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9 Springer-Verlag 1985
Absence of environment-specificity
in morphine tolerance acquired in non-distinctive environments:
habituation or stimulus overshadowing ?
Richard Dafters and Laura Bach
Psychology Department, Adam Smith Building, Glasgow University, Glasgow Gt2, Scotland
102
differential effects of environment-change observed by
Kesner and Cook (1983), and indeed, an explicit demonstration of overshadowing in the morphine analgesia paradigm has recently been reported (Walter and Riccio 1983).
The first experiment to be reported here sought to evaluate the overshadowing interpretation by attempting to diminish the ability of injection-ritual cues to act as conditioned stimuli and hence to overshadow environmental
stimuli during tolerance development. Rats were pre-exposed to injection-ritual cues (placebo injections) prior to
tolerance acquisition in distinctive or non-distinctive environments and a subsequent environment-change test. Such
pre-exposure to the conditioned stimulus normally retards
subsequent development of the conditioned response - a
phenomenon known as latent inhibition (Lubow 1973). To
the extent that this procedure is effective in preventing overshadowing, and in contrast to the results of Kesner and
Cook (1983), attenuation of tolerance should occur even
in that group which acquired tolerance in a non-distinctive
environment. The experiment also examined the effect on
tolerance of a 14-day " r e s t " period in the colony room
during which all experimental treatments were discontinued. If tolerance reflects habituation of the drug effect it
might be expected, on the basis of previous habituation
research (Groves and Thompson 1970), that some spontaneous recovery of the analgesic response would occur on
the subsequent drug administration test. If tolerance reflects
the development of conditioned responses, no recovery of
the drug effect is to be expected in the absence of explicit
extinction training.
Experiment 2 sought to confirm the finding of Kesner
and Cook (1983) that under standard conditions, where
no pre-exposure to the injection ritual is given, the responsiveness of tolerance to an environment change depends
upon the distinctiveness of the acquisition environment. In
addition, it compared the effect on tolerance of a 14-day
rest period in the colony room with that of an explicit extinction procedure in which injection-ritual stimuli (the putative conditioned stimulus), in the form of placebo injections, were administered unaccompanied by drug (the putative unconditioned stimulus). To the extent that injectionritual stimuli have control over the tolerance response, as
the conditioning theory maintains, tolerance will be attenuated in the extinction group but not in the rested group.
The use of extinction tests in this way to confirm the existence of an environment-drug association has previously
been reported in studies of morphine (Siegel 1975; Siegel
1977) and alcohol tolerance (Crowell et al. 1981).
Materials and method
E x p e r i m e n t 1 - injection ritual p r e - e x p o s u r e . Twelve male
hooded Lister rats obtained from Bantin and Kingman
Ltd., Aldbrough, Hull, England and weighing 400-580 g
at the beginning of the experiment were used. They were
maintained in individual cages on a freely available food
and water schedule. The rats were assigned at random to
two groups ( N = 6) at the start of the experiment. The analgesia-testing apparatus consisted of a standard rat operant
chamber (30 x 30 x 30 cm) with a grid floor composed of
0.32-cm diameter metal rods, through which electric shock
could be delivered from a shock generator (Model 521C,
Campden Instruments Ltd., London) via a constant current
scrambler (Model 521S, Campden Instruments Ltd., Lon-
don) for 0.5 s. The apparatus was located in a room immediately adjacent to the colony room. All injections were
subcutaneous in the dorsal surface of the neck. The dose
used throughout the experiment was 10 mg/kg morphine
sulphate in 10 mg/ml solution of 0.9% physiological saline.
The volume of the physiological saline (placebo) injection
was I ml/kg.
The design of the experiment is shown in Table 1. During the initial pre-exposure phase (14 sessions) all rats were
weighed daily in the colony room, administered a placebo
injection, and immediately returned to the home cage. On
each daily session of the tolerance acquisition phase (14 sessions) rats were weighed and injected in the colony room
and then immediately transported in a mobile cage-rack
into the adjacent room containing the testing apparatus.
On alternate sessions this room constituted the distinctive
and non-distinctive environment. On distinctive-environment sessions the room was illuminated with flashing light
from a Griffin xenon stroboscope (2 flashes/s) and a white
noise stimulus of 70 dB was continuous; on non-distinctive
sessions the room was illuminated with white light from
an overhead 150-W light source identical to that in the
colony room, and the white noise was absent.
The rats remained undisturbed in the test room for
30 min before being placed in the analgesia-testing apparatus. During the test each rat was placed on the shock grid,
and after a 60-s adaptation period, received an ascending
series of 0.5-s footshocks. The intershock interval was usually 6 s but shocks were only administered when all four
paws were in contact with the grid. Shocks started at
0.5-mA intensity and increased in 0.5-mA steps until a vocalisation response (defined as any detectable vocalisation)
was obtained. At this point the rat was removed from the
test box and returned to its cage and the shock intensity
recorded. If a rat failed to make the criterion response when
a current of 2.5 mA was delivered, the trial was terminated
and an intensity of 2.5 mA recorded. Vocalisation threshold
was used because it has been reported as being a m o r e
sensitive measure in this apparatus than either flinch or
jump thresholds (Kesner and Cook 1983). After testing,
the rats remained in the test room until a total of 60 rain
had elapsed since they first entered it, and were then returned to the colony room. The only difference between
the treatments of groups M-DIST and M - N O N D I S T in
this phase of the experiment concerned the relationship of
drug and environment. Thus, on distinctive-environment
sessions group M-DIST received morphine and group MN O N D I S T received placebo; on non-distinctive-environment sessions, group M-DIST received placebo and group
M - N O N D I S T received morphine.
On successive days following the tolerance acquisition
phase, each group experienced an environment-change test.
On the first test (T1) both groups were adinistered morphine and tested in the distinctive environment - this constituted an environment-change condition for group MN O N D I S T ; on the second test (T2) both groups were administered morphine and tested in the non-distinctive environment - this constituted an environment-change condition for group M-DIST. For the six sessions following these
tests the subjects were again exposed to the regimen of
alternating distinctive and non-distinctive sessions which
they had experienced during tolerance acquisition. This
reacquisition phase was intended to counteract any disturbances to the established environment-drug associations
103
Table 1. Experimental design
Injection-ritual
pre-exposure
(14 sessions)
Tolerance
acquisition (A)
(14 sessions)
Environment-specific tests
(T1)
(T2)
M-DIST
P-COLONY ROOM
M-DIST
M-NONDIST
M-NONDIST
P-COLONY ROOM
M-DIST/
P-NONDIST
M-NONDIST/
P-DIST
M-DIST
M-NONDIST
Group
Reacquisition
(six sessions)
Post-rest
tolerance test
(PRT)
M-DIST/
P-NONDIST
M-NONDIST/
P-DIST
M-DIST
M-NONDIST
[] M-DIST
"] TEST
/
M-NONDISTJ
DAYS
9
H
M-
DIST
MORPHINE
M-DIST
PLACEBO
I1- 9
7-.5
M-NONDIST
MORPHINE
M- NONDIST
PLACEBO
e-e
Results
As Fig. 1
shows, tolerance (a decrease in vocalisation threshold)
clearly develops in both groups during the morphine sessions of tolerance acquisition. There appears to be no consistent change in either group in placebo sessions. Analysis
of variance of vocalisation scores with Group (M-DIST
vs M-NONDIST), Treatment (morphine vs placebo), and
Sessions as factors revealed significant main effects of
Treatment [F(1,10)=93.0,
P<0.001]
and Sessions
[F(6,60)=10.9, P<0.001] and a significant Treatment x
Sessions interaction [F(6,60)=12.9, P<0.001]. No other
main effects or interactions approached significance ( P >
0.05 in all cases). A Test for Simple Main Effects revealed
that the Treatment x Sessions interaction was due to a decrease in vocalisation thresholds over morphine sessions
[F(6,60)=22.72, P<0.001] but not over placebo session
(P>0.1).
Experiment
injection-ritual
pre-exposure.
~o
1.5
~"
z %0
.o
N
~ o,5
o
o.o
I
A
J. .L
TI T2
I
R
.L
PAT
quisition sessions; 7"1 environment-change test 1 ; T2 environmentchange test 2; R reacquisition sessions; P R T post-rest test. Test
data shown + SEM
Figure 1 shows that both groups demonstrated a
marked attenuation of tolerance (increased vocalisation
thresholds) when tested in an environment-change condition (T1 for group M - N O N D I S T ; T2 for group M-DIST).
The attenuation was examined statistically by analysis of
variance with Group and Test-environment as factors (the
latter is a comparison of scores on the environment-change
test with those on the last drug session of the acquisition
phase). The analysis revealed a significant main effect of
Test-environment [F(I,10) = 23.7, P < 0.001] but no main
effect of Group or interaction of factors (P>0.05 in each
case).
To check that tolerance levels in the two groups had
not been differentially affected by the test sessions and were
therefore equal prior to the final phase of the experiment,
analysis of variance was conducted on the morphine sessions of the reacquisition phase with Group and Sessions
as factors. Neither main effect nor their interaction was
104
9 EXTINCTION
separate analyses of placebo and morphine sessions, to derive from a significant group difference over placebo sessions
[F(6,60)=4.2, P<0.01] but not over morphine sessions
A REST
[] N I - D I S T
O NI-NONDIST
N ~ I ! Nl- D I ST
MORPHINE
N--IIM-DIST
PLACEBO
2.5
~Nl-
NONDIST
MORPHINE
O - O M - NONDISsT
PLACEBO
q
I
~2.0
O
I11
n.
~1.0
N
< 0.5
(J
O
>
0.G
.L J.
T1 T2
I J..L
PRT PET
Fig. 2. Mean vocalization threshold scores in Experiment 2. A acquisition sessions; T1 environment-change test 1 ; T2 environmentchange test 2; R reacquisition sessions; P R T post-rest rest; P E T
post-extinction test. Test data shown_+ 1 SEM
significant ( P > 0 A in all cases). The effect of the final
14-day " r e s t " phase was assessed by analysis of variance
with Group and Stage as factors (the latter comparing
scores on the post-rest tolerance test (PRT) with those on
the last morphine session of reacquisition). No main effects
or interactions were significant ( P > 0.1 in all cases).
Thus, in Experiment t the groups differed neither in
the acquisition of tolerance nor in the degree to which tolerance was attenuated by a change in the environmental stimuli accompanying the drug administration. There was no
evidence of spontaneous recovery of the analgesic response
after the 14-day rest period in either group.
E x p e r i m e n t 2 - no p r e - e x p o s u r e . As Fig. 2 shows, tolerance
developed in both groups and analysis of variance with
Group (M-DIST vs M-NONDIST), Treatment (morphine
vs placebo), and Sessions as factors revealed significant
main effects of Treatment [F(1,10)=81.8, P<0.001] and
Sessions [F(6,60)=23.04, P<0.001] but not Group ( P >
0.05). A significant Treatment Sessions interaction
[F(6,60)=5.48, P<0:001] was found in a subsequent test
for Simple Main Effects to be due to a decrease in thresholds over morphine sessions [F(6,60)= 23.5, P < 0.001] but
not over placebo sessions (P>0.05). There was an unexpected Group x Sessions interaction [F(6,60)= 2.5, P < 0.05]
and a significant Group x Treatment x Sessions interaction
[F(6,60) =2.68, P < 0.05]. These interactions were found, on
(e > 0.1).
In contrast to Experiment 1, no evidence of attenuation
of tolerance on the environment-change test (T1 and T2)
was obtained. Analysis of variance with G r o u p and Testenvironment as factors showed both main effects and their
interactions to be non-significant (P > 0.2 in all cases).
The difference between the effects of the environmentchange in Experiments 1 and 2 attests to the efficacy of
injection-ritual pre-exposure in modifying stimulus control
of tolerance. However, this conclusion is weakened somewhat by the fact that it requires an interexperiment comparison. Stronger evidence would be provided by combining
the environment-change data from the two experiments and
performing a single, factorial analysis - a legitimate procedure since the Subjects in the two experiments were treated
identically except for the variable under examination (the
presence or absence of injection-ritual pre-exposure). Thus,
an Analysis of Variance was performed on the combined
data with Pre-exposure condition (pre-exposure/no pre-exposure), Drug-environment (M-DIST/M-NONDIST), and
Test-environment (same/changed) as factors. There was a
significant main effect of Test-environment IF(l,20) = 5.07,
P < 0.05], and a significant interaction of Pre-exposure condition Test-environment IF(I,20)= 14.1, P<0.001]. No
other main effects or interactions reached significance (P >
0.2) in all cases. The significant interaction was examined
further by means of Simple Main Effects analysis, which
showed that the environment-change manipulation was effective in reducing tolerance only in the injection-ritual preexposed Subjects (F(1,20) = 9.13, P < 0.01].
As in Experiment 1, analysis of morphine session scores
during reacquisition in Experiment 2 showed that in neither
group had tolerance been affected by the environmentchange tests, and that both groups were demonstrating
equivalent levels of tolerance prior to the rest phase [Group,
Sessions, and Group Sessions effects were all non-significant (P>0.05 in each case)]. Again, as in Experiment 1,
a 14-day rest period in the colony room did not affect tolerance levels in either group - Group and Stage main effects
were non-significant, as was the Group Stage interaction
(P>0.1 in all cases). However, as is clear from Fig. 2, a
difference between the Extinction and Rest groups emerged
on the final post-extinction tolerance test (PET). Analysis
of variance of the extinction phase data, with Group of
origin (M-DIST/M-NONDIST), Treatment (extinction/
rest), and Stage (pre/post Treatment) as factors, revealed
significant main effects of Treatment [F(1,8)=5.26, P <
0.05] and Stage [F(1,8)=7.69, P<0.05], and a significant
Treatment x Stage interaction [F(1,8) = 7.69, P < 0.05]. A
test for Simple Main Effects on the interaction revealed
that groups EXT and REST differed following, but not
prior to, the extinction/rest phase [F(1,8)= 37.5, P < 0.001],
and that pre- vs post-treatment scores differed only for
group EXT [F(1,20=46.16, P<0.001].
Discussion
Experiment 1 showed that following prior exposure to injection-ritual cues in the absence of drug, stimulus control
of tolerance, as assessed by responsiveness to an environment-change test, is gained by the drug-associated environ-
105
ment irrespective of its distinctiveness. This lends support
to the view that, in studies where pre-exposure to injectionritual cues has not been given (e.g. Kesner and Cook 1983),
failures to demonstrate environment-specific tolerance may
be due to the overshadowing of relatively non-distinctive
environmental stimuli by injection-ritual stimuli, rather
than to the operation of an independent habituation process. The same experiment found that a 14-day rest period
in the colony room did not affect tolerance levels in groups
trained in either distinctive or non-distinctive environments
a result in accord with a classical conditioning account
of tolerance and which argues against an habituation interpretation. According to the latter, loss of tolerance should
occur in the non-distinctive group due to spontaneous recovery of the habituated analgesic response; according to
the former, loss of tolerance would not be expected in either
group in the absence of explicit extinction procedures (i.e.
exposure to the drug-associated stimuli in the absence of
drug).
Experiment 2 provided further confirmation of the conditioning/overshadowing analysis by showing that where
the salience of injection-ritual stimuli is not reduced by a
pre-exposure procedure such stimuli do indeed gain control
of the tolerance response and overshadow drug-associated
environmental stimuli - a conclusion derived from the ineffectiveness of the environment-change manipulation observed in this experiment. Although it had been predicted,
from the results of Kesner and Cook (1983), that such overshadowing might only occur in the group made tolerant
in the non-distinctive environment, in the present experiment it occurred in both groups. This discrepancy may be
due to differences in the relative salience of environmental
and injection-ritual stimuli between the experiment of
Kesner and Cook (1983) and the present experiments it is likely, for example, that the salience of injection-ritual
stimuli will be affected by Experiment-specific factors such
as the roughness of handling and the degree of discomfort
caused by the injection itself. Such factors were, of course,
held constant in the present experiments, and the only variable which can plausibly explain the differential effectiveness of the environment-change in Experiments 1 and 2 is
the presence or absence of injection-ritual pre-exposure.
Experiment 2 confirmed the finding of the first experiment that 14 days' rest in the colony room, in the absence
of all experimental treatments, did not attenuate tolerance
in either group, but showed that subjects receiving an additional 14 days of extinction (placebo injections) did show
reduced tolerance, unlike subjects receiving a further period
of rest. This finding also lends support to the conditioning/
overshadowing view - the compensatory conditioned response thought to mediate tolerance, like all conditioned
responses, should be reduced by an extinction procedure
but not by mere passage of time.
The results reported here should be taken as indicative
of an associative mechanism in tolerance (as opposed to
a non-associative one), and not as evidence for classical
conditioning as opposed to habituation. One reason for
emphasizing this distinction is that the classical conditioning model requires evidence, independent of a reduced drug
effect per se, of a learned response that is compensatory
for the initial drug effect. Such evidence was not obtained
or sought in the present experiments and has proved to
be elusive in the literature generally (Hughes and Bardo
1978; Morris et al. 1981 ; La Hoste et al. 1980; Tiffany et al.
1983): A second reason for emphasizing an associative versus non-associative distinction as opposed to a conditioning
versus habituation one is that a recently proposed theory
of tolerance suggests that associative tolerance phenomena,
such as the environment-specificity effect and the overshadowing effect reported here, which have traditionally been
taken as evidence for the classical conditioning theory, can
be explained by an habituation model (Baker and Tiffany
1985). This model also emphasizes the difference between
tolerance acquired in distinctive and non-distinctive environments but accounts for these differences in terms derived
from a powerful theory of habituation proposed by Wagner
(1976, 1979, 1981). According to this theory, habituation
occurs to a stimulus which is already represented or
"primed" in short-term memory, and an important feature
of the theory is that a stimulus may be primed in memory
either non-associatively, by a prior recent presentation of
the stimulus (drug) itself (self-generated priming), or assodatively, by presentation of stimuli (such as a distinctive
context) previously paired with the stimulus (associative
priming). According to this analysis, then, tolerance acquired in a non-distinctive environment primarily reflects
a non-associative habituation process; tolerance in a distinctive environment primarily reflects an associative habituation process. The advantages of this theory over the classical conditioning account are that it does not depend on
the demonstration of compensatory conditioned responses,
and it can account for some features of tolerance which
are not predicted by the conditioning account (Baker and
Tiffany 1985).
Whether or not the habituation theory proves to be
a more satisfactory account of tolerance than the classical
conditioning model, an important implication of the experiments reported here is that no conclusion regarding the
associative or non-associative properties of tolerance may
be drawn from the outcome of an environment-change test
unless precautions are taken to prevent unauthorised procedural stimuli (such as injection ritual cues) from entering
into an association with the drug.
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