Placebo-induced analgesia in an operant pain model in rats
Todd A. Nolana, Donald D. Priceb, Robert M. Caudleb, Niall P. Murphyc, John K. Neuberta,⇑
aCollege of Dentistry, Department of Orthodontics, University of Florida, Gainesville, FL, USA
bDepartment of Oral Surgery, University of Florida, Gainesville, FL, USA
cDepartment of Psychiatry and Biobehavioral Sciences, UCLA, Los Angeles, CA, USA
Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article.
a r t i c l e i n f o
Received 10 January 2012
Received in revised form 28 March 2012
Accepted 24 April 2012
a b s t r a c t
Analgesia is particularly susceptible to placebo responses. Recent studies in humans have provided
important insights into the neurobiology underlying placebo-induced analgesia. However, human studies
provide incomplete mechanistic explanations of placebo analgesia because of limited capacity to use cel-
lular, molecular, and genetic manipulations. To address this shortcoming, this article describes the devel-
opment of a rat model of conditioned analgesia in an operant pain assay. Specifically, rats were
conditioned to associate a placebo manipulation with the analgesic effect of 1 mg/kg morphine (subcu-
taneously) on facial thermal pain. We found that conditioned (placebo) responding bore 3 of the hall-
marks of placebo-induced analgesia: (1) strong interanimal variability in the response, (2) suppression
by the opiate antagonist naloxone (5 mg/kg subcutaneously), and (3) a positive predictive relationship
between the unconditioned analgesic effect and the conditioned (placebo) effect. Because of the operant
nature of the assay and the use of only a mild noxious thermal stimulus, we suggest that these results
provide evidence of placebo-induced analgesia in a preclinical model that utilizes an affective behavioral
end point. This finding may provide opportunities for invasive preclinical studies allowing greater under-
standing of placebo-induced analgesia, thus paving the way for avenues to harness its benefits.
? 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
Placebo effects have been observed across diverse clinical stud-
ies, including immune responses [1,26,49], Parkinson disease ,
drug abuse,andin particular
[2,4,9,10,18,19,35,38–40,50,51,54,60,62,65]. In this context, the
placebo effect is usually defined as a sham or simulated medical
intervention that improves a given outcome, such as pain relief
. Although placebo effects are often viewed as confounding in
clinical trials , understanding this phenomenon could provide
valuable insight into psychosomatic effects, which in turn could
be harnessed for therapeutic benefit.
The causes of placebo effects have been the subject of much
discussion. Major mechanistic factors include learning, percep-
[2,5,9,14,22,50,52]. However, one factor that seems to apply to
placebo responses across species is that of conditioning, including
pain and analgesia
Pavlovian conditioning. Indeed, Gliedman et al.  forwarded
this account as early as 1957. Subsequently, Herrnstein  pro-
vided early preclinical evidence supporting this hypothesis, show-
ing that a conditioned stimulus (i.e., placebo) paired with the
amnesic agent scopolamine causes animals to perform poorly in
a memory test. Certainly, when viewed in the context of classical
conditioning, many examples of such placebo responses can be
found throughout the fields of learning, memory, and addiction
studies have focused on placebo-induced analgesia. Thus, we
present here evidence of placebo-induced analgesia in a preclini-
As far as we are aware, there have been only 2 major preclinical
studies of placebo-induced analgesia using animal models. Both
studies used reflex-based pain assays and relatively high doses of
opiate drugs [11,29]. In the first, Bryant et al. showed that placebo
induced analgesia in mice conditioned with the potent opiate fen-
tanyl, measured using a hot plate . In the second, Guo et al.
showed that a cue paired with morphine (MOR) or aspirin elicited
analgesic responses, also on a hot plate .
Although these studies provide important foundations for fur-
ther studies of placebo-induced analgesia, we reason that they are
0304-3959/$36.00 ? 2012 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.
⇑Corresponding author. Address: University of Florida, 1600 SW Archer Road,
P.O. Box 100444, Room D7-44, Gainesville, FL 32610-0444, USA. Tel.: +1 352 273
5687; fax: +1 352 846 0459.
E-mail address: JNeubert@dental.ufl.edu (J.K. Neubert).
?153 (2012) 2009–2016
not equipped to assess aversive behaviors indicative of affective
components of pain. The affective or emotional components of pain
are particularly important to chronic pain sufferers and constitute
one of the most common and debilitating aspects [42,57]. In light
of this, we report here the use of an operant-based pain assay for
studying placebo-induced analgesia. In what is essentially a con-
flict-reward assay, rodents express their willingness to withstand
thermalpainto obtain a sweetreward [46–48,56].Because this par-
adigm tests mild noninjurious pain and uses operant responses, we
reason that, unlike the more reflexive tests of other models, this
model better incorporates cognitive and affective components of
pain. As such, this assay could support detailed neurobiological
investigationsof bothspinalandbrainmechanismsof placeboanal-
gesia, including cortical mechanisms known to be critically impor-
tant in humans [6,10,17,21,22,36,50–52,54].
Hairless male Sprague-Dawley rats (250 to 300 g, Charles River,
Raleigh, NC) were used and housed with a cage mate in a standard
12:12-hour light/dark cycle. Water and standard laboratory chow
were available ad libitum when not being tested, and weights were
recorded weekly. Experiments were conducted in accordance with
the guidelines of the Committee for Research and Ethical issues of
the International Association for the Study of Pain on using labora-
tory animals . The University of Florida Committee for the Care
and Use of Animals approved all experimental procedures.
Morphine sulfate (15 mg/mL, uncorrected for molecular salt
and water content, Baxter Healthcare Corporation, Deerfield, IL)
was diluted in phosphate-buffered saline (PBS), pH 7.4 and injected
subcutaneously (SC) at a dose of 1 mg/kg. Naloxone (NLX) hydro-
chloride dihydrate (Sigma, St. Louis, MO) was dissolved in PBS at
25 mg/mL (uncorrected for molecular salt and water content)
and injected subcutaneously at a dose of 5 mg/kg. All drugs were
diluted to a final volume of 0.3 mL with PBS before injection.
2.3. Thermal facial operant testing
A detailed description of the reward-conflict operant testing
paradigm used to assess facial analgesia can be found in our previ-
ous reports [47,56]. Briefly, this used an acrylic testing chamber
with an adjustable slit (approximately 1 to 2 cm wide) opening
lined with aluminum tubing. The tubing served as an adjustable
thermode by circulating heated water via polyethylene tubing. A
standard rodent water bottle containing diluted (1:2 with water)
sweetened condensed milk solution at room temperature (Carna-
tion, Nestlé USA, Glendale, CA) was mounted outside the cage as
a palatable reward to incentivize rats to expose their faces to the
thermode. Room temperature was maintained at 22?C ± 1?C for
all behavioral tests.
Unrestrained animals were placed individually in testing cham-
bers. The reward bottle was positioned such that access to the
sweetened milk reward was possible contingent on facial contact
with the thermode. The metal spout of the watering bottle was
connected to a 13-V DC power supply and, in series, to a data
acquisition module (WinDaq Lite Data Acq DI-194, DATAQ Instru-
ments, Inc., Akron, OH). When licking from the bottle, the skin of
the rat’s muzzle contacted the thermode, and the tongue contacted
the metal spout, thus completing 2 separate electrical circuits that
registered as analog signals referred to as contact and licking
events, respectively [45–47,56].
2.4. Acquisition of operant responding
Animals were fasted for 16 hours before testing sessions. All
sessions were 20 minutes long, separated by 48 hours, and were
conducted between 08:00 and 12:00. Animals were first trained
with the thermodes set to the nonnoxious temperature of
37?C ± 1?C (see Fig. 1A for overview) for 6 sessions to reach criteria
previously described . Subsequently, animals underwent a sin-
gle training session at 48?C ± 1?C (termed BASE48). This session
served to introduce animals to the noxious temperature at which
analgesia testing would ultimately be performed, as well as to ob-
tain baseline measurements for subsequent data normalization.
This session was followed by 1 additional session at 37?C ± 1?C.
2.5. Induction of placebo-induced analgesia
The first 2 sessions after the training described previously were
performed with the thermode set at 48?C and were designed to
condition animals to associate the handling procedure with mor-
phine-induced analgesia (Fig. 1B). The handling procedure itself
consisted of the SC injection procedure and light shaking of the
animal for 3 seconds. These 2 sessions were referred to as COND1
and COND2, respectively. After this, a final session referred to as
TEST was performed (also at 48?C), in which rats were adminis-
tered PBS (SC) or naloxone (SC). Throughout all 3 sessions (COND1,
COND2, and TEST), rats were injected PBS or drugs 30 minutes be-
fore exposure to the training apparatus, during which time they re-
mained in their home cages. The main experimental groups are
described in Fig. 2B. Additional groups of rats were included to
compare any conditioned effects of morphine with unconditioned
effects. In this case, rats were injected with either morphine or
PBS (controls) in their home cages 24 hours before COND1 and
COND2 training sessions.
2.6. Data and statistical analyses
The primary outcome measure in this study was the ratio of
successful licks to lick attempts, termed a success ratio. This ratio
reflected the number of successful lick events occurring coincident
with facial contact with the thermode. We have previously re-
ported that this measure is sensitive to manipulations altering pain
perception. For instance, increasing the thermode temperature de-
creases the number of successful licks, whereas analgesics increase
the number of successful licks . Here, success ratios during
COND1, COND2, and TEST are presented normalized as a percent-
age of those during BASE48 within animals to account for be-
tween-subject variability in unconditioned responding.
Various statistical analyses were applied to investigate the ef-
fects of drug treatment, the relationship between responding at
various time points, and the probability of specific responses
occurring. These included repeated-measures analysis of variance,
linear regression,v2tests, Student t tests, and F-tests. The rationale
for applying specific methods of analyses is introduced in the rele-
vant sections later. All statistical analyses were made using Stat-
view (Abacus Concepts, Berkeley, CA), Microsoft Excel, or online
resources. Unless otherwise stated, data are presented as the
mean ± standard error of the mean (SEM) and probability (P) val-
ues <.05 were considered statistically significant.
3.1. Effects of drug treatment on responding during conditioning
Based on our previous studies, rats were considered trained
upon reaching 1000 successful licks during a 20-minute testing
T.A. Nolan et al./PAIN
?153 (2012) 2009–2016
session with the thermode heated to 37?C . In the current
study, rats attained a mean of 2443 ± 571 licks per 20-minute ses-
sion when considered across all groups before drug treatments. A
single rat was removed from the MOR ? NLX group because of ex-
tremely high (649%) responding during COND1, which proved to
be an outlier according to the Grubbs test.
Fig. 2A shows the normalized success ratios during COND1,
COND2, and TEST in rats conditioned to 1 mg/kg SC morphine. Data
Fig. 1. (A) Schematic representation of the behavioral paradigm used. Boxes represent exposure to the operant training apparatus wherein rats had access to palatable reward
contingent on contacting their muzzles to a heated thermode stimulus. Variations of phosphate-buffered saline (PBS) vehicle, morphine (MOR, 1 mg/kg), or naloxone (NLX,
5 mg/kg) were administered 30 minutes before each of the final 3 sessions. (B) Summary of major groups in the current study.
Fig. 2. (A) Mean operant licking responses for palatable reward under various drug treatment conditions as described in Fig. 1B. Dashed line indicates responding in the
PBS ? PBS-treated group during COND1 as a point of reference. Data are expressed as responses normalized to mean responding during BASE48 as depicted in Fig. 1A within
each treatment group. Data are expressed as mean ± SEM. Group sizes (n): PBS ? PBS, 8; MOR ? PBS, 19; MOR ? NLX, 10. (B) P values for t test, Mann-Whitney U and F-test
comparisons for responses at all time points shown in A. P values (t test or F-test) <.05 are in boldface type.
T.A. Nolan et al./PAIN
?153 (2012) 2009–2016
are shown normalized to responding in the BASE48 session. Taking
responding as the dependent measure over the 3 testing sessions,
repeated-measures analysis of variance over the 3 sessions showed
a statistically significant main effect of treatment (F2,34 = 4.501,
P = .0184), but no effect of session (F2,68 = 2.582. P = .0831) or
P = .1746). The results of post-hoc t test and Mann-Whitney U com-
parisons between the effects of specific treatments within sessions
are shown in Fig. 2B. These showed statistically significant eleva-
tions in responding in both groups of morphine-treated rats (la-
beled MOR ? PBS and MOR ? NLX in Fig. 2A) during COND2
relative to PBS-treated animals. Notably, the mean success ratio in-
creased approximately 30% between COND1 and COND2 in both
groups of morphine-treated rats. However, as shown in Table 1,
no correlation was found in responding between these sessions.
An additional control experiment was performed to test
whether morphine administration 48 hours before testing influ-
ences operant pain responding per se. For this purpose, morphine
or PBS were injected in the home cage 48 hours before operant fa-
123.2% ± 31.0% and 100% ± 14.5% (n = 6 per group) for morphine-
treated and vehicle-treated rats, respectively. There was no statis-
tically significant difference between these responses (t test,
P = .127).
Animals treated with morphine during COND1 and COND2 and
then treated with PBS during TEST showed approximately 50%
more responding during TEST than animals treated with PBS
throughout. However, t test comparison yielded a P value of .356
due to large variation in responding in the MOR ? PBS group. A
statistically significant difference in the distribution of responding
between the MOR ? PBS and animals treated with PBS throughout
was confirmed using an F-test, which yielded a P value of .045
when comparing these 2 groups during TEST (Fig. 2B). Animals
treated with morphine during COND1 and COND2 and then treated
with NLX during TEST showed slightly less responding during TEST
than animals treated with PBS throughout, suggesting that nalox-
one reversed the enhanced responding during TEST seen in
MOR ? PBS-treated animals. A separate group of rats that did
not undergo conditioning sessions COND1 and COND2 but treated
with 5 mg/kg naloxone (n = 6) showed no difference in responding
per se compared with PBS (n = 7, 101.5 ± 46.7 vs 100.0 ± 20.7, t test,
P = .941), suggesting that the suppressive effects of naloxone pre-
sented in Fig. 2A depended on previous morphine treatment. To-
gether, these data suggest that the enhanced responding seen
during TEST in the MOR ? PBS-treated group depended on in-
creased endogenous opioid activity.
Given the large variability in responses during TEST in
MOR ? PBS-treated rats, a more refined analysis was performed
to determine the characteristics of the distribution of the re-
sponses. Fig. 3 shows the responses of individual rats in the 3
groups of rats during TEST. Comparing the distribution of re-
sponses between MOR ? PBS and PBS ? PBS rats shows that sev-
andsession (F4,68 = 1.639,
success ratios were
eral rats stand out within the MOR ? PBS group as displaying
exceptionally high responses. Given this, we sought to determine
the statistical likelihood of such high responses occurring based
on the assumption that the responses of PBS ? PBS-treated rats
would follow a normal distribution.
First, Anderson-Darling normality testing showed the distribu-
tion of responses during the TEST session of the PBS ? PBS and
MOR ? PBS groups to have P values of .4051 and .0146, respec-
tively (Table 2). This indicated that the PBS ? PBS group displayed
a normal distribution. In contrast, MOR ? PBS-treated animals did
not. The apparent lack of normality in the distribution of responses
in the MOR ? PBS-treated group was most likely due to skewing
toward higher values. This was reflected in higher skewness and
kurtosis values in the MOR ? PBS group (1.48 and 2.23, respec-
tively) than the PBS ? PBS group (1.07 and 1.12, respectively).
Subsequently, the probability of the occurrence of rats respond-
ing at various levels during TEST in the MOR ? PBS relative to
PBS ? PBS-treated rats was estimated. To achieve this, the mean
responding and standard deviation during TEST in PBS ? PBS-trea-
ted rats was calculated (shown as horizontal dashed lines in Fig. 3).
This value was used to predict the expected number of responders
in any given range of standard deviations from this mean based on
a normal distribution, as shown in Table 3. The expected number of
responders was statistically compared to the actual number of
responders using an v2test (Table 3). Based on this method of
Pearson product-moment correlation coefficient (r) and P values for comparisons within groups during specific sessions as depicted in Fig. 1A.
Treatment group COND1 vs COND2COND1 vs TESTCOND2 vs TESTCOND2-COND1 vs TEST
r PBS ? PBS
MOR ? PBS
MOR ? NLX
PBS ? PBS
MOR ? PBS
MOR ? NLX
P values (Pearson test) <.05 are in boldface type.
COND1 = first conditioning session; COND2 = second conditioning session; MOR = morphine; NLX = naloxone; PBS = phosphate-buffered saline; TEST = test for conditioned
Fig. 3. Operant licking responses for palatable reward in individual rats during the
TEST session as described in Fig. 1A. Solid horizontal line indicates mean responding
in PBS ? PBS-treated rats. Dashed horizontal lines indicate increasing standard
deviations (SD) away from the mean responding for the PBS ? PBS-treated group.
T.A. Nolan et al./PAIN
?153 (2012) 2009–2016
responders up to 6 SD from the mean, yielding extremely low P
values using an v2test. No such effects were seen in the
MOR ? NLX group. It is important to note that this analysis was
based on the prediction that TEST responding in the MOR ? PBS
group would be higher than in the PBS ? PBS-treated group, as
would be expected of a placebo response.
duringTEST,MOR ? PBS-treated animalsincluded
3.2. Relationships between operant responding between sessions
Linear regression (Pearson correlation) showed weak nonsignif-
icant relationships between responding during COND1 and TEST
within all groups (Table 1). Additionally, there were no statistically
significant correlations between responding between COND1 and
COND2. However, responding in COND2 was significantly posi-
tively correlated (r = 0.459. P = .048) with responding during the
TEST session in MOR ? PBS-treated animals (Fig. 4), indicating that
on a subject-by-subject basis, animals showing the strongest re-
sponse during COND2 showed the strongest responding during
TEST. Moreover, despite a lack of any significant correlation be-
tween COND1 and TEST, the strongest and most statistically signif-
icant correlation (r = 0.516, P = .024) with TEST was found when
comparing the difference between COND2 and COND1 with TEST.
This indicated that those animals showing the largest increase in
morphine-induced analgesic responses between COND1 and
COND2 showed the strongest conditioned (placebo) response dur-
As far as we are aware, this report is the first presenting placebo
effects in a preclinical operant pain assay. These results suggest
that rats, like humans, show opioid-conditioned placebo analgesia.
We believe this finding provides evidence that, in addition to noci-
ceptive reflexes [42,57], operant responses involving cognitive
functions are also susceptible to placebos. Because these responses
better index the affective dimension of pain, they are especially
relevant clinically [38,40]. We suggest that the approach used here
may provide preclinical researchers a means for studying the affec-
tive dimension of placebo-induced analgesia using an animal
4.1. Characteristics of placebo responses observed in the current study
Placebo effects have several notable features. Among them is
high variability in response size [9,34,53]. To a large extent, the
apparent degree of this variability depends on the criteria used
for defining the placebo response. Over the years, there have been
numerous nuanced definitions of placebo responses , but most
agree that any sham or inert intervention with a beneficial effect
beyond changes that occur due to natural history may be called a
placebo effect [5,22,52]. Defining how large that effect needs to
be is matter of discussion. Some studies specify a specific magni-
tude of symptom change and claim to identify placebo responses
in individual subjects. However, rather than define a specific crite-
rion, an alternative and objective approach is to study effect size, of
which a d statistic is particularly common. In a recent discussion of
this approach applied to meta-analytic findings on placebo effects
in clinical settings, effects sizes for placebo effects of 0.24 and
above were observed . In this study, we estimate the effect size
(d) to be 0.45 (as defined in ), although it is important to note
that relative to clinical studies, our preclinical study relied on con-
siderably fewer subjects, thus decreasing the reliability of effect
Herein, rather than defining a specific criterion, we determined
the likelihood of responses of given magnitudes arising naturally.
We based our analysis on the assumption that responses within
control rats follow a normal (Gaussian) distribution. Indeed, nor-
mality testing suggested this was the case for PBS ? PBS-treated
rats. However, our group sizes were relatively small, which may
have impacted the reliability of the statistical analysis. Nonethe-
less, as such, our analysis showed incidences of responding during
TEST in the MOR ? PBS group that statistically-speaking were ex-
tremely unlikely to occur by chance. We suggest that these rats,
which fell many standard deviations from the mean, could be con-
sidered placebo responders. It is also important to note that
although our analysis was biased toward identifying animals that
responded positively, 1 animal responded more than 1 SD below
the mean of the group treated with PBS throughout.
A second major characteristic of placebo responses is that they
often have strong relationships with genuine (unconditioned) re-
sponses [2,8]. Indeed, we found a statistically significant correla-
tion between responding in the COND2 and TEST sessions in
MOR ? PBS-treated animals. It was notable, and somewhat sur-
prising, that no such correlation existed between COND1 and TEST
in this group. On first look, that correlations between COND1 or
COND2 and TEST sessions might exist may be unremarkable, be-
cause presumably animals should be consistent in their responses
if experimental conditions remain constant. However, this was not
the case in the current study because no correlations were found
between sessions in animals treated with PBS throughout. This
may reflect small but significant day-to-day changes in experimen-
tal conditions beyond the control of the experimenter. If so, that a
correlation was found in morphine-treated animals reinforces the
view that this relationship can overcome daily variation and may
be physiologically important. This relationship may directly reflect
individual differences in the analgesic efficacy of morphine or ef-
fects on learning capability.
A slightly stronger correlation was found between changes in
responding between COND1 and COND2 sessions and responding
during TEST, albeit it only marginally more statistically significant
than the correlation discussed previously. This is interesting when
viewed in the context of recent theories in the psychobiology of
substance abuse, one of which emphasizes that sensitization to
cues predicting the availability of rewarding drugs is critical to
the development of addiction [55,59]. The increased responding
between COND1 and COND2 sessions (rather than tolerance) seen
in both morphine-treated groups is reminiscent of such sensitiza-
tion. Furthermore, that such sensitization best predicted placebo
responding suggests an involvement of incentive learning and
expectancy in acquiring placebo responses. Indeed, our previous
studies show that sensitization of locomotion and mesolimbic
dopamine (a neural pathway considered fundamental to reward
prediction) is predictive of the rewarding effects of drugs .
A third known feature of placebo responses is sensitivity to opi-
oid antagonism [21,29]. We also confirmed this in our operant fa-
cial analgesia model, revealing the involvement of endogenous
opioids by using the opiate antagonist naloxone. As discussed ear-
lier, many have reasoned that placebo effects are fundamentally
Results of Anderson-Darling normality testing for distributions of responses during
COND1, COND2, and TEST.
Treatment group COND1COND2TEST
PBS ? PBS
MOR ? PBS
MOR ? NLX
COND1 = first
MOR = morphine; NLX = naloxone; PBS = phosphate-buffered saline; TEST = test for
Statistically significant P values are in boldface type.
conditioning session;COND2 = secondconditioning session;
T.A. Nolan et al./PAIN
?153 (2012) 2009–2016
similar in their neurobiology to other expectancy effects [8,39]. In-
deed, recent human imaging studies show that the expectancy of
reward is accompanied by increased endogenous opioid activity
in limbic and cortical areas . Additionally, opiate antagonists
such as naloxone and naltrexone block various conditioned effects
on pain [3,21,29]. Genetic deletion of some (but not all) endoge-
nous opioids attenuates the expectancy of rewards [15,23,37,41].
However, an involvement of endogenous opioids does not appear
universal to all placebo effects. For example, Amanzio and Bened-
etti found that naloxone blocks placebo analgesia induced by opi-
oid conditioning but not by conditioning with the nonopioid
ketorolac . Among the few preclinical studies of placebo-in-
duced analgesia, Guo et al. reported that placebo-induced analge-
sia was sensitive to naloxone, but that induced by aspirin was
insensitive . This raises the possibility, as suggested by human
imaging studies discussed earlier, that placebo-induced analgesia
stems from the brain simulating the original (unconditioned) effect
of the drug, an idea also put forward by Guo et al. In the case of
morphine, this would be increased endogenous opioid release, thus
being susceptible to naloxone.
4.2. Application to human placebo responses
Conditioning is one of several causes of human placebo analge-
sic responses, yet other causes also exist [5,9,14,52]. Indeed, recent
studies by Bryant et al. suggest that rodents show analgesic re-
sponses simply by being exposed to neighboring rodents exposed
to opiates . Thus, an operant model of placebo analgesia has
the potential to analyze cognitive and brain mechanisms related
to several causes/types of placebo analgesia, such as the social
observational learning just mentioned, for mice  as well as hu-
4.3. Alternative explanations and limitations
Although this study suggests that rats show placebo responses
in an operant facial pain assay, other factors may be involved. First,
the assay used incorporates several psychological processes that
may act synergistically or competitively before being emitted as
a behavioral output. These include not only pain, but also reward,
incentive motivation, learning, memory recall, and appetite. For in-
stance, previous studies show that opiates can modulate appeti-
tiveness and learning related to rewards [20,33,43,44]. In this
regard, our previous studies indicate that at the dose administered
and over the time scale applied, morphine does not influence lick-
ing per se under nonpain conditions, although more complex inter-
actions may occur. Second, other studies show that morphine can
stimulate locomotion [30,58]. Although we did not perform an
exhaustive analysis here, studies elsewhere show that morphine
can increase locomotion in Sprague-Dawley rats at doses as low
as 1 mg/kg (e.g., ). Such increases in locomotion could encour-
age rats to approach the reward, although these seems unlikely be-
cause again, under nonpain conditions, morphine as applied here
does not influence licking behavior.
4.4. Summary and future implications
Our study introduces a novel preclinical model for studying
placebo-induced analgesia that shows canonical features of pla-
cebo effects observed in humans. Because of its operant nature
and use of mild noxious stimuli only, this paradigm may better
address the emotional aspects of placebo-induced analgesia and
executive cortical control placed over them. In doing so, these
experiments provide the foundation for methodologies that
would be extremely difficult in human studies, such as single
neuron recordings, neurotransmitter depletion, focal lesions, and
Estimation of probabilities (P) of the number of subjects responding at given levels relative to PBS ? PBS-treated rats during TEST given groups size and a normal distribution by v2test.
Subjects during TEST in
PBS ? PBS group
PBS ? PBS (n = 8)
MOR ? PBS (n = 19)
MOR ? NLX (n = 10)
Expected number of
Number of subjects
Expected number of
Number of subjects
Expected number of
Number of subjects
4 ? 10?8
4 ? 10?77
3 ? 10?4
3 ? 10?4
2 ? 10?6
5 ? 10?6
3 ? 10?6
8 ? 10?9
2 ? 10?8
1 ? 10?8
See Results section for complete explanation of analysis method. P values (v2test) <.05 are in boldface type.
MOR = morphine; NLX = naloxone; PBS = phosphate-buffered saline; TEST = test for conditioned responding.
T.A. Nolan et al./PAIN
?153 (2012) 2009–2016
molecular/genetic manipulations. As an example, human studies
have shown that, unlike conditioning with morphine, placebo
analgesia (on a tourniquet tolerance test) generated by condition-
ing with ketorolac was not prevented by naloxone  but was
prevented by a specific cannabinoid antagonist, CB1 . This dis-
tinction could be tested with the operant assay used here and
could lead to its molecular and neuropharmacological character-
ization. Another possibility is that the genetic distinctions be-
tween placebo responders and nonresponders might also be
characterized using this same behavioral assay.
Conflict of interest statement
RC, NPM and JKN are employees of Velocity Laboratories, a com-
pany that provides fee-for-service behavioral testing using operant
T.A.N., N.P.M., and J.K.N. are funded by National Institutes of
Health grant 5R21DA027570-02.
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