The effects of neurohypophyseal hormones on tolerance to the hypothermic effect of ethanol

alcohol. Vol. 2, pp. 567--574, 1985. ©Ankho InternationalInc. Printed in the U.S.A.

0741-8329/85 $3.00 + .00

The Effects of Neurohypophyseal Hormones on Tolerance to the Hypothermic Effect of Ethanol GYULA

SZAB0,

G,~BOR L. KOVACS, S,6,NDOR SZI~KELI AND GYULA TELEGDY t

Institute o f Pathophysiology, University Medical School, S e m m e l w e i s u. 1. P . O . B . 531, Szeged, Hungary 6701 R e c e i v e d 19 O c t o b e r 1984 SZAB0, G., G. L. KOVACS, S. SZI~KELI AND G. TELEGDY. The effects ofneurohypophyseal hormones on tolerance to the hypothermic effect of ethanol. ALCOHOL 2(4) 567-574, 1985.--Mice were made tolerant to the hypothermic effect of ethanol by repeated administration of ethanol (4 g/kg, 25% v/v, IP) on three consecutive days. The colonic temperature was measured in individually-housed animals immediately before and 45 rain after ethanol treatment. Peptide treatments with various schedules were made SC 2 hr before the first ethanol challenge. The decrease in hypothermic response was accepted as a tolerance phenomenon, which developed in control animals by day 2. A single injection of oxytocin (OXT) or lysine vasopressin (LVP [0.1 or I 1U peptide] animal) before the first ethanol injection did not change the initial sensitivity to ethanol. This absence of acute interactions is also reflected in the sleep onset and sleep duration after 5 gtkg ethanol (IP). In contrast, both OXT and LVP affected the development of tolerance. Repeated treatments with graded doses of OXT (0.5-2 IU) or LVP (0.25-1 IU) every day for 3 days blocked the development of tolerance. 0.002 IU LVP facilitated the development of hypothermic tolerance. The remaining doses of the peptides were ineffective. A high dose of LVP (1 IU) facilitated hypothermic tolerance if the peptide was injected when tolerance to ethanol had developed fully without previous peptide treatment. OXT, on the other hand, was ineffective in this particular experimental model. The data suggest that both neurohypophyseal hormones (LVP and OXT) block the early developmental phase of tolerance to ethanol. On the other hand, LVP facilitated the expression of tolerance if the peptide was given to mice with fully developed tolerance. Oxytocin

Vasopressin

Ethanol hypothermia

Ethanol tolerance

E A R L I E R data indicate that the neurohypophyseal hormones oxytocin (OXT) and'vasopressin (VP) exert opposite effects on the central nervous system. VP facilitates the consolidation and retrieval of memory processes [3, 20, 21]. Under appropriate experimental conditions OXT may have behavioral effects opposite to those induced by VP [10,12]. The opposite effect is also observed in electrophysioiogical parameters at the level of the septo-hippocampal complex [22], a likely target area for the opposite actions of these peptides [11]. The ability o f a living organism to develop tolerance to addictive drugs (narcotic analgesics, ethanol, etc.) has likewise been associated with learning and memory processes (of. [8]). VP facilitates the development of tolerance to the analgesic effect of morphine [15,23]. OXT, on the other hand, attenuates acute and chronic tolerance to morphine in mice [14]. DGAVP, a behaviorally active analog of VP enhanced ethanol intake if the peptide was given during the acquisition of the drinking habit [4]. VP treatment enhanced the retention of ethanol tolerance [5, 6, 16, 17], while OXT, the C-terminal tripeptide of OXT (Pro-Leu-Gly-NH~) and cycio-Leu-Gly [16] were devoid of activity on ethanol

tolerance if the animals were fed ethanol in a liquid diet, and the level o f residual tolerance was assessed thereafter by repeated IP administration of ethanol. In these studies the environmental cues were different in the tolerance testing environment compared to the conditions of ethanol administration [19]. However, when a multiple peptide-ethanol injection paradigm was used, AVP tended to delay the development of tolerance to ethanol [7]. Under these experimental conditions conditioned environmental factors (injection procedure, route o f administration, etc.) play a major role in the development of tolerance to ethanol [ 19]. Due to the fact that VP improves adaptation processes [3, 20, 21] a facilitatory effect was anticipated on ethanol tolerance too. Based on the aforementioned experiments, the present study investigates the effects of graded doses of LVP and OXT on tolerance to ethanol using multiple peptide/ethanol injections in different testing paradigms. METHOD

Animals Random-bred C F L P albino, male mice weighing 30-35 g

~Requests for reprints should be addressed to Professor G. Telegdy, Institute of Patbophysiology, University Medical School, P.O.B. 531., Szeged, Hungary 6701.

567

SZABO E l AL.

568 ETHANOL

TOLERANCF

MICE

IN

ETHANOL Z,g/kg (~p)

PEPTIDE (sc)

l

-120'

" O

0'

45'

1 C

O

C

t.d rr

"c t.l a. 38' z~ laJ t--- 37'

TT t

£

t TOLERANCE I---

(_) t.d rY

36"

Statistical Analysis

6 TOLERANCE

injected. Forty-five min later the colonic temperature was again measured. This was achieved with a rectal probe lubricated with paraffin and inserted 3 cm into the colon. An analog read-out (Unitherm Animal Thermometer ~) was used. Measurements were made between 10.00 and 13.00 hr each day (Fig. l) and were repeated on three consecutive days. In two further sets of experiments, animals were pretreated with a single injection of neurohypophyseal peptide either before the first ethanol injection (0.5 IU OXT or LVP) or on day 3, when tolerance to the hypothermic effect of ethanol had already developed (0.01, 0.1, I IU OXT or LVP), A separate group of mice was injected with either 4 g ethanol/kg (25% v/v) or vehicle. Temperatures were measured every 15 min for 2 hr (Fig. 2). Only a few animals had low temperatures before ethanol injection or became ill due to repeated measurements. These animals were eliminated from evaluation.

4's

= DECREASING --

M,N

HYPOTHERMIA

With the exception of the first experiment, where Student's paired t-test was used, the data were analysed by A N O V A or Kruskal-Wallis test. A probability level of p<0.05 was accepted as a significant difference.

I DAY

--- 2 DAY ..... 3 D A Y

FIG. 1. Treatment schedule used in the measurement of tolerance to ethanol.

were used in the experiments. The animals were maintained on a 12 hr light/12 hr dark cycle (light on from 6.00 to 18.00 hr).

Sleep Onset and Duration Folhm'ing Ethanol Injection Mice were pretreated with different doses (0. i and 1 IU) of either oxytocin (OXT, Richter, Budapest) or lysine vasopressin (LVP, Sandoz, Basle) and were returned to their home cages for a 120 min pretesting interval. The peptides were diluted in physiological saline and injected SC in a volume of 0.2 ml/animal. Control mice were treated with vehicle. At the end of this time 5.0 g ethanol/kg (25% v/v) was administered IP. Within 150 seconds the animals fell asleep and were placed on their backs. The ability of the animal to right onto all four paws twice within one minute was accepted as a demonstration of the righting reflex. Sleep onset time was measured as the time between the injection of ethanol and the loss of the righting reflex. Sleep duration was assessed as the time between the loss and the regaining of the righting reflex.

Ethanol-Induced Hypothermia Temperature studies were carried out on mice housed in individual cages throughout the experiments and kept at constant environmental temperature (24+_1°C). Animals were pretreated with either OXT (2, 1,0.5, 0.25, 0.02, 0.002 IU) or LVP (1, 0.5, 0.25, 0.02, 0.002, 0.0002 IU) SC 2 hr before the first temperature measurement. Control animals received vehicle. Two hours after pretreatment the colonic temperature was measured and 4 g ethanol/kg (25% v/v) was

