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ORIGINAL ARTICLE
Acute nicotine treatment enhances compulsive-like
remifentanil self-administration that persists despite
contextual punishment
Sarah C. Honeycutt | Morgan S. Paladino | Rece D. Camadine |
Ashmita Mukherjee | Gregory C. Loney
Program in Behavioral Neuroscience,
Department of Psychology, State University of
New York, University at Buffalo, Buffalo, New
York, USA
Correspondence
Gregory C. Loney, Program in Behavioral
Neuroscience, Department of Psychology,
State University of New York, University at
Buffalo, 214 Park Hall, Buffalo, NY 14260,
USA.
Email: gcloney@buffalo.edu
Funding information
National Institute on Alcohol Abuse and
Alcoholism, Grant/Award Number: T32:
AA007583; National Institute on Drug Abuse,
Grant/Award Number: DA048336
Abstract
Opioid use disorder (OUD) and opioid-related deaths remain a significant public
health crisis having reached epidemic status globally. OUDs are defined as chronic,
relapsing conditions often characterized by compulsive drug seeking despite the del-
eterious consequences of drug taking. The use of nicotine-containing products has
been linked to increased likelihood of prescription opioid misuse, and there exists a
significant comorbidity between habitual nicotine use and opioid dependence. In
rodent models, nicotine administration nearly doubles the amount of opioids taken in
intravenous self-administration paradigms. Here, we examined the effect of acute
systemic nicotine administration in male rats on responding for the synthetic opioid
remifentanil (RMF) in a contextual punishment paradigm using either an exterocep-
tive punisher (foot-shock) or an interoceptive punisher (histamine). Nicotine adminis-
tration, relative to saline, increased RMF intake in both unpunished and punished
contexts, regardless of form of punishment, and resulted in significantly higher moti-
vation to obtain RMF in the previously punished context, as measured by progressive
ratio breakpoint. Additionally, regardless of context, nicotine-treated rats were
slower to extinguish RMF responding following drug removal and displayed higher
levels of cue-induced reinstatement than saline-treated controls. Furthermore, these
data support that, compared with histamine adulteration, contingent foot-shock is a
more potent form of punishment, as histamine punishment failed to support contex-
tual discrimination between the unpunished and punished contexts. In contrast to
RMF administration, augmentation of responding for an audiovisual cue by nicotine
pretreatment was lost following contextual punishment. In conclusion, acute nicotine
administration in adult male rats significantly enhances compulsive-like responding
for RMF that persists despite contingent punishment of drug-directed responding.
KEYWORDS
comorbidity, conditioning, histamine, motivation, opioid, polysubstance
Received: 18 August 2021 Revised: 31 January 2022 Accepted: 8 March 2022
DOI: 10.1111/adb.13170
Addiction Biology. 2022;27:e13170. wileyonlinelibrary.com/journal/adb © 2022 Society for the Study of Addiction 1of14
https://doi.org/10.1111/adb.13170
1|INTRODUCTION
Nearly 16 million individuals meet diagnostic criteria for an opioid use
disorder (OUD), and opioid-related overdoses contribute to approxi-
mately 120 000 deaths per year worldwide.
1,2
Of note, it is estimated
that approximately 83–95% of individuals currently enrolled in an opi-
oid treatment programme (OTP) are habitual users of nicotine, and
concurrent use of nicotine significantly increases the likelihood of
misusing prescription opioids.
3–5
Furthermore, we have recently dem-
onstrated in rodents that acute nicotine administration prior to opioid
intravenous self-administration (IVSA) nearly doubles the amount of
opioids taken.
6
As such, it is imperative to elucidate mechanisms con-
tributing to the development and maintenance of OUDs and the facili-
tation of opioid addiction liability by nicotine.
A critical component of the development and maintenance of sub-
stance use disorders (SUDs), including OUD, is the formation and recall
of drug-associated memories, including learning about the discrete and
contextual cues associated with opioid use. The reinforcing and aver-
sive properties of drugs of abuse can become associated with various
cues and contexts, and these associations later serve to coordinate
motivated behaviours to either approach or avoid associated stimuli
and even drugs themselves.
7–10
There is ample evidence to demon-
strate that nicotine administration incentivizes approach to cues asso-
ciated with the positive, reinforcing properties of subsequently
administered drugs
11–14
as well as limits avoidance of cues associated
with the aversive consequences of drug administration.
14–20
Given
that SUDs are primarily characterized as chronic, relapsing conditions
and that a major hurdle to sustained abstinence is the high likelihood
of relapse when exposed to environmental cues previously associated
with drug use,
21
it follows that this high approach, low avoidance phe-
notype to drug-associated cues induced by nicotine treatment may
exacerbate the liability for continued drug-seeking in the face of the
aversive consequences of drug use.
Generally speaking, successful recovery from SUDs in humans is
largely dependent on self-imposed abstinence, as opposed to forced
extinction resulting from the complete removal of abused substances
and associated contexts.
22,23
The perceived imbalance between the
adverse and reinforcing properties of continued drug use is often a
significant contributor to self-imposed abstinence.
24
Recently, many
studies have successfully modelled this self-imposed abstinence
through an adaptation of the ABA renewal paradigm, in which absti-
nence is achieved through explicit punishment of drug taking within a
distinct context.
10,25–28
Here, we employed a further adaptation of
this model, in which we alternated the contexts daily rather than
imposing abstinence through repeated presentations of the punished
context at the end of the intravenous self-administration (IVSA) ses-
sions. Specifically, we were interested in the degree to which nicotine
administration would enhance remifentanil (RMF) IVSA in the face of
contingent punishment and what effect, if any, nicotine would have
on the ability to discriminate between unpunished and punished con-
texts. To this end, male rats were tested for RMF self-administration
within two distinct contexts, one in which RMF IVSA was unpunished
and a second in which RMF IVSA resulted in contingent punishment.
Nicotine or saline was acutely administered prior to IVSA sessions.
