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Pharmacological Research Communications, Voi. 16, No. 5, 1984 495 MORPHINE PREFERENCE IN RATS PREVIOUSLY MORPHINE DEPENDENT Dai, S., Hui, S-C.G. and Ogle, C.W. Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong. Received in final form 5 December 1983 SUMMARY Morphine preference and tendency to relapse to morphine tolerance and dependence were studied in rats which were previously made morphine dependent. Tolerance to, and physical dependence on, morphine were initially produced by administration of increasing concentrations of morphine sulphate in 5% sucrose solution for 3 weeks. A test for drinking preference was performed 4 days after the rats had been successfully detoxified and showed no significant sig~Is of morphine dependence. It was found that, while control animals drank only negligible amounts of morphine solution, previously morphine-dependent rats consumed significantly larger volumes of morphine solution and had recurrence of morphine tolerance and dependence. The present findings show that chronic administration of morphine in drinking fluid produces tolerance and physical dependence as well as addiction in rats; the latter definition is exemplified by these animals having a high tendency to relapse after successful drug withdrawal. Introduction It has been suggested that the term addiction should be re-defined as a behavioural pattern of drug use which 0031-6989/84/050495-17/$03.00/0 © 1984 Thel~lianPharmacologJcalSociety

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Page 1: Morphine preference in rats previously morphine dependent

Pharmacological Research Communications, Voi. 16, No. 5, 1984 495

MORPHINE PREFERENCE IN RATS PREVIOUSLY MORPHINE DEPENDENT

Dai, S., Hui, S-C.G. and Ogle, C.W.

Department of Pharmacology, Faculty of Medicine, University of Hong Kong, 5 Sassoon Road, Hong Kong.

Received in final form 5 December 1983

SUMMARY

Morphine preference and tendency to relapse to morphine

tolerance and dependence were studied in rats which were

previously made morphine dependent. Tolerance to, and

physical dependence on, morphine were initially produced

by administration of increasing concentrations of morphine

sulphate in 5% sucrose solution for 3 weeks. A test for

drinking preference was performed 4 days after the rats

had been successfully detoxified and showed no significant

sig~Is of morphine dependence. It was found that, while

control animals drank only negligible amounts of morphine

solution, previously morphine-dependent rats consumed

significantly larger volumes of morphine solution and had

recurrence of morphine tolerance and dependence. The

present findings show that chronic administration of morphine

in drinking fluid produces tolerance and physical dependence

as well as addiction in rats; the latter definition is

exemplified by these animals having a high tendency to

relapse after successful drug withdrawal.

Introduction

It has been suggested that the term addiction should

be re-defined as a behavioural pattern of drug use which

0031-6989/84/050495-17/$03.00/0 © 1984 Thel~lianPharmacologJcalSociety

Page 2: Morphine preference in rats previously morphine dependent

496 Pharmacological Research Communications, Vo/. 16, No. 5, 1984

is characterized by overwhelming involvement with the use

of a drug, the securing of its supply, and a high tendency

to relapse after withdrawal. By this definition, the

term addiction cannot be used interchangeably with

physical dependence. Thus, it is possible to be physically

dependent on drugs without being addicted to them, and,

in some special circumstances, to be addicted without being

physically dependent (Jaffe, 1980).

The production of tolerance to, and physical dependence

on, morphine under laboratory conditions has been successfully

accomplished in various experimental animals (Longo and

Napolitano, 1953; Huidobro and Maggiolo, 1956; Martin, 1961;

Gunne, 1963; Stolerman and Kumar, 1970; Bhargova and Way,

1972; Fuentes et al., 1978; Gellert and Holtzman, 1978;

Badawy et al., 1982). However, information regarding the

tendency to relapse after successful withdrawal is lacking.

Thus, if re-definition of the term addiction is accepted,

past findings cannot be said to show clearly that the

experimental animal can indeed be addicted to morphine.