RESULTS

While the body temperature of vehicle-treated control animals remained constant, a single dose of ethanol caused a rapid fall in colonic temperature 15 rain after treatment. The greatest fall was obtained between 15 and 30 rain q~<0.01, paired t-test) and the colonic temperature reached a constantly low level from 45 rain on, so this time point was selected for measurement of the hypothermic effect of ethanol in further studies on ethanol tolerance (Fig. 2). The hypothermic effect was still present 2 hr after ethanol injection. In hypothermia studies 4 g ethanol/kg was used since a high incidence of mortality was observed after repeated administration of 5 g ethanol/kg. Sleep onset and sleep duration after a single dose of 5 g ethanol/kg were not affected by two doses of either OXT or LVP given 2 hr before the ethanol dose (Fig. 3), i.e.. the initial sensitivity to ethanol was not changed by the peptides in the doses investigated. Six doses of OXT were investigated on tolerance to ethanol (Table 1). Pretreatment with 2.0, 1.0 or 0.5 IU OXT/animal on three consecutive days before repeated ethanol administration blocked the development of tolerance to the hypothermic effect of ethanol. Lower doses of OXT, on the other hand, had no effect on ethanol tolerance, i.e., the tolerance developed in latter groups to the same degree 0.25 IU/animal, F(2,16)=17.61, p < 0 . 0 0 0 1 : 0 . 0 2 IU/animal, F(2,18)=4.14, p<0.003; 0.002 IU/animat, F(2,16)=7.28, p<0.006) as in the control group, F(2,36)=20.35, p<0.0001. Treatments with different doses of OXT (2.0-1.(~0.5 IU/animal) resulted in a significantly greater hypothermic effect on days 2 (F(6,68)=3.75, p<0.01 for 2 IU OXT: F(6,68) = 2.77, p <0.05 for i I U OXT, and F(6,68) = 2.70, p < 0.05 for 0.5 IU OXT) and 3 (F(6,68)= 10.80,p<0.001 for2 IU OXT: F(6,68)=4.43, p<0.002 for 1 IU OXT: and F(6,68)=4.75, p<0,002 for 0.5 IU OXT), than in the control group on days 2 and 3, respectively. On day 1, however, there was no difference in the hypothermic response to ethanol either in peptide treated or in the control groups.

N E U R O H Y P O P H Y S E A L PEPTIDES

569

*C 39

(5) 37-

~i (5)

0

15

30

&5

60

" CONTROL

75

9f~

105

120

MIN

• ETHANOL (/.g/kgl

FIG. 2. The effect of a single dose of ethanol on body temperature in mice (*p
SLEEP-DURATIONTIME

SLEEP-ONSETTIME MIN 20:

MIN 100'

"I" " ,.~ \ N

10.

31122 17123

[]

CONTROL

I [ ] tO tU LVP

[ ] 10 tU. 0XT

FIG. 3. Sleep-onset and sleep-duration times in mice after 5 g ethanol/kg (IP). Peptide treatments were given SC two hours before starting the experiments. Statistical analysis was made by ANOVA and Kruskal-Wallis tests.

The effect of LVP, repeated on three consecutive days, on development of tolerance to the hypothermic effect of ethanol is depicted in Table 2. Injection of graded doses of LVP (1.0, 0.5 or 0.25 IU/animal) blocked the development of tolerance to ethanol. Lower doses of LVP were ineffective, thus tolerance developed in these groups by day 3, (0.02 IU/animal, F(2,34)=6.99, p<0.004: 0.002 IU/animal, F(2,16)=7.49, p<0.006; 0.0002 IU/animal, F(2,18)=6.08, p<0.01 ), as iT, the control animals, (F(2,56)=20.17, p<0.0001. Since the previous calculation does not permit comparing the level of tolerance between control and LVP (0.02-0.0002 IU/animal) treated groups, the data were further analysed by direct comparison of the hypothermic response between various groups on a given day of treatment. Kruskal-Wallis

analysis o f these data revealed that LVP pretreatment did not influence the hypothermic response to ethanol on days 1 and 2. On day 3, however, a dose-dependent dual effect of LVP was observed in response to ethanol; while in mice treated with high doses of LVP (0.5-0.25 IU/animal) greater hypothermic response to ethanol was observed, in the group treated with 0.002 IU/animal of LVP, ethanol induced smaller degree of hypothermia than in the control group. It is likely that a small dose o f 0.002 IU LVP facilitated the development o f tolerance to ethanol. The data obtained in the preceding two experiments were plotted as percentages o f the control level (control=i00%) on the last testing day (Fig. 4). OXT pretreatment facilitated the expression of the hypothermic response to ethanol in

