injected intraperitoneally (i.p.) with a 2 g / k g dose of ethanol to evaluate tolerance to its poikilothermic effects [13,16]. Rectal temperatures were recorded 5 min before and 30, 60, 90, 120, 150, and 180 min after injection. These tests were conducted at room temperature (22°C). Statistical analysis of ethanol-induced hypothermia was by means of a three-way ANOVA with rat strain (Sprague-Dawley versus Brattleboro) as the first factor, ethanol pretreatment (chronic ethanol inhalation versus ethanol naive) as the second factor, and time of temperature sample (5 min before and 30, 60, 90, 120, 150, or 180 rain after ethanol injection) as the third factor. As dictated by a significant interaction in the three-way ANOVA, additional two-way ANOVAs were run separately on the data for Sprague-Dawley and Brattleboro rats. These analyses used ethanol pretreatment (chronic ethanol inhalation versus ethanol naive) as the first factor and time of temperature sample (5 min before and 30, 60, 90, 120, 150, or 180 rain after ethanol injection) as the second factor.

Results

Effects of ethanol injection and rat strain on IR A VP levels, IR fl-endorphin levels, blood alcohol levels, body weights, and brain tissue weights Across strains there are no statistically reliable dose dependent effects of acute ethanol on IR Arg-vasopressin levels or IR/3-endorphin levels. However, there are highly significant differences in these variables, and in body and tissue weights, between the two strains (Table I). At the same age, Sprague-Dawley rats weigh more than either homozygous or heterozygous Brattleboro rats, probably as a partial consequence of the metabolic expenditures accompanying the excessive water intake of Brattleboros [9]. Wet weights of the hypothalamus, pituitary, and rest of brain are

36 TABLE I Mean body and tissue weights, IR peptide content, and blood alcohol levels for Sprague-Dawley and Brattleboro rats Rat strain Sprague-Dawley

P Brattleboro Heterozygous

n Body weight(g)

16

16

Homozygous 16

313

-+ 6

216

÷ 3

154

± 3

<0.001

Hypothalamusweight (mg)

76

--+ 2

72

± 2

64

÷ 2

<0.01

Pituitary weight (mg)

11.0 ± 0.5

Rest of brain weight (mg)

1746

+ 23

8.0 -+ 0.3 1568

-+ 32

7.0 ± 0.2 1458

~- 33

<0.001 < 0.001

IR fl-endorphin ( n g / m g wet weight) Hypothalamus Pituitary Rest of brain ( p g / m g )

0.78 -+ 0.08 179 -+ 15 10 -+ 0.7

0 . 4 8+- 0.04 116 -+ 5 9.5 -+ 0.7

0 . 5 9+- 0.08 110 + 8 9.5 --+ 0.7

<0.01 <0.001 ns

IR Arg-vasopressin ( n g / m g wet weight) Hypothalamus Pituitary Rest of brain ( p g / m g )

1.21-+ 0.09 191 -+25 6.0 ± 0.1

0.53± 0.05 61 ± 14 3.0 ± 0.1

0.07± 0.06 nd nd

<0.001 <0.001 <0.01

Mean blood alcohol level (mg%) 10-120 min after ethanol (4 rats/point) 0.50 g / k g 1.25 8 / k g 2.00 g / k g

22 98 200

--+ 4 ± 9 ± 12

19 65 138

± 3 ± 7 -+20

24 55 124

-+ 4 -+ 8 ± 9

Weight and peptide data are pooled over control and acute ethanol treated rats, since these values are not significantly altered by ethanol administration. (nd = not detectable; lower limit of assay = 3-5 pg).

also significantly higher in the Sprague-Dawley rats. As expected, even corrected for their higher brain tissue weights, the Sprague-Dawley animals exhibit higher levels of hypothalamic, pituitary, and rest of brain IR Arg-vasopressin. In addition, Sprague-Dawley animals have higher levels of hypothalamic and pituitary IR fl-endorphin than either heterozygous or homozygous Brattleboro rats. Sprague-Dawley rats also exhibit higher blood alcohol levels to the same doses of ethanol. Subsequent investigations have shown that this result is probably not due to innate differences in the rat strains, but to the fact that the same g/kg ethanol dose produces higher blood alcohol levels in heavier rats than lighter rats [1]. Heavier rats have more body fat and a lower percentage of body water; thus, they have a lower relative volume for distribution of ethanol.

