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