This study examines the extent of morphine preference

and the tendency for recurrence of morphine tolerance and

dependence in rats which have successfullybeen withdrawn

from the drug. It attempts to evaluate whether morphine

addiction can be produced in experimental animals.

Materials and methods

I. General

Male Sprague-Dawley rats, weighing 140-160 g, were used.

They were kept in groups of 3-4 per cage in a rat battery,

Page 3: Morphine preference in rats previously morphine dependent

Pharmacological Research Communications, Vol. 16, No. 5, 1984 497

and allowed free access to a standard laboratory diet of

Purina rat chow (Ralston Purina Co., U.S.A.) and to

drinking water. The animals were housed in an air-conditioned

room where temperature was maintained at 23±IOC and relative

humidity at 60-70%, and were exposed to a constant dark-

light cycle.

2. Administration of morphine

In order to mask the bitter taste of morphine sulphate,

5% w/v sucrose in ordinary tap water was used as drinking

fluid. Morphine sulphate (Macfarlan) was chronically

administered in this drinking fluid which was provided

ad libitum. The drug was given in increasing concentrations

(48 h apart) of 0.i, 0.2, 0.3, and finally 0.4 mg/ml

(expressed as the salt). The rats continued to receive

the final concentration of morphine sulphate until the end

of the 3-week experimental period. Control animals received

sucrose solution, but without morphine sulphate as drinking

fluid.

Body weight was measured in all rats. Fluid and food

intakes were estimated for each animal hy taking the average

consumption of the 3 or 4 rats in each cage.

3. Morphine tolerance

The tail-immersion test was used for detecting the

development of morphine tolerance (Badawy et al., 1982).

Morphine sulphate 2, 4, or 8 mg/kg, or an equivalent

volume of 0.9% w/v NaCI solution (saline), was injected

intraperitoneally into the control and chronically morphine-

treated rats. Twenty minutes later, the terminal 3 cm of

Page 4: Morphine preference in rats previously morphine dependent

498 Pharmacological Research Communications, Vol. 16. No. 5, f984

their tails were immersed in water at 55oC. The time

elapsing between immersion and flicking of the tails was

then recorded as the reactien time. This test was done on

day 16 of the 3-week period used for inducing dependence

on the drug.

4. Morphine withdrawal

The method of Collier et al. (1974), with slight

modifications, was adopted for assessing the withdrawal

syndrome. Immediately after intraperitoneal injection of

naloxone HCI (Endo) I mg/kg (expressed as the salt), the

following behavioural parameters were observed for 20 min-

wet-dog shakes, head shakes, diarrhoea, ptosis, chattering

teeth, writhing, chewing, paw tremor and irritability to

touch and handling. The number ~ of parameters shown by

each rat was recorded as the number of withdrawal signs.

The amount of faeces excreted and body weight loss, at 20 min

and 4 h respectively, after naloxone injection were also

measured. No fluids or food were allowed before these

measurements were completed. Thereafter, the animals

were allowed free access to rat chow and to ordinary tap

water without sucrose or morphine sulphate. Naloxone

was administered to both control and chronically morphine-

treated rats; an equivalent volume (2 ml/kg) of saline was

injected into separate groups of control and chronically

morphine-treated animals. The first naloxone challenge,

to begin morphine withdrawal (0 h), was given on the last

day of the 3-week period used for induction of drug

dependence.

Page 5: Morphine preference in rats previously morphine dependent

Pharmacological Research Communications, VoL 16, No. B, 1984 499

In order to find out when the animals had completely

lost their morphine dependence, the naloxone-precipitated

withdrawal syndrome was tested for once daily, starting

at 10.15 a.m., in 12 control and 20 chronically morphine-

treated rats until 96 h (the 4th day) following withdrawal.

5. Drinking preference

Drinking preference was observed over a period of

i0 days, starting from the 4th day following withdrawal,

in 20 rats previously used as controls (Group I) and in

20 animals previously morphine treated (Group II). The

latter group was considered to be free of morphine

dependence, when no more withdrawal signs were seen after

repeated daily challenges with naloxone. During the test

period, both groups were allowed free access to two bottles

of 5% sucrose solution, one of which contained morphine

sulphate 0.4 mg/ml. Daily fluid intake from each bottle

was recorded.