37

Chronic ethanol exposure by inhalation Through the 21 days of ethanol treatment, Brattleboro rats exhibit an average blood alcohol level of 211 +--18 mg%, a level not significantly different from the average 193 +-- 15 mg% of Sprague-Dawley rats.

Hypothermic effects of ethanol injection in chronically exposed and ethanol naive Brattleboro and Sprague-Dawley rats The three-way A N O V A reveals several significant treatment effects in the hypothermia experiments. Across all rats, there are reliable time-dependent changes in body temperature as a result of acute ethanol injection ( P < 0 . 0 0 1 ) . Of greater interest, there is a significant difference in the hypothermic response of Sprague-Dawley and Brattleboro rats ( P < 0 . 0 0 1 ) , with the latter exhibiting consistently lower temperatures at all time points after ethanol administration. This does not appear attributable to higher blood alcohol levels or decreased ethanol metabolism in Brattleboro rats because their blood alcohol levels are actually less than that of age-matched Sprague-Dawley rats given the same ethanol dose (Table I).

39

38

CHRONIC

SO

e,,

37 (1.

~'~"~ '

W F-

SD ETHANOL ~'~. NAIVE'~

.... L

g 36 Z

BRAT~ ETHANOL

~J

35 BRAT~ CHRONIC ETHANOL

I

I

I

I

I

0

30

60

90

120

I

150

|

180

MINUTES POST-INdECTION

Fig. 1. Hypothermic effect of acute ethanol (2 g / k g IP) in ethanol naive ( © ) and chronic ethanol ( e ) Sprague-Dawley ( . . . . . . ) and Brattleboro ( ) rats. Tolerance tests were conducted at 22°C in a different room from that where ethanol was chronically administered.

38 Finally, the three-way ANOVA reveals a significant difference between the two rat strains in the way ethanol interacts with chronically-exposed as opposed to ethanolnaive rats. The basis for this significant strain versus ethanol pretreatment interaction ( P < 0.05) is made clear in requisite two-way ANOVAs. For Sprague-Dawley rats, previous chronic ethanol inhalation results in significant hypothermic tolerance ( P < 0.01) (Fig. 1). In Brattleboro rats, however, previous ethanol inhalation fails to produce tolerance to the hypothermic effects of ethanol; indeed, after acute intraperitoneal ethanol the temperature drop of chronic ethanol and ethanol-naive Brattleboros is virtually identical (Fig. 1). The use of A scores (in which the temperature at any given time after ethanol is subtracted from the baseline temperature to yield °C temperature drop) gives the same results (i.e., Sprague-Dawley rats show hypothermic tolerance, Brattleboro rats do not) since baseline temperatures of both Sprague-Dawley groups (ethanol treated and ethanol naive) are nearly identical. The same is true for both Brattleboro groups. Post-hoc data analyses do not indicate any statistically reliable differences between D I / D I and D I / + rats in their inability to become tolerant to ethanol's hypothermic action.

Discussion Our results indicate that both homozygous and heterozygous Brattleboro rats show significantly reduced levels of hypothalamic IR AVP and a significantly reduced ability to develop tolerance to the hypothermic effects of ethanol when compared to rats of the Sprague-Dawley strain. Some important points should be considered, however. Other workers have focused on the ability of exogenously administered AVP and AVP analogues to prolong tolerance [2,3,5,6]. Thus, it is possible that Brattleboro rats do in fact develop ethanol tolerance, but the tolerance wanes so rapidly that it could not be observed in our experiments: rats were withdrawn from ethanol for 48h before tolerance testing to ensure complete clearance in "fill rats of blood alcohol from chronic inhalation exposure. It would be of interest to determine whether or not Brattleboro rats manifest behavioral symptoms of withdrawal during the first few days after chronic ethanol exposure, since this might suggest that they are also capable of developing a rapidly-waning tolerance. We also believe it would be a mistake to believe our data necessarily prove a causal relationship between AVP deficiency and failure to develop or maintain tolerance: Brattleboro rats also exhibit a number of other abnormalities [9,22]. For example, the present experiments indicate that both heterozygous and homozygous Brattleboro rats have lower levels of hypothalamic and pituitary IR B-endorphin than do Sprague-Dawley rats of similar age. Since peptides of the endorphin family have also been implicated in aspects of conditioning and memory [3], it is possible that the failure of Brattleboro rats to develop or maintain tolerance is as much related to deficits in IR fl-endorphin as to deficits in IR AVP. However, there is a growing body of evidence relating AVP to various tolerance phenomena [2,12-15]. In particular, administration of AVP or fragments of this

39 molecule to mice enhances and prolongs tolerance to alcohol [6,14]. The Brattleboro rat appears to provide a natural model for extending and confirming these results from normal animals: if AVP excesses prolong tolerance, then AVP deficits in Brattleboro rats should diminish or prevent tolerance. Our data support such reasoning and strongly suggest a functional role for AVP in the development or the maintenance of tolerance to ethanol's hypothermic effects. The mechanisms by which AVP might enhance tolerance development remain obscure. AVP may participate directly in pharmacologic changes involved in tolerance development or it may act indirectly through mechanisms of conditioning which have been implicated in tolerance development [10,19,23]. With respect to the latter point, however, it is worth noting that tolerance testing in our experiments was conducted in a wholly different environment from that where rats had been chronically exposed to ethanol. Moreover, the route of chronic ethanol administration was by inhalation, whereas hypothermic tolerance was tested by acute intraperitoneal injection. Under a conditioning theory of tolerance, the environment where ethanol is chronically administered and the route of chronic administration may become cues relevant to the tolerance response. To the extent that these cues were not present, and in fact were changed, in our tests of hypothermic tolerance, the rats should have shown diminished tolerance. Nonetheless, the Sprague-Dawley rats exhibited a clear and statistically reliable tolerance to ethanol-induced hypothermia. Of course, in our study and those of others, the drug-state induced by ethanol still remains a salient and consistent cue regardless of the ambient environment or route of drug administration. Brattleboro rats in our experiments not only failed to develop hypothermic tolerance to ethanol, but also exhibited an increased sensitivity to the hypothermic effects of acute ethanol. After 2 g / k g ethanol, their body temperature was lower than that of Sprague-Dawley rats at all time points measured, a difference which proved to be highly significant. This result is not explicable by higher blood alcohol levels in the Brattleboro rats; on the contrary, Brattleboro rats have lower blood alcohol levels than age-matched Sprague-Dawley controls given the same g / k g ethanol dose. Subsequent experiments [1] suggest that these lower blood alcohol levels are not due to a metabolic deficit so much as to an inadequacy of g / k g formulations to provide equivalent blood alcohol levels in rats of different weights. Thus, the significantly greater hypothermia exhibited by Brattleboro compared to Sprague-Dawley rats occurs despite lower blood alcohol levels in the former group. It is possible that AVP, or some undefined factor lacking or reduced in Brattleboro rats may be required for defense of body temperature against the hypothermic effects of ethanol. In this respect, it would be of interest to determine the response of Brattleboro rats to other poikilothermic agents [c.f. 21]. Finally, our data indicate that even a 50-60% deficiency in AVP levels, as found, for example, in heterozygous Brattleboros, may be sufficient to induce clear deficits in acquisition or maintenance of tolerance phenomena. The corollary, that AVP injection might restore an ability to develop tolerance in Brattleboros, is eminently testable. Differences in levels of brain AVP or other peptides may be responsible, in part, for the well-known differences among different strains of rats or mice to the behavioral and pharmacological effects of ethanol [8,20].

40

Acknowledgements Supported by NIAAA 03504. QJP supported by MRC (Canada). We thank R. Azad for expert technical assistance with the peptide assays.

References 1 Bloom, F., Lad, P.J., Pittman, Q.J. and Rogers, J., Blood alcohol levels in rats: non-uniform yields from IP doses based on body weight, Brit. J. Pharmacol., 75 (1982) 251-254. 2 De Wied, D. and Versteeg, D.H.G., Neurohypophyseal principles and memory, Fed. Proc., 38 (1979) 2348-2352. 3 De Wied, D., Neuropeptides in normal and abnormal behaviour. In E. Start, G.B. Makara and E. Endroczi (Eds.), Adv. Physiol. Sci. 13, Endocrinology, Neuroendocrinology. Pergamon Press/Akad. Kiado, New York, 1981, pp. 23-38. 4 Finkelberg, F., Kalant, H. and Le Blanc, A., Effect of vasopressin-like peptides on consumption of ethanol by the rat, Pharmacol. Biochem. Behav., 9 (1978) 453-458. 5 Hoffman, P.L., Ritzmann, R.F., Walter, R. and Tabakoff, B., Arginine vasopressin maintains ethanol tolerance, Nature, 276 (1978) 614- 616. 6 Hoffman, P.L., Ritzmann, R.F., and Tabakoff, B., Neurohypophyseal peptide influences on ethanol tolerance and acute effects of ethanol, Pharmacol. Biocbem. Behav., 13 (1980) 279-284. 7 Iversen, L.L., Iversen, S.D., and Bloom, F.E., Opiate receptors control vasopressin release from nerve terminals in rat neurohypophysis, Nature, 284 (1980), 350-351. 8 Kakihana, R., Alcohol intoxication and withdrawal in inbred strains of mice: behavioural and endocrine studies, Behav. Neural Biol., 26 (1979) 97-105. 9 Laycock, J.F., The Brattleboro rat with hereditary hypothalamic diabetes insipidus, Gen. Pharmacol., 8 (1977) 297-302. 10 Le, A.D., Poulos, C.X., and Cappell, H., Conditioned tolerance to the hypothermic effect of ethyl alcohol, Science, 206 (1979) 1109-1110. 11 Mucha, R.F. and Kalant, H., Effects of des-glycinamide-lysine-vasopressin and prolyl-leucyl-glycinamide on oral ethanol intake in the rat, Pharmacol. Biochem. Behav., 10 (1979) 229-234. 12 Rigter, H. and Crabbe, J.C., Alcohol: modulation of tolerance by neuropeptides. In M. Sandier (Ed.), The Psychopharmacology of Alcohol, Raven Press, New York, 1980, pp. 179-189. 13 Rigter, H., Cral~b¢, J.C. and Schonbaum, E., Hypothermia in mice as an index of the rapid development of tolerance to ethanol. In B. Cox, P. Lomax, A.S. Milton and E. Schonbaum (Eds.), Thermoregulatory Mechanisms and Their Therapeutic Implications, Karger, Basel, 1980, pp. 216-219. 14 Rigter, H., Dortmans', C. and Crabbe, J.C., Jr., Effects of peptides related to neurohypophyseal hormones on ethanol tolerance, Pharmacol. Biochem. Behav., 13 (1980) 285-290. 15 Rigter, H., Rijk, H. and Crabbe, J.C., Tolerance to ethanol and severity of withdrawal in mice are enhanced by a vasopressin fragmextt, Eur. J. Pharmacol., 64 (1980) 53-68. 16 Ritzmann, R.F. and Tabakoff, B., Body temperature in mice: a quantitative measure of alcohol tolerance and physical dependence, J. Pharmacol. Exp. Ther., 199 (1976) 158-170. 17 Rogers, J., Wiener, S.C. and Bloom, F.E., Long-term ethanol administration methods for rats: advantages of inhalation over liquid diets, Behav. Neural Biol., 27 (1979) 466-486. 18 Rossier, J., Rogers, J., Shibasaki, T., Guillemin, R., and Bloom, F.E., Opioid peptides and a-melanocyte-stimulating hormone in genetically obese (ob/ob) mice during development, Proc. Natl. Acad. SCi. U.S.A., 76 (1979) 2077-2080. 19 Siegel, S., Morphine tolerance acquisition as an associative process, J. Exp. Psychol. (Animal Behav.), 3 (1977) 1-13. 20 Sorensen, S., Palmer, M., Dunwiddie, T. and Hoffer, B., Electrophysiological correlates of ethanol-induced sedation in differentially sensitive lines of mice, Science, 210 (1980) 1143-1145.

41 21 Tache, Y., Pittman, Q. and Brown, M., Bombesin induced poikiiothermy in rats, Brain Res., 188 (1980) 525-530. 22 Valtin, H., Sawyer, W.H. and Sokol, H.W., Neurohypophysial principles in rats homozygous and heterozygous for hypothalamic diabetes insipidus (Brattleboro strain), Endocrinology, 77 (1965) 701-705. 23 Wenger, J.R., Berlin, V. and Woods, S.C., Learned tolerance to the behaviourally disruptive effects of ethanol, Behav. Neural Biol., 28 (1980) 418-430.