570

SZAB0 ET AL TABLE 1 T H E E F F E C T OF OXYTOCIN ON E T H A N O L T O L E R A N C E IN MICE

Hypothermia Induced by Ethanol (°C) Days

control

2 IU

1 IU

0.5 IU

0.25 IU

0.02 IU

0.002 IU

Day 1 Day 2

2.42 ± 0.20* 1.33 ± 0.22

2.91 ± 0.43 2.83 _+ 0.30

2.34 ± 0.24 2.62 ± 0.26

2.40 ± 0.39 2.56 _ 0.31

3.30 ± 0.36 2.16 _+ 0.20

2.12 ± 0.34 1.40 _ 0.24

2.14 ± 0.31 1.29 _ 0.21

Statistical Analysis (Anova") NS contr, vs. 2 IU~: c o n t r , vs.

1 IU + c o n t r , vs.

Day 3

1.04 ± 0.12

2.87 ± 0.17

2.21 ± 0.29

2.21 ± 0.20

1.38 _+ 0.17

1.23 _+ 0.14

0.64 _+ 0.23

0.5 IU+ contr, vs. 2 IU ¢ contr, vs. 1 IU§ c o n t r . VS.

0.5 1U§ n

19

19

l0

l0

9

l0

9

NS

day l vs. 2 day l vs. 3 day 2 vs. 3 -t

day 1 vs. days 2 and 3 -~

day l vs. days 2 and 3 t

day l vs. days 2 and 3 ANOVAh t

NS

NS

* mean ± SEM (°C). ANOVA a one-way analysis of variance: comparison between treatments, ANOVAb two-way analysis of variance: comparison within a treated group (the least significant difference [p<0.05] is indicated). n number of animals used. t p<0.05. :~p<0.01. § p<0.02. ¶p<0.01. NS not significant.

dose-dependent manner, which effect could be characterized by a linear regression line (y=92x+ l 1l, r=0.90), while LVP pretreatment resulted in a linear plot only for doses of 0.0002-0.5 IU LVP (y=358x+80, r=0.96). The most efficient common dose at which 3 consecutive injections of both OXT and LVP blocked the development of tolerance to ethanol was 0.5 IU peptide/animal. This dose was therefore studied as a single treatment given prior to the fast ethanol challenge. A single dose of either OXT or LVP before the first ethanol injection did not block the development of tolerance to ethanol (Table 3). In another set of experiments the animals which were not treated with peptides for 2 days were rendered tolerant to the hypothermic effect of ethanol by repeated administration of ethanol as described in the Method section. When tolerance had developed, a single injection of either OXT or LVP was given 2 hr before ethanol treatment. In this experiment model OXT was ineffective in changing the expression of fully-developed tolerance to ethanol in the doses investigated (Table 4). On the other hand, the expression of tolerance to ethanol was facilitated by a high dose of LVP ( 1 IU/animal, F(2,18)= 19.59,p<0.0001; Table 5), as indicated by the observation that 1 IU LVP caused a significantly lower temperature decrease than that measured before peptide treatment (day 2).

DISCUSSION

The data presented here show that tolerance to the hypothermic effect of ethanol can develop rapidly. This is in agreement with the findings of Crabbe et al. [2]. Animals tolerant to the hypothermic effect of ethanol after peripheral administration were also found to be tolerant to the direct injection of ethanol into the central nervous system, suggesting that ethanol-induced hypothermia is mediated by central nervous processes [18]. Although VP alters temperature regulation in fever [9,13] the effect of neurohypophyseal hormones on normal body temperature depends on the time and route of administration and species differences can also account for diversity for different data [1]. When, however, administered 2 hr prior to ethanol, neither VP; OXT nor the C-terminal tripeptide fragment of OXT altered the hypothermic response to ethanol (unpublished observations). Earlier data [6] showed a post-injection decline in body temperature after peptide treatment, and core temperature returned to baseline within 2 hours. Prior to evaluating the effects of OXT and LVP on the development of hypothermic tolerance the influence of peptides on the initial response to ethanol administration was tested. Neurohypophyseal hormones did not alter the acute

NEUROHYPOPHYSEAL

PEPTIDES

571 TABLE 2

THE EFFECT OF LYSINE-VASOPRESSIN ON ETHANOL TOLERANCE

Hypothermia Induced by Ethanol (°C) Days

control

1 IU

0.5 IU

0.25 IU

0.02 1U

0.002 IU

0.0002 IU

Day 1 Day 2 Day 3

2.41 -+ 0.20* 1.31 -+ 0.16 1.15 -+ 0.13

1.43 _+ 0.32 1.33 -+ 0.30 1.19 _ 0.42

3.40 +- 0.32 2,38 +- 0.45 2.90 _ 0.39

2.04 ± 0.33 1.81 ± 0.49 2.09 _+ 0.19

2.41 _+ 0.30 1.34 _ 0.26 1.23 _ 0.22

1.74 - 0.20 1.06 -+ 0.34 0.51 -+ 0.28

2.01 - 0.14 1.50 +- 0.25 1.05 +- 0.28

29

8

5

8

18

9

10

day 1 vs. days 2 and 3 t

day i vs. day 3

NS

day I vs. days 2 and 3 t

n ANOVA h

day 1 vs. days 2 and 3 t

NS

NS

Statistical Analysis (Kruskal (Wallis test ~) NS NS contr, vs. 0.5 IU;t contr, vs. 0.25 IU$ contr, vs. 0.002 I U t

t

* mean +_ SEM (°C). " comparison between treatments. ANOVA" two-way analysis of variance: comparison within a treated group. n number of animals used. t p<0.05. :~p<0.01. NS not significant.

TABLE 3 THE EFFECT OF A SINGLE DOSE OF NEUROHYPOPHYSEAL PEPTIDE ON THE DEVELOPMENT OF ETHANOL TOLERANCE Hypothermia Induced by Ethanol (°C) Days Day 1 Day 2 Day 3 ANOVA ~'

control

0.5 IU OXT

0.5 IU LVP

Statistical Analysis (ANOVA a)

2.74 -+ 0.31" (8) 1.76 -+ 0.31 (8) 1.24 _+ 0.39 (8)

2.02 _+ 0.23 (16) 1.27 _ 0.19 (16) 1.24 _+ 0.15 (16)

2.37 +- 0.19 (20) 1.78 +_ 0.18 (20) !.51 _+ 0.20 (20)

NS NS NS

day 1 vs. days 2 and 3 t

day 1 vs. days 2 and 3 ~

day ! vs. days 2 and 3 t

* mean _+ SEM (°C) (number of animals used). ANOVA '' one-way analysis of variance: comparison between treatments. ANOVA" two-way analysis of variance: comparison within a treated group. NS not significant. t p<0.05. Peptide treatment was made on day 1 before the first ethanol challenge.

r e s p o n s e o f C F L P a l b i n o mice to e t h a n o l in o u r e x p e r i e n c e . A c c o r d i n g l y , t h e r e was n o c h a n g e in e i t h e r sleep o n s e t o r sleep duration after the injection of different doses of OXT a n d L V P . Similar d a t a w e r e p u b l i s h e d e a r l i e r [6] with differe n t strain o f mice. P r e t r e a t m e n t for 3 d a y s with n e u r o h y p o p h y s e a l p e p t i d e s b l o c k e d t h e d e v e l o p m e n t o f t o l e r a n c e to the h y p o t h e r m i c effect o f e t h a n o l , p a r t i c u l a r l y a f t e r h i g h e r close o f p e p t i d e s . Animals pretreated with graded doses of neurohypophyseal h o r m o n e s b e f o r e e v e r y e t h a n o l c h a l l e n g e for 3 d a y s s h o w e d

close c o r r e l a t i o n b e t w e e n t h e level o f h y p o t h e r m i c r e s p o n s e to e t h a n o l a n d t h e i n h i b i t i o n o f t h e d e v e l o p m e n t o f t o l e r a n c e o n t h e last t e s t i n g day. O X T facilitated t h e e x p r e s s i o n o f h y p o t h e r m i c r e s p o n s e to e t h a n o l a n d b l o c k e d t h e d e v e l o p m e n t o f t o l e r a n c e in a d o s e - d e p e n d e n t m a n n e r . L V P , o n t h e o t h e r h a n d , s h o w e d a b i m o d a l a c t i o n ; 0.002 I U L V P facilitated t h e d e v e l o p m e n t o f t o l e r a n c e to e t h a n o l , while high d o s e s o f L V P , similar to O X T , b l o c k e d this p r o c e s s . E a r l i e r d a t a s h o w e d t h a t A V P t r e a t m e n t g i v e n e i t h e r during e t h a n o l i n t o x i c a t i o n a n d w i t h d r a w a l [5] or d u r i n g with-

572

SZAB0 ET AL TABLE 4 THE EFFECTOF SINGLE GRADEDDOSES OF OXYTOCINON ETHANOLTOLERANCE IN MICE Hypothermia Induced by Ethanol (°C) Days .

control

1 IU

0. l IU

0.01 IU

Statistical Analysis (ANOVA")

2.56 ± 0.38 (8) 2.23 _+ 0.19 (8) 2.15 _+ 0.18 (8)

NS NS NS

m

Day l Day 2 Day 3 ANOVA k,

3.20 _ 0.30* (9)

3.06 ± 0.43 (9)

3.23 -+ 0.27 (10)

1.90 __+0.37 1.12-+ 0.34

1.86 ± 0.17 (9) 1.92 ± 0.54 (9)

2.40 _+ 0.29 (10) 1.86_+ 0.31 (I0)

day I vs. days 2 and 3 +

day l vs. day 3

(9) (9)

day 1 vs. days 2 and 3 i-

t

day 1 vs. day 3 +

* mean _+ SEM (°C) (number of animals used). ANOVA" one-way analysis of variance: comparison between treatments. ANOVAh two-way analysis of variance: comparison within a treated group. NS not significant. * p<0.05. Peptide treatment was made on day 3 when the tolerance to ethanol has developed.

%

0×I

200

I0Q

r : 090 y : 92x -111 n:6

IT

U

002 05 0002 025 i

CONTROL

2

IU

r =096

LVP

y : 358x.80 200.

n:5

r

IOC

0002

--

1

025

00002 002

05

CONTROL

IU

0

FIG. 4. Hypothermic reslxmse to ethanol 14 g/kg) for 3 days after repeated administration of OXT and LVP on day 3. The temperature differences were plotted in percent of control (control= ItXY'/~). For OXT linear regression plots were obtained in all doses investigated. for LVP the dose range of 0.0002-0.5 IU was linear.

drawal [6] retained the decay of residual tolerance to ethanol. Rigter and Crabbe [17] found an increased residual tolerance to ethanol when AVP was infused during only the induction of tolerance to and dependence on ethanol. Wide

dose-ranges of OXT were ineffective in these studies [5,6]. In these experiments oral ethanol intake [5,6] or ethanol vapor [17] was used to render the animals tolerant to and dependent on ethanol. The residual tolerance to ethanol was measured after dissipating the overt withdrawal signs and the experimental conditions were different during development and testing of tolerance. In our study, however, nondependent animals were used for the testing of development of hypothermic tolerance to ethanol and assessment of tolerance occurred in the same condition as peptide or ethanol treatment. These factors may account for the diversity in the data. The development of tolerance can represent a conditioned response to ethanol and environmental cues. It seems likely, if given before the first ethanol challenge, both OXT and LVP block the development of tolerance to the hypothermic effect of ethanol. The inhibition of tolerance cannot be attributed to an altered sensitivity to ethanol, since neither LVP nor OXT changed the initial response to ethanol (hypothermia, sleep onset and sleep duration) after the first ethanol challenge, as found by Hoffman et al. [6]. It seems unlikely that the tolerance development was affected by vascular responses (vasoconstriction, hypertension) elicited by peripheral administration of neurohypophyseal peptides since l IU LVP and OXT blocked the development of tolerance given before every ethanol challenge, while LVP facilitated it when the tolerance had fully developed and the peptide was injected thereafter. The same dose of OXT was ineffective in the latter experiment. The background for the different modes of action of the two peptides in these experimental conditions needs further investigations. A single bolus injection of OXT or LVP, given before the Fwst ethanol challenge, was not sufficient to block the development of tolerance. After single injection of 0.5 IU peptide/animal on day 1, the hypothermic tolerance was found to be fully developed by day 3, similar to control animals. It is known that neurohypophyseal peptides cause long lasting effect in neurochemical and behavioral parameters [3, 20, 21] though their effects are not likely to persist for 72 hr after administration.

NEUROHYPOPHYSEAL

PEPTIDES

573 TABLE 5

THE EFFECT OF SINGLE GRADED DOSES OF LYSINE VASOPRESSIN ON ETHANOL TOLERANCE IN MICE Hypothermia Induced by Ethanol (°C) Days Day 1 Day 2 Day 3 ANOVA"

control

1 IU

0.1 IU

0.01 IU

Statistical Analysis (ANOVA ,')

3.20 _ 0.30* (9) 1.90 ___0.37 (9) 1.12 ± 0.34 (9)

2.33 _+ 0.25 (10) 1.41 __ 0.34 (10) 0.83 _+ 0.33 (10)

2.42 __ 0.34 (9) 1.63 ± 0.30 (9) 1.59 ± 0.30 (9)

2.29 ± 0.35 (9) 1.56 - 0.31 (9) !.46 ± 0.22 (9)

NS NS NS

day 1 vs. days 2 and 3

day 1 vs. days 2 and 3 day 2 vs. 3

day 1 vs. days 2 and 3

day I vs. days 2 and 3

t

t

t

t

* mean _+ SEM (°C) (number of animals used). ANOVA" one-way analysis of variance: comparison between treatments. ANOVA ~' two-way analysis of variance: comparison within a treated group. NS not significant. t p<0.05. Peptide treatment was made on day 3 when the tolerance to ethanol has developed.

In c o n c l u s i o n , t h e d a t a s u g g e s t t h a t O X T b l o c k s the early d e v e l o p m e n t a l p h a s e o f t o l e r a n c e to e t h a n o l . L V P c a u s e s b i m o d a l effect o n t h e d e v e l o p m e n t o f t o l e r a n c e : a small d o s e facilitated while h i g h e r d o s e s i n h i b i t e d the h y p o t h e r m i c t o l e r a n c e . W h e n t h e effect o f the t w o p e p t i d e s was investig a t e d in fully t o l e r a n t a n i m a l s , L V P facilitated t h e e x p r e s sion o f t o l e r a n c e , while O X T w a s ineffective. T h u s different p h a s e s o f e t h a n o l t o l e r a n c e are differentially affected b y

n e u r o h y p o p h y s e a l h o r m o n e s . In g e n e r a l , the t w o p e p t i d e s exerted an opposite action on ethanol tolerance. ACKNOWLEDGEMENTS This work was supported by the Scientific Research Council, Hungarian Ministry of Health (No. 16/4-10/502/'I"). We are indebted to Dr. Krisztina Boda for computing the data and to Mrs. Katalin Kov:~cs for skilled technical assistance.

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