Additionally, we tested the efficacy of two different forms of punish-
ment in supporting contextual discrimination between the unpunished
and punished environments. In one experiment, we used an extero-
ceptive punisher (foot-shock), and in the second, we used an intero-
ceptive punisher (histamine adulteration). Finally, because nicotine
has been shown to enhance responding for audiovisual
(AV) stimuli,
29–31
we conducted a third experiment in nicotine- and
saline-treated rats wherein we tested the effect of contingent foot-
shock punishment on responding for saline infusions paired with the
AV cue.
2|METHODS
2.1 |Animals and housing
Sixty adult male Long–Evans rats (Envigo; Indianapolis, IN) weighing
250–275 g upon arrival were individually housed on a reverse light
cycle in standard polycarbonate rat cages in a humidity- and
temperature-controlled vivarium. Because the stage of oestrus cycle
has been shown to affect responsivity in foot-shock conditioning
paradigms,
32
we chose to solely use males in this initial characteriza-
tion of the effect of nicotine on compulsive-like responding for RMF.
Following arrival to the facilities, rats were handled daily for 3 days
and given at least 1 week to acclimate prior to surgical procedures. All
procedures were approved by the University at Buffalo Institutional
Animal Care and Use Committee and were carried out in accordance
with all relevant guidelines and regulations, including applicable Ani-
mal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.
2.2 |Surgical procedures
Rats were surgically implanted with chronically indwelling catheters in
the right jugular vein under isoflurane anaesthesia (1–3%). Briefly, a
15-cm catheter line (C30PU-RJV1402, Instech) was implanted into the
right external jugular vein, and the opposite end was connected to an
externalized vascular access button (VABR1B/22, Instech). Each access
button was implanted just posterior to the scapular region on the dor-
sal side of the rat. Catheters were flushed with 0.1 ml of heparinized
saline and enrofloxacin (Baytril, Bayer HealthCare LLC; Shawnee Mis-
sion, KS) before and after self-administration on test days. Catheter
patency was verified at the conclusion of the study by flushing 0.1 ml
of ketamine (10 mg/ml) and verifying the expression of ataxia.
2.3 |Chemical stimuli
Nicotine tartrate (NIDA DSP) was dissolved in sterile saline at a con-
centration of 0.4 mg/ml (freebase) and the pH was adjusted to 7.4
with dilute NaOH. All nicotine injections were administered subcuta-
neously (s.c.) at a dose of 0.4 mg/kg. Remifentanil hydrochloride
2of14 HONEYCUTT ET AL.
(RMF; NIDA DSP) was dissolved in sterile saline such that each intra-
venous infusion of RMF was delivered at a dose of 3.2 μg/kg. In
Experiment 2, histamine dihydrochloride (Sigma Aldrich) was dissolved
in RMF solutions such that each infusion of 3.2 μg/kg RMF resulted in
simultaneous delivery of 1.0–4.0 mg/kg
33,34
of histamine.
2.4 |Behavioural procedures
2.4.1 | Acquisition of intravenous remifentanil self-
administration
Prior to beginning operant training, rats were assigned to receive either
nicotine (0.4 mg/kg) or saline (1.0 ml/kg) in a weight-balanced fashion.
For 2 days prior to starting training, rats were given a single non-
contingent injection of their assigned stimulus in their home cages in
order to acclimate the rats to the effects of nicotine and the injection
procedures. Throughout all training and testing sessions, rats were
administered their assigned treatment (nicotine or saline, s.c.), returned
to home cages for 15 min, and then transported to the operant experi-
ment room, where they immediately began the behavioural procedures.
Rats were trained in 2-h sessions to press a retractable lever in
standard operant cages (Med Associates; St Albans, VT) for delivery of
a drug reinforcer. Each operant box was equipped with a standard red
house-light, a sound attenuating fan, two retractable levers, and two
stimulus lights located directly above each lever. Behaviour was
shaped such that one press of the active lever resulted in retraction of
both levers, illumination of the cue light, and intravenous delivery of
RMF (3.2 μg/kg). The cue light remained illuminated during the infusion
period (3 s). Presses of the inactive lever had no programmed conse-
quence. Lever assignments (active/inactive) were counterbalanced
across rats. Once stable responding was established (5 days), rats were
advanced to an FR-2 schedule of reinforcement (4 days). Upon comple-
tion of each 2-h session, the house-light was turned off, both levers
were retracted, and rats were promptly returned to their home cages.
A control group underwent identical acquisition training, however,
infusions contained only sterile saline (0.9%) rather than RMF.
2.4.2 | Contextual punishment of remifentanil self-
administration with contingent foot-shock
Following acquisition of operant RMF self-administration, rats were
trained such that, in one context, they were able to freely administer
RMF on an FR-2 schedule with no consequence while, in a second
context, RMF infusions were punished with a 1-s foot-shock on a
RR-2 schedule. Contexts were distinguished by visual and tactile stim-
uli: grid vs. bar flooring, addition of black stripes to the cage walls, and
alternating house light orientation from the back of the cage to the
front. These 2-h self-administration sessions alternated daily between
the punished and unpunished contexts. The intensity of the foot-
shock was systematically increased across punished sessions (0.0–
0.6 mA). Contextual conditioning occurred over 12 days, so that each
rat was exposed to a total of 6 days in each of the alternating con-
texts. The order of first context presentation (i.e., punished or
unpunished) was counterbalanced across all rats. Prior to contextual
conditioning, rats were exposed to the contexts for 30 min in order to
acclimate them to the new chamber orientation. No levers were pres-
ented during these acclimatization sessions and rats were not allowed
to self-administer RMF during the 30-min session.
2.4.3 | Contextual punishment of remifentanil self-
administration with histamine adulteration
Contextual conditioning of histamine punishment was identical to that
of foot-shock punishment with the exception that no foot-shock was
delivered and RMF solutions were adulterated with histamine in the
punished context. Briefly, rats were trained in alternating contexts,
such that in one context they were free to administer RMF with no
additional consequence, whereas in the alternate context RMF infu-
sions contained histamine. Contextual presentations were alternated
daily and the concentration of histamine was systematically increased
across conditioning sessions (0.0–4.0 mg/kg/infusion). Conditioning
took place across 12 days such that each rat was exposed to a total of
6 days in each of the alternating contexts. The order of context pre-
sentation was counterbalanced across all rats.