Tests for morphine tolerance were made on the 8th day

using the tail-immersion test, and for naloxone-

precipitated withdrawal effects at the end of the 10-day

observation period. For comparative purposes, the same

tests were carried out on a group of 20 naive rats.

. Statistical analysis

The data were analysed by means of Student's t-test.

Results

i. Production of morphine tolerance and dependence

Figure I shows the daily fluid intake throughout

Page 6: Morphine preference in rats previously morphine dependent

500 Pharmacological Research Communications, VoL 16, No. 5, 1984

11111

7o

,g

2O

10 a t _ t _ • t , I t

1 2 3 4 5 6 7 $ 9

¢ t t s I l I m • J ~,

10 11 12 /3 1# ]5 15 ]7 18 19 20 21

DAY

FIGURE I

Fluid intake by morphine-treated rats.

o--o, control (n=24); e ~ e , morphine-treated rats (n=36). Values plotted are the means±S.E.M. *P < 0.05, **P < 0.02 when compared with the corresponding control values.

the 3-week experimental period. At the commencement of

the experiment, the daily fluid intakes of the two groups

of animals were essentially similar. Starting from day 5

when the concentration of morphine sulphate in the drinking

sucrose solution was increased to 0.3 mg/ml, the daily

intake of morphine solution by the test group became

less than the intake of plain 5% sucrose solution by the

controls'; statistical significance of the differences

was seen on days 6, 7, 9, I0, 13, 15, 16, and 20. The

average daily intake of morphine sulphate 0.4 mg/ml

solution (from day 7 onwards) was 51.53 ml/rat; the

estimated daily intake of morphine sulphate by the test

group during this period was. therefore, about 82.45 mg/kg.

Page 7: Morphine preference in rats previously morphine dependent

Pharmacologica/ Research Communications, Vol. 16, No. 5, 1984 501

10

9 o l

8

7

5

4

3

- , I I

NS 2 4 6 8

MORPHINE SULPHATE (~/Ko)

FIGURE 2

Effects of acutely administered morphine on the reaction time of the rats using the tail-immersion test.

o--o, control (n=6 for each dose): • e, morphine- treated rats (n=9 for each dose). NS = normal saline. Values plotted are the means±S.E.M. *P < 0.05, **P < 0.01 when compared with the saline control of the same group. +P < 0.01, ++P < 0.001 when compared with the corresponding control values.

Both control and test rats exhibited steady increases

in food intake and body weight over the 3-week period

(data not shown). There were no significant differences

between these two groups of animals except towards the

end of the experimental period when the body weights of

morphine-treated rats appeared to be lighter; statistical

significance was seen only on day 19.

The reaction time to pain, determined by the tail-

immersion test, is shown in fig. 2. Intraperitoneal

injection of morphine sulphate 4 or 8 mg/kg significantly

Page 8: Morphine preference in rats previously morphine dependent

502

Table i.

Pharmacological Research Communications, Vol. 16, No. 5, 1984

Naloxone-precipitated withdrawal effects in rats chronically treated with morphine in drinking fluid.

Faecal weight % Body at 20 min weight

No. Number of after loss at 4 h Treatment of withdrawal injection after

(i.p.) Dose rats signs (g) injection

Controls

Saline 2 ml/kg 12 0.54±0.16 0.67±0.29 3.31±0.43

Naloxone I mg/kg 12 0.75±0.20 0.76±0.12 3.14±0.37

Chronic morphine treatment

Saline 2 ml/kg 16 0.72±0.25 0.84±0.15 3.74±0,57

Naloxone i mg/kg 20 5.04±0.505 4.23±0.575 8.16±0.875

i.p. = intraperitoneal injection. The values are the means ± S.E.M. *P < 0.001 when compared with the corresponding values in

naloxone-injected controls. +P < 0.001 when compared with the corresponding values in

morphine-treated rats injected with saline.

lengthened the reaction time only in the control animals.

The various doses of morphine appeared not to influence the

reaction time of the morphine-treated rats, thus, indicating

that these animals had already developed tolerance to the

analgesic action of acutely administered morphine by day 16.

Table i shows that intraperitoneal injection of

naloxone I mg/kg precipitated marked withdrawal effects

in rats chronically treated with morphine in their drinking

fluid. When compared with the control animals injected with

Page 9: Morphine preference in rats previously morphine dependent

Pharmacological Research Communications, VoL 16, No. 5, 1984 503

saline or naloxone, or with the chronically morphine-

treated rats injected with saline, this group of animals

showed a significantly larger number of withdrawal signs

as well as greater faecal excretion and body weight loss.

It is reasonable to conclude that these rats were physically

dependent on morphine.

2. Morphine withdrawal

At 24 h following the replacement of morphine

sulphate 0.4 mg/ml in 5% sucrose solution with ordinary

tap water as the drinking fluid, intraperitoneal injection

of naloxone i mg/kg again precipitated the withdrawal

syndrome to a significant extent (Table 2). However, the

number of withdrawal signs and the amount of body weight

loss were significantly less than those recorded at

0 h. Repeated injections of naloxone at 48, 72, and

96 h failed to produce significant withdrawal effects

when compared with the control group whose drinking

fluid had also been changed from 5% sucrose solution

to ordinary tap water at 0 h. These findings suggest

that the rats previously treated chronically with

morphine were beginning to show no significant signs

of morphine dependence at about 48 h after drug

withdrawal.

The body weight of each rat was monitored during

this period. It was found that the body weight of the

previously morphine-treated animals significantly

decreased from 265.4±5.2 g to 245.8±5.6 g during the

first 24 h after morphine withdrawal (P < 0.02), whereas

Page 10: Morphine preference in rats previously morphine dependent

Table 2

Naloxone-precipitate4 withdrawal effects in morphine-dependent rats

following removal of morphine from drinking fluids

~n

O 4~

Time after

withdrawal

(h)

Number of withdrawal signs

Morphine-treated

Controls

rats

Faecal weight at 20 min

after injection (g)

Morphine-treated

Controls

rats

% Body weight loss at

4 h after injection

Morphine-treated

Controls

rats

0

0.75±0.20

5.04±0.50 ***

0.76±0.12

4.23±0.57 ***

3.14±0.37

24

0.48±0.16

2.40±0.45"*

0.55±0.14

3.45±0.74"*

3.14±0.36

x]

48

0.70±0.26

1.00±0.37 I I

0.81±0.25

1.06±0.32 'i'!

2.84±0.45

72

0.54±0.20

0.80±0.28 ;I

0.68±0.18

1.38±0.51 ++

3.16±0.40

96

0.56±0.18

0.30±0.15 I I

0.66±0.20

0.53±0.10 ++

3.20±0.46

8.16±0.87 ***

4.77±0.53 ÷

3.60±0.51 ++

2.25±0.52 ++

2.69±0.32 4-+

n=12 in control; n=20 in morphine-treated rats.

The values are the means ± S.E.M.

*P < 0.05, **P < 0.01, ***P < 0.001 when compared with the corresponding values

in naloxone-injected controls.

+P < 0.01, ++P < 0.001 when compared with the corresponding values at 0 h

after withdrawal.

Page 11: Morphine preference in rats previously morphine dependent

Pharmacological Research C.~mmunications, VoL 16, No. 5, 1984 505

-5o

10

1 2 3 ~ 5 6 7 8 9 10

~Y

FIGURE 3

Drinking preference of rats after morphine withdrawal.

Group I = rats previously used as control before the test for drinking preference. Group II = rats previously treated chronically with morphine in drinking fluid before morphine withdrawal and the test, for drinking preference, n=20 in each group, o o, intake of 5% sucrose solution by group I; • •, intake of 5% sucrose solution by group II;

A, intake of morphine sulphate 0.4 mg/ml in 5% sucrose solution by group I; A A, intake of morphine sulphate 0.4 mg/ml in 5% sucrose solution by group II. Values plotted are the means ± S.E.M. *P < 0.02, **~ < 0.01, ***P < 0.001 when compared with the corresponding values in group I. +P < 0.05, ++P < 0.001 when compared with the corresponding values in the same group drinking 5% sucrose solution.

the body weight of the control group changed only

slightly from 275.0±5.4 g to 272.4±5.2 g. In the

following 72 h, no more significant body weight changes

were nbticed in either group of rats.

3. Drinking preference

Figure 3 shows the drinking preference of rats

after morphine withdrawal. When given a choice, the

animals which wer~ previously used as controls (Group I)

Page 12: Morphine preference in rats previously morphine dependent

506 Pharmacological Research Communications, VoL 16, No. 5, 1984

6

S

2

NAIVE RAT

!

II

Z

its 2 q 6 8

MORP~Ii~£ S~LPHATE t'~/Kg)

FIGURE 4

Effects of acutely administered morphine on the reaction time of rats in the tail-immersion test following the test for drinking preference.

Group I = rats previously used as control before the test for drinking preference. Group II = rats previously treated chronically with morphine in drinking fluid before morphine withdrawal and the test for drinking preference, o o, naive rats; • e, group I; A 4, group II. n=5 for each dose in all groups. NS = normal saline. Values plotted are the means ± S.E.M. • P < 0.05, **P < 0.01, ***P < 0.001 when compared with the saline control of the same group. +P < 0.001 when compared with the corresponding values in naive rats. WP < 0.001 when compared with the corresponding values in group I.

showed a..significant preference for 5% sucrose solution

without morphine (P < 0.001 throughout the whole test

period). This behaviour significantly contrasted with

that of rats which were previously.treated chronically

with morphine in their drinking fluid (Group II), These

animals showed preference for 5% sucrose solution only

Page 13: Morphine preference in rats previously morphine dependent

Pharmacological Research Communications, Vol. 16, No. 5, 1984 507

in the first 2 days. Thereafter, the daily fluid intake

from either bottle was essentially equal. Towards the end

of the 10-day test period, consumption from the bottles

containing morphine sulphate 0.4 mg/ml in 5% sucrose

solution appeared to be greater than that from the

bottles containing plain 5% sucrose solution; statistical

significance was, however, reached only on day 8. When

compared with group I, the animals in group II drank

significantly less plain 5% sucrose solution, but

significantly more morphine sulphate 0.4 mg/ml in 5%

sucrose solution; the average daily intake of morphine

sulphate 0.4 mg/ml solution was 36.85 ml/rat in group II

and 4.70 ml/rat in group I. The estimated daily intake

of morphine sulphate during this period was about

59 mg/kg in group II but only about 7.5 mg/kg in group I.

4. Recurrence of morphine tolerance and dependence

The tail-immersion test was repeated on the 8th day

after the start of the test for drinking preference.

Acutely administered morphine 2, 4, or 8 mg/kg significantly

prolonged the reaction time in a dose-dependent manner in

both the naive rats and animals in group I (Fig. 4)

There were no significant differences in reaction time

between these two batches of rats. On the contrary,

those animals in group II showed no significant responses

to acutely administered morphine, and their reaction time

was significantly shorter than that of naive rats and

of the animals in group I. This finding suggests that

the rats in group II had again developed morphine

tolerance.

Page 14: Morphine preference in rats previously morphine dependent

508 Pharmaco/ogica/ Research Comrnun/cat/ons, Vo/. f6, No. 5, 1984

Table 3.

Naloxone-precipitated withdrawal effects in rats after examination for drinking preference.

Group

Number of withdrawal

signs

Faecal weight at 20 min

after injection (g)

% Body weight loss at 4 h

after injection

Naive rats 0.35±0.12 0.75±0.18 3.42±0.36

I 0,45±0.16 0.97±0.21 3.33±0.33

II 4.50±0.565 4.67±0.695 5.93±0,525

Group I -- rats previously used as control before the test for drinking preference.

Group II-- rats previously treated chronically with morphine in drinking fluid before morphine withdrawal and the test for drinking preference.

n=20 in each group. The values are the means ± S.E.M. *P < 0.001 when compared with the corresponding values

in naive rats. +P < 0.001 when compared with the corresponding values

in group I.

Naloxone challenge was carried out at the end of the

10-day experimental period. Compared with the naive

rats, those in group I showed no significant withdrawal

signs following the injection of the antagonist.

However, in the case of the group II animals, naloxone

induced a markedly larger number of withdrawal signs;

significantly greater faecal praduction and body weight

loss were also seen (Table 3), indicating that physical

dependence on morphine had recurred in these rats.

Page 15: Morphine preference in rats previously morphine dependent

PharmacologicalResearchCommunica~n~ ~L 1~ No.~ 1984

Discussion

Most investigators have produced morphine tolerance

and dependence in experimental animals by repeated

injections of the drug or by implanting it in pellet

form. However, it has been suggested that oral

administration of morphine in drinking water is a better

method of inducing morphine tolerance and dependence

(Fuentes et al., 1978; Badawy et al., 1982). Experimental

animals, however, often refuse to consume significant

amounts of morphine solution, presumably because of its

bitter taste. Attempts have been made to overcome this

problem, and these include scheduled provision of the

drinking solution (Gellert and Holtzman, 1978), masking

the taste of the solution by sucrose (Fuentes et al.,

1978), or a regimen of starting with low concentrations

of morphine solution and gradually increasing the drug

content (Badawy et al., 1982). In the present study,

the latter two methods were combined and found to be

very effective in producing morphine tolerance and

dependence in rats. Sucrose solution was used in this

investigation as the basic vehicle because taste masking

was required during the test for drinking preference,

but sucrose itself is not an essential component of

this morphine tolerance and dependence producing

combination (Khavari and Risner, 1973; Badawy et al.,

1982).

Khavari and Risner (1973) have reported that sucrose

enhances morphine preference by rats. The test for

drinking preference used in the present study showed that

509

Page 16: Morphine preference in rats previously morphine dependent

510 PharmacologfcalResearch Communicat/ons, Vol. 16 No.~ 1984

the control rats drank only negligible amounts of morphine

solution. Thus, it appears that the presence of sucrose

in the morphine solution does not attract animals which

have not previously been morphine-dependent, when they

are given a choice. This suggests that naive rats

dislike and refuse to consume morphine not only because

of its bitter taste but also because of the unpleasant

drug effects, or for other reasons; the latter two

possibilities are more likely if we assume that the

bitter taste of the drug had been completely masked by

the 5% concentration of sucrose solution. The finding

that animals previously treated with morphine, and shown

to be free of drug dependence, consumed a significantly

greater volume of morphine solution during the test for

drinking preference implies that these rats have a high

tendency to relapse if the drug is made available to

them, provided that its bitter taste is masked. It is

interesting to note that even morphine dependent rats

will refuse an 0.4 mg/ml morphine solution if sucrose

is not present (unpublished findings); this agrees with

the observations of Khavari and Risner (1973). This

study also permits the conclusion that morphine addiction,

as defined by Jaffe (1980), can be produced in rats by

chronic administration of morphine solution in drinking

fluid.

Acknowledgements

The authors wish to thank Endo Lab. , N.Y. for the

gift of naloxone, and are grateful to Miss S.Y.N. Lee

and Mr. G.S.K. Man for their technical assistance.

Page 17: Morphine preference in rats previously morphine dependent

Pharmacological Research Communications, Vol. 16, No. 5, 1984 511

References

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