2.4.4 | Progressive ratio responding for remifentanil
in both the punished and unpunished contexts
Following contextual conditioning sessions, all rats were subjected to
two progressive ratio (PR) test sessions: one in the unpunished con-
text and one in the punished context. Here, the number of lever pres-
ses required to earn RMF infusions was systematically increased
following each earned reinforcer.
35
Importantly, RMF infusions were
not punished in this phase in order to examine the degree to which
the context itself would serve to limit RMF IVSA. Each session was
terminated either when the rat failed to complete the current ratio
requirement within 1 h of earning their last RMF infusion or after a
maximum session duration of 4 h. The order of the PR sessions
(punished context vs. unpunished context) followed the same
counterbalanced assignment as the conditioning sessions.
2.4.5 | Operant responding during drug withdrawal
Following the last PR session, rats were tested for responding under
extinction conditions. In these 2-h sessions, pressing the active
(or inactive) lever had no programmed consequences. First, rats were
tested for 10 days in the unpunished context and then once in the
punished context. Rats continued to receive their assigned drug pre-
treatment (nicotine or saline) during these sessions. Three rats were
removed due to the development of illness prior to completion of the
extinction tests (n=45).
HONEYCUTT ET AL.3of14
2.4.6 | Cue-induced reinstatement following
contextual punishment of foot-shock
Following extinction tests, a subset of rats (n=19) from the foot-
shock experiment were tested for responding in a 2-h cue-induced
reinstatement test. Here, rats were tested either in the unpunished
(n=10) or punished context (n=9) such that successful completion
of FR-2 on the active lever resulted in illumination of the cue light but
no RMF delivery. Rats continued to receive their assigned drug pre-
treatment (nicotine or saline) during these sessions.
2.4.7 | Contextual punishment of responding for an
AV reinforcer with contingent foot-shock punishment
Contextual punishment of AV responding with contingent foot-shock
was identical to that of RMF punishment. Briefly, nicotine- and saline-
treated rats responded on an FR-2 schedule for an IV infusion of
saline paired with lever retraction and cue-light illumination. Contexts
were alternated daily and the first presented context was
counterbalanced across both groups. The intensity of foot-shock was
increased across sessions in the punished context (0.0–0.6 mA). Con-
ditioning lasted for 12 days, six sessions in either context.
2.4.8 | Progressive ratio responding for an AV
reinforcer in both the punished and unpunished
contexts
Following conditioning, rats were given two PR sessions, one in the
punished context and one in the unpunished context. All experimental
conditions mirrored those of the RMF PR sessions with the exception
that no RMF was delivered upon completion of the ratio, only saline
and the AV cue.
2.5 |Data analyses
For the acquisition of RMF and saline self-administration, active and
inactive lever presses as well as earned reinforcers were indepen-
dently analysed with mixed-measures analyses of variance (ANOVAs)
with Drug administration (nicotine or saline) as a between-subjects
factor and Session as a within-subjects factor.
During conditioning for both foot-shock and histamine punished
experiments, active lever presses, infusions, and latencies were inde-
pendently analysed with mixed-measures ANOVAs with Drug as a
between-subjects factor and Session and Context as a within-
subjects factors. Breakpoints from the PR sessions were analysed
with Drug and form of Punishment (foot-shock or histamine) as
between-subjects factor and Context as within-subjects factor.
Breakpoints from the PR session in control rats were analysed with
Drug as between-subjects factor and Context as within-subjects
factor.
Responding on the active and inactive levers during withdrawal
sessions were analysed with mixed-measures ANOVAs with Drug and
Punishment as between-subjects factors and Session as a within-
subjects factor.
Responding on the active and inactive levers during the reinstate-
ment test was analysed with ANOVAs with Drug and Context as
between-subjects factors.
Significant main and interactive effects were further explored
with Newman–Keuls post hoc tests where appropriate.
3|RESULTS
3.1 |Nicotine enhances the acquisition of
remifentanil self-administration
Pretreatment with nicotine, relative to saline, produces significantly
higher drug-directed responding resulting in significantly higher RMF
intake across the 9-day acquisition phase (Figure 1). Analysis of active
vs. inactive lever responding in a three-factor ANOVA revealed a
FIGURE 1 Pretreatment with nicotine significantly increases
intravenous self-administration of remifentanil. Rats were trained in
2-h sessions for 5 days on an FR-1 schedule of reinforcement to press
a retractable lever in standard operant chambers for IVSA of RMF
before being moved to an FR-2 schedule for the remaining 4 days.
(A) Rats treated with nicotine, relative to those treated with saline,
made significantly more active lever presses on days 6–9 of training.
There were no differences between groups on inactive lever presses.
(B) As a result, nicotine pretreatment resulted in significantly more
earned RMF infusions on day five of acquisition training. *s indicate
significant group differences (P< 0.05)
4of14 HONEYCUTT ET AL.
significant Lever Session Drug interaction (F
(8,368)
=2.81,
P< 0.01). Post hoc analyses demonstrate that nicotine-treated rats
significantly elevated their active lever presses above inactive lever
presses on the fourth session, whereas saline-treated rats did not reli-
ably do so until the fifth session. Furthermore, nicotine-treated rats,
relative to saline-treated rats, responded more on the active lever
starting on session six and continued to remain reliably elevated
throughout the remaining sessions. There were no statistical differ-
ences in responding on the inactive lever between nicotine- and
saline-treated rats. Consistent with these results, a two-factor
ANOVA conducted on the number of earned infusions revealed a sig-
nificant Session Drug interaction (F
(8,368)
=2.04, P< 0.05), indicat-
ing that nicotine-treated rats took more infusions of RMF, relative to
saline-treated rats.
3.2 |Nicotine continues to enhance remifentanil
self-administration despite contextual punishment of
drug-taking with contingent foot-shock
While contextual administration of contingent foot-shock punish-
ment substantially reduced RMF intake in nicotine- and saline-
treated rats, intake in nicotine-treated rats remained elevated rela-
tive to saline-treated rats (Figure 2). Importantly, nicotine pre-
treatment did not result in differential sensitivity to foot-shock
punished drug seeking, as nicotine-treated rats reduced their RMF
intake at a rate similar to that of saline-treated rats. A three-factor
ANOVA conducted on the number of earned RMF infusions across
sessions as a function of drug-pretreatment and contextual punish-
ment revealed that nicotine-treated rats took significantly more
RMF than saline-treated rats, regardless of context (main effect of
Drug; F
(1,25)
=7.21, P< 0.05). Administration of contingent foot-
shock in the punished context significantly reduced drug-intake
within that context, with no effect on intake in the unpunished con-
text (Context Session interaction; F
(5,125)
=18.83, P< 0.0001).
While nicotine-treated rats took larger overall amounts of RMF,
they did decrease their RMF intake as a function of increasing foot-
shock intensity at a rate largely similar to that of saline-treated rats
yet continued to remain significantly elevated in the unpunished
context (Context Drug interaction; F
(5,125)
=5.77, P< 0.05). In
addition, we found a significant Session Drug interaction
(F
(5,125)
=2.54, P< 0.05). In general, nicotine-treated rats took more
RMF but were similarly impacted by escalating foot-shock intensity
(Figure 2C,D). Nearly identical effects were observed when ana-
lysing the number of active lever presses (Figure 3A,B: Drug;
F
(1,25)
=7.20, P< 0.05; Context Drug; F
(5,125)
=5.78, P< 0.05;
Session Drug; F
(5,125)
=2.52, P< 0.05).
Next, we analysed the latencies to the first earned infusion as a
function of nicotine treatment and context (Figure 3A,B). This three
factor ANOVA revealed a significant Context Session Drug inter-
action (F
(5,125)
=2.30, P< 0.05). Consistent with the earned infusion
data summarized above, we found no reliable change in the latency to
earn the first infusion in the unpunished context, indicating that pun-
ishment in the opposite context did not significantly impact
responding in the unpunished context; furthermore, there were no
differential effects of nicotine treatment. Conversely, the latency to
first infusion in the punished context increased with escalating shock
intensity across sessions. Interestingly, this increase was reduced and
delayed in nicotine-treated rats relative to saline-treated rats. Specifi-
cally, saline-treated rats significantly increased their latency to first
FIGURE 2 Despite administration of
foot-shock punishment contingent on
remifentanil infusions, nicotine-treated
rats took significantly more remifentanil
than saline-treated. (A) Nicotine-treated
rats, compared with saline-treated rats,
pressed significantly more on the active
lever in the unpunished context, with no
differences in presses on the inactive
lever, resulting in (B) significantly more
earned RMF infusions in the unpunished
context. (C) Nicotine-treated rats,
compared with saline-treated rats,
pressed significantly more on the active
lever in the punished context but reduced
lever pressing as a function of escalating
foot-shock intensity at a rate similar to
that of saline-treated rats, ultimately
resulting in (D) significantly more earned
RMF infusions across the conditioning
sessions. *s indicate significant main
effect of nicotine (P< 0.05)
HONEYCUTT ET AL.5of14
infusion in the punished context on the session following conditioning
with 0.4-mA foot-shock whereas nicotine-treated rats did not reliably
do so until the final day of conditioning. Furthermore, the latency to
first-earned infusion in the punished context was significantly lower
in nicotine-treated rats, relative to saline treatment, on session six of
conditioning, with a statistical trend on session 5 (P=0.06). Similar
analyses conducted on the latency to first earned punisher in the
punished context revealed a significant Session Drug interaction
(F
(4,100)
=3.18, P< 0.05). Here, we found that nicotine-treated rats
were significantly quicker than saline-treated rats to earn their first
punished RMF infusion on sessions five and six during conditioning
with 0.5 and 0.6 mA, respectively (Figure 3C).
3.3 |Nicotine continues to enhance remifentanil
self-administration despite contextual punishment of
drug taking with contingent histamine administration
Similar to foot-shock punishment of RMF intake, contingent hista-
mine administration significantly lowered RMF self-administration
FIGURE 3 Latency to self-administer remifentanil within the punished context increased as a function of foot-shock punishment but less so
in nicotine-treated rats. (A) There was no difference in latency to first infusion in the unpunished context, and latency in the unpunished context
was not affected by punishment in the alternate context. (B) Saline-treated rats reliably increased their latency to first RMF infusion following
conditioning with 0.4-mA foot-shock, while nicotine-treated rats did not do so until the final conditioning session. Additionally, nicotine-treated
rats were significantly faster than saline-treated rats to earn their first infusion during the final conditioning session (0.6 mA). (C) Similarly,
compared with nicotine-treated rats, saline-treated rats reliably increased their latency to first earned punisher following conditioning with
0.4-mA foot-shock, while nicotine-treated rats did not do so until the final conditioning session. Nicotine-treated rats were significantly faster
than saline-treated rats to earn their first punisher during the final two conditioning sessions. *s indicate significant group differences (P< 0.05)
FIGURE 4 Similar to that seen with
foot-shock punishment, punishment of
remifentanil intake by histamine
adulteration significantly lowered RMF
self-administration, yet nicotine-treated
rats continued to earn significantly more
RMF. (A) Nicotine-treated rats pressed
significantly more on the active lever than
saline-treated rats in the unpunished
context. There were no group differences
in responding on the inactive lever.
(B) Likewise, nicotine, relative to saline,
increased RMF intake. (C) Adulteration of
the RMF solution with increasing
concentrations of histamine across
sessions resulted in a concentration-
dependent decrease in active lever
presses in the punished context, yet
nicotine-treated rats continued to press
more on the active lever and (D) earned
more RMF infusions across the six-day
conditioning paradigm. *s indicate
significant main effect of nicotine
(P< 0.05)
6of14 HONEYCUTT ET AL.
(Figure 4) in the punished context, yet RMF intake in nicotine-
treated rats remained elevated, relative to saline-treated rats,
throughout the conditioning paradigm. Despite nicotine-treated rats
taking more RMF than saline-treated rats, they were equally sensi-
tive to the reduction in intake induced by increasing histamine con-
centration. A three-factor ANOVA conducted on the number of
earned RMF infusions across sessions as a function of drug-
pretreatment and context revealed that nicotine-treated rats self-
administered more RMF, regardless of context, than saline-treated
rats (Figure 3C,D; main effect of Drug; F
(1,19)
=5.51, P< 0.05).
Adulteration of the RMF solution with increasing concentrations of
histamine across sessions resulted in a concentration-dependent
decrease in RMF intake in the punished context, with no effect on
intake in the unpunished context (Context Session interaction;
F
(5,95)
=6.05, P< 0.0001). There were no interactive effects
between Drug and Context, indicating that nicotine-treated rats
were not differentially sensitive to increasing concentrations of his-
tamine. Nearly identical effects were observed when analysing the
number of active lever presses (Figure 3A,B: Drug; F
(1,19)
=5.48,
P< 0.05; Context Session; F
(5,95)
=6.10, P< 0.0001).
Additionally, we analysed the latencies to first earned infusion as a
function of nicotine treatment and context (Figure 5). Here, we found
main effects of Context (F
(1,19)
=8.38, P< 0.01) and Session
(F
(5,95)
=4.10, P< 0.01). Importantly, unlike what was found with foot-
shock punishment, regardless of nicotine treatment, rats punished with
histamine did not systematically increase their latency to the first
earned infusion, even in the punished context (Figure 5B). Thus, despite
serving to significantly reduce RMF self-administration, histamine pun-
ishment did not support learning the contextual discrimination.
3.4 |Despite a previous history of contextual
punishment, nicotine enhances the motivation to
obtain remifentanil regardless of form of punishment
The breakpoint in responding for RMF infusions was significantly
higher in nicotine-treated rats, relative to saline-treated rats,
FIGURE 5 Regardless of drug
pretreatment, rats punished with
histamine did not systematically increase
their latency to the first earned infusion,
even within the punished context.
(A) Latency to first earned RMF infusion
did not differ between nicotine- and
saline-treated rats in the unpunished
context. (B) Likewise, latency to first
infusion did not differ between nicotine-
and saline-treated rats in the punished
context. There was no effect of
punishment history on the latency to earn
an RMF infusion in either context
FIGURE 6 Nicotine administration significantly elevated the breakpoint in responding for RMF infusions, regardless of context or form of
punishment. (A) Following punishment of RMF intake with foot-shock, the breakpoint for RMF administration was significantly higher in nicotine-
treated rats than saline-treated rats in both the unpunished and punished contexts. (B) Similarly, nicotine produced significantly higher
breakpoints in RMF responding following punishment of RMF intake with histamine adulteration. Regardless of drug pretreatment, histamine
punishment of RMF did not affect the breakpoint for RMF in the punished context. *s indicate significant group differences (P< 0.05)
HONEYCUTT ET AL.7of14
regardless of context and form of punishment (Figure 6). A three-
factor ANOVA conducted on the breakpoint in responding for RMF in
both the punished and unpunished contexts as a function of nicotine
treatment and form of punishment revealed that nicotine, relative to
saline pretreatment, enhanced the motivation to obtain RMF (main
effect of Drug; F
(1,36)
=17.48, P< 0.001). Additionally, we found a
Context Punishment interaction (F
(1,36)
=24.64, P< 0.0001). Post
hoc analyses on this interaction showed that punishment with foot-
shock resulted in significantly lower breakpoints in the punished con-
text, relative to the unpunished context (Figure 6A), whereas there
were no differences in the breakpoint between punished and
unpunished contexts following conditioning with histamine punish-
ment (Figure 6B).
3.5 |Treatment with nicotine enhances
remifentanil seeking under extinction conditions in
both punished and unpunished contexts
Following removal of RMF administration, responding on the active
lever continued to be higher in nicotine-treated rats, relative to saline-
treated rats, regardless of context. A four-factor ANOVA conducted
on both active and inactive lever presses across the 10-session extinc-
tion phase in the unpunished context revealed a significant main
effect of Punishment (F
(1,37)
=6.86, P< 0.05) and a significant
Lever Session Drug interaction (F
(9,369)
=6.90, P< 0.0001). Post
hoc analyses revealed that responding during extinction was higher
following contextual punishment with histamine and that, regardless
of form of punishment, nicotine-treated rats responded significantly
more on the active lever during the first five sessions, relative to
saline-treated rats (Figure 7A). There were no significant group differ-
ences in responding on the inactive lever.
Similarly, a three-factor ANOVA conducted on the active and
inactive lever presses in the histamine- and shock-punished contexts
as a function of nicotine pretreatment revealed a significant main
effect of Punishment (F
(1,37)
=12.91, P< 0.001) and a significant Lev-
er Drug interaction (F
(1,37)
=10.00, P< 0.01). Post hoc analyses of
these effects revealed that responding was significantly higher in the
histamine-punished rats relative to shock-punished rats and that,
regardless of punishment type, nicotine-treated rats responded signif-
icantly more than saline-treated rats on the active lever (Figure 7B).
There were no significant group differences in responding on the inac-
tive lever.
3.6 |Nicotine-treated rats displayed equally high
levels of cue-induced reinstatement, regardless of
context
Following extinction training, nicotine-treated rats showed signifi-
cantly higher cue-induced reinstatement of responding on the
active lever, relative to saline-treated rats. A three-factor ANOVA
conducted on the active and inactive lever presses in either the
unpunished or punished context as a function of nicotine treat-
ment revealed a Lever Drug interaction (F
(1,15)
=11.22, P< 0.01).
Post hoc analysis showed that, regardless of context, nicotine-
treated rats responded more on the active lever, as opposed to
the inactive lever, relative to saline-treated rats (data not shown).
A similar main effect of nicotine was found when examining the
number of earned cue presentations (F
(1,15)
=9.96, P< 0.01;
Figure 8). We only tested rats from the foot-shock experiment
because rats from the histamine experiment did not differ in their
motivation to obtain RMF between the formerly unpunished and
punished contexts.
FIGURE 7 Nicotine-treated rats, relative to saline-treated controls, displayed significantly higher rates of remifentanil seeking in extinction,
regardless of punisher or context. (A) Regardless of punishment type, nicotine-treated rats, compared with saline-treated rats, were slower to
extinguish RMF seeking in the unpunished context, as evidenced by higher active lever presses under extinction conditions across the first
5 days. (B) Likewise, regardless of form of punishment, nicotine-treated rats pressed significantly more on the active lever than saline-treated rats
within the punished context. *s indicate significant group differences (P< 0.05)
8of14 HONEYCUTT ET AL.
3.7 |Nicotine enhances the acquisition of saline
self-administration paired with an audiovisual cue
Pretreatment with nicotine, relative to saline, enhanced responding
on the active lever with no effect on the inactive lever. Analysis of
active vs. inactive lever responding across the 9-day acquisition
period with a three-factor ANOVA revealed a significant
Lever Session Drug interaction (F
(8,80)
=2.80, P< 0.01). Group
differences between saline- and nicotine-treated rats in responding
on any given day failed to survive post-hoc correction. There were no
statistical differences in responding on the inactive lever between
nicotine- and saline-treated rats. Analyses of the number of rein-
forcers earned revealed a significant main effect of Session
(F
(8,80)
=3.89, P< 0.001) as earned reinforcers increased across ses-
sions, however, there were no significant group differences between
nicotine- and saline-treated rats on number of reinforcers earned
across the 9-day acquisition phase.
3.8 |Nicotine-induced differences in responding
for an audiovisual cue are lost following contingent
punishment with foot-shock
The observed increase in responding for an audiovisual cue induced
by nicotine was lost following contingent punishment with footshock.
Furthermore, both nicotine- and saline-treated rats were more likely
to generalize between the two contexts as reinforcers earned
decreased, and latency to respond increased, in both contexts
(Figure 10). A three-factor ANOVA conducted on the number of
earned reinforcers across sessions as a function of drug-pretreatment
and contextual punishment revealed a significant Session Drug
interaction (F
(5,38)
=5.19, P< 0.001). Post-hoc analyses revealed that
nicotine-treated rats earned more reinforcers in the first session in
both contexts, but these differences were lost following the first day
of contingent foot-shock punishment (Figure 10C,D). We also found a
significant main effect of context (F
(1,38)
=5.57, P< 0.05) such that all
rats earned more reinforcers in the unpunished context. Identical
effects were observed when analysing the number of active lever
presses (Session Drug; F
(5,38)
=5.26, P< 0.001; Context;
F
(1,38)
=5.74, P< 0.05).
Next, we analysed the latencies to the first earned infusion as a
function of nicotine treatment and context (Figure 11A,B). This three
factor ANOVA revealed a main effect of Session (F
(5,38)
=4.30,
P< 0.01) but no significant differences between nicotine- and saline-
treated rats in latency to receive first infusion. Furthermore, there
were no main or interactive effects of context demonstrating that, in
contrast to Experiment 1, the increase in latency to earn a reinforcer
increased in both the unpunished and punished contexts. Similar ana-
lyses on the latency to earn the first shock revealed a main effect of
FIGURE 8 Nicotine-treatment, relative to saline, resulted in
greater levels of cue-induced reinstatement. Additionally, following
presentation of the RMF cue, nicotine treated rats relapsed to RMF
seeking levels in the formerly foot-shock punished context
comparable to the unpunished context. Regardless of context,
nicotine-treated rats earned significantly more cue presentations,
compared with saline-treated rats. *s indicate significant group
differences (P< 0.05)
FIGURE 9 Nicotine-treatment, relative to saline, enhanced
acquisition of responding for an AV reinforcer. (A) Nicotine-treated
rats responded significantly more on the active lever compared with
saline-treated rats. There were no effects of nicotine administration
on responding on the inactive lever. (B) Nicotine-treated rats earned
more AV reinforcers but this difference did not reach statistical
significance within this 9-day period. *s indicate a significant main
effect of nicotine
HONEYCUTT ET AL.9of14
Session (F
(4,39)
=5.26, P< 0.01) but, again, no main or interactive
effects of nicotine administration (Figure 11C).
3.9 |Nicotine administration failed to enhance
motivation for an audiovisual cue following contextual
punishment conditioning
The breakpoint in responding for audiovisual reinforcers was similar in
nicotine- and saline-treated rats regardless of context (Figure 12). A
two-factor ANOVA conducted on the breakpoint in responding in
both the punished and unpunished contexts as a function of nicotine
treatment revealed a main effect of Context (F
(1,10)
=13.99,
P< 0.01), indicating that regardless of pretreatment condition, rats
were less motivated to respond for the cue in the punished context.
There were no main or interactive effects of nicotine administration.
4|DISCUSSION
Prior nicotine use is often associated with the development of a num-
ber of subsequent substance use disorders.
5,40–43
Although nicotine
and alcohol interactions have been extensively studied, the role that
nicotine may play in liability for OUD has been largely overlooked
despite equally high rates of comorbidity.
37,39,41
In a previous study,
we demonstrated that nicotine administration prior to RMF and
FIGURE 10 The enhancement of
responding for an AV reinforcer by
nicotine administration was lost following
contextual punishment with foot-shock.
(A,B) Regardless of context, nicotine-
treated rats, relative to saline-treated,
pressed significantly more on the active
lever on the first day of testing.
Responding on the active lever decreased
in both contexts across sessions (C,D)
Likewise, nicotine-treated rats earned
more AV reinforcers, regardless of
context, on the first day of testing, but
this difference was lost following
administration of foot-shock punishment
FIGURE 11 Latency to earn an AV reinforcer increased in both the punished and unpunished contexts equally in nicotine- and saline-treated
rats. (A) In contrast to observations from testing with RMF administration, the latency to earn an AV reinforcer increased across sessions in the
unpunished context following punishment in the alternate context. (B) The latency to earn an AV reinforcer increased in the punished context
equally in nicotine- and saline-treated rats. (C) Similarly, the latency to earn the first foot-shock increased equally in nicotine- and saline-treated
rats
10 of 14 HONEYCUTT ET AL.
morphine IVSA sessions substantially increased the amount of drug
consumed
6
relative to that rat's own baseline intake. In the current
study, we have replicated and extended those previous findings by
demonstrating for the first time that nicotine enhances compulsive-
like responding for RMF across multiple behavioural measures. More
specifically, nicotine pretreatment enhanced RMF intake despite
ongoing punishment and nicotine-treated rats, relative to saline-
treated, remained more motivated for RMF regardless of prior punish-
ment or extinction conditions, an effect that was not observed in rats
responding for the AV cue alone. In addition, we compared the effi-
cacy of an exteroceptive (foot-shock) and interoceptive (histamine)
form of punishment and found that nicotine continued to enhance
motivation for RMF, regardless of form of punishment. While both
forms of punishment served to limit RMF intake as a function of
increasing intensity, our data support that contingent foot-shock is a
more potent form of punishment compared with histamine adultera-
tion, as only foot-shock punishment supported the development of
discrimination between the unpunished and punished contexts.
Successful recovery from substance use disorders is largely depen-
dent on volitional abstinence of drug taking, which often results from
assessing the deleterious consequences of continued drug use.
22,23
Here, we found that nicotine pretreatment significantly enhanced RMF
self-administration (Figure 1) and that this increased intake in nicotine-
treated rats, relative to saline-treated, persisted regardless of form of
punishment (Figures 2 and 4). The number of active lever presses
(Figures 2A and 4A) and resultant RMF intake (Figures 2B and 4B) was
significantly higher following nicotine administration, relative to saline,
in the unpunished context across both experiments. Likewise, active
lever presses (Figures 2C and 4C) and RMF intake (Figures 2D and 4D)
were significantly higher in nicotine-treated rats in the context
punished with either foot-shock or histamine adulteration, despite
administration of the punisher serving to limit RMF intake in an
intensity-dependent fashion. There were no differences in responding
on the inactive lever across any portion of either experiment.
Comparison of the latencies to first infusion and first earned foot-
shock revealed that the latency to first RMF infusion in the unpunished
context was unaffected by administration of punishment in context B
and that latencies to first RMF infusion did not reliably differ between
nicotine- and saline-treated rats in the unpunished context (Figure 3A).
Conversely, administration of foot-shock significantly increased the
latency to first RMF infusion and yet nicotine-treated rats were signifi-
cantly faster to earn their first RMF infusion and their first foot-shock
punisher in the punished context (Figure 3B). In contrast, we found no
effect of histamine adulteration on the latency to earn RMF infusions
in either the unpunished or punished contexts (Figure 5A,B, respec-
tively), and there were no effects of nicotine administration on latency
to earn RMF infusions. Examination of the breakpoint for RMF in the
progressive ratio paradigms revealed that motivation for RMF was sig-
nificantly higher in nicotine-treated rats, relative to saline-treated rats,
regardless of context or form of punishment (Figure 6A,B). Similar to
that revealed by the latency measures, we found no effect of context
on the motivation to obtain RMF following punishment with histamine
adulteration. Specifically, former punishment with foot-shock in con-
text B resulted in significantly lower breakpoints compared with those
observed in the unpunished context (Context A). In contrast, the moti-
vation to obtain RMF following punishment with histamine adulteration
did not differ between context A and B in either nicotine- or saline-
treated rats. Considering that histamine adulteration of RMF also did
not significantly affect latency to RMF infusion in context B in the
histamine-adulteration experiment, these data support that foot-shock
is a much more potent form of punishment than histamine adulteration.
That is, despite both methods serving to significantly limit RMF intake
during ongoing punishment, histamine adulteration failed to support
the development of contextual discrimination within the present study.
We found that nicotine not only enhanced compulsive-like RMF
IVSA but also significantly enhanced compulsive-like RMF seeking in
the unpunished context that persisted under extinction conditions
longer in nicotine-treated rats compared with saline-treated rats,
regardless of punishment condition (Figure 7A). This enhancement of
RMF seeking by nicotine was also clearly evident when tested under
extinction conditions in the formerly punished context (Figure 7B),
again regardless of form of previous punishment. Additionally, we
tested a subset of rats in a cue-induced relapse test following prior
contextual punishment with foot-shock. Here, completion of the FR-2
schedule resulted in the illumination of the RMF cue light but no
delivery of RMF. Nicotine-treated rats, relative to saline-treated rats,
displayed a higher propensity to relapse to drug-seeking behaviour
upon re-exposure to the RMF cue, regardless of context. When com-
paring the efficacy of cue-induced reinstatement, we found that addi-
tion of the RMF cue did not significantly increase RMF seeking in
saline-treated rats above that of their last day of extinction condition-
ing but significantly enhanced RMF seeking in nicotine-treated rats.
Furthermore, nicotine-treated rats displayed no difference in the cue-
FIGURE 12 Nicotine failed to enhance motivation to earn an AV
reinforcer following contingent foot-shock punishment. There were
no differences in the breakpoint for responding for an AV reinforcer
between nicotine- and saline-treated rats in either the punished or
unpunished context
HONEYCUTT ET AL.11 of 14
induced reinstatement between the unpunished and punished con-
texts (Figure 8). Therefore, the formerly punished context was suffi-
cient to limit relapse to RMF seeking in saline-treated rats following
re-exposure to the RMF cue but not in nicotine-treated rats.
Because nicotine can enhance responding for nonpharmacological
reinforcers, we examined these effects of nicotine in a group of rats
trained to respond for presentation of an AV reinforcer. Consistent
with previous reports,
29–31
we found that nicotine administration
enhanced responding for an AV reinforcer alone, in the absence of
RMF infusions (Figure 9). In contrast to what was observed in rats
responding for RMF, the nicotine-induced augmentation of
responding for an AV cue was lost following the first day of contin-
gent foot-shock punishment (Figure 10). Furthermore, it appeared
that the AV cue was devalued in both the punished and unpunished
contexts, an effect that was not observed in rats responding for RMF
infusions. Specifically, the number of earned AV reinforcers decreased
across days in both contexts (Figure 10B,D). Likewise, the latency to
earn an AV reinforcer increased in both contexts equally in both
nicotine- and saline-treated rats (Figure 11A,B). Consistent with the
notion that the AV cue was equally devalued, we found no effect of
nicotine on motivation to earn AV reinforcers in a PR task (Figure 12),
no differences in responding under extinction conditions (Figure S1),
nor differences in reinstatement of AV cue responding (Figure S2). It
has been demonstrated previously that the reinforcer enhancing
effects of nicotine are modulated by the relative value of the rein-
forcer.
30,44
Our data support and extend those previous findings such
that the degree to which nicotine enhances a punishment-resistant
phenotype is also modulated by the value of the reinforcer.
It is important to note that we did not find any evidence of differ-
ential sensitivity to the punishment itself in either experiment.
Despite nicotine-treated rats demonstrating higher overall RMF intake
compared with saline-treated controls, both nicotine- and saline-
treated rats decreased their consumption of RMF at comparable rates
as a function of increasing punishment intensity in both the foot-
shock (Figure 2) and histamine (Figure 4) experiments. This is an
important consideration given that nicotine has been demonstrated to
have antinociceptive properties.
36,45
The fact that both forms of pun-
ishment were equally as effective in nicotine- and saline-treated rats
at reducing RMF administration from baseline as a function of increas-
ing intensity demonstrates that the persistent differences in RMF
intake following nicotine administration were unlikely due to any inoc-
ulating effect of nicotine on the aversive physiological effects of the
punishers during their administration. Moreover, the lack of effect of
nicotine administration, relative to saline, in enhancing foot-shock
punished responding for an AV reinforcer further precludes the con-
clusion that the effects were entirely driven by any antinociceptive
properties of nicotine. Rather, it appears more likely that the differ-
ences in RMF intake between nicotine- and saline-treated rats were
due to an enhancement in the motivational efficacy of RMF induced
by nicotine, a deficit in learning the contextual discrimination between
the unpunished and punished environments when RMF was available,
or perhaps a combination of the two. In support of the former, nico-
tine has been shown to enhance the motivational properties of
subsequently administered substances across multiple classes of drugs
of abuse,
6,14,17,38,46–50
an effect demonstrated here with RMF. Nico-
tine administration not only increased total RMF intake but also
increased the breakpoint for obtaining RMF infusions (Figure 6) and
significantly delayed extinction of RMF seeking under extinction con-
ditions (Figure 7). Support for the latter interpretation comes from
studies demonstrating that nicotine pretreatment interferes with con-
textual conditioning with other commonly abused drugs.
6,15,17,20
In
the present study, the context associated with foot-shock punishment
was sufficient to significantly limit RMF intake in the cue-induced
reinstatement paradigm in saline-treated rats whereas nicotine-
treated rats displayed relapse to RMF seeking identical to that seen in
rats tested in the unpunished context (Figure 8). Furthermore, previ-
ous studies have demonstrated that contextual relapse to drug seek-
ing following volitional abstinence is dependent on neural activity
within the insular cortex,
25,26,51
such that rats that were more likely to
relapse to alcohol seeking within a punished context displayed more
fos-like immunoreactivity within the insula.
28
Previously, we have
shown that nicotine delivered directly to the insular cortex is suffi-
cient to interfere with insular-dependent contextual drug
conditioning,
6
suggesting that nicotinic modulation of insular function
may impair the ability to withhold drug-directed responding in con-
texts previously associated with punishment. Given that nicotine gen-
erally enhances contextual fear conditioning with foot-shock,
52–54
it
follows that these effects are at least in part dependent on the avail-
ability of the reinforcing properties of RMF, a conclusion supported
by the lack of effect of nicotine when rats were responding for the
AV reinforcer alone. Consistent with this notion, nicotine pre-
treatment interferes with the ability to voluntarily suppress intake of a
reinforcing saccharin solution following its pairing with commonly
abused drugs but not following pairing with the purely emetic stimu-
lus lithium chloride
17
that has no known reinforcing properties.
As a whole, these data support that nicotine administration sub-
stantially increases the expression of a number of compulsive-like
behaviours directed towards obtaining opioids. This enhancement
may arise from the augmentation of the motivational properties of
RMF, such that the incentive to self-administer RMF outweighs the
aversive consequences of the punishers, or from a reduced capacity
to learn that a specific environmental context results in further dele-
terious consequences when a reinforcing drug is available. It is also
possible that the enhanced resistance to punishment induced by
nicotine may be due to the elevated RMF intake observed prior to
punishment conditioning. Future studies should be conducted in
which the presence of nicotine administration is systematically
altered alongside the various phases of this experiment. Regardless
of the underlying causes, our findings provide the initial characteri-
zation of a novel mechanism that may help to explain the extreme
comorbidity of nicotine and opioid misuse. OUDs are primarily char-
acterized by the compulsive use of opioids despite harmful or aver-
sive consequences and a high rate of relapse to misuse despite
efforts to remain abstinent. An extended history of drug taking
results in an escalation of drug self-administration and ultimately
produces insensitivity to punishment of drug-seeking for that
12 of 14 HONEYCUTT ET AL.
particular drug.
10,34,55
To the best of our knowledge, these are the
first data to demonstrate that acute, noncontingent administration
of nicotine is sufficient to engender a punishment-resistant pheno-
type in animals responding for opioids. As such, it is likely that nico-
tine use may be an antecedent risk factor for the development of
OUDs. Nicotine has been shown to modulate the thalamacortical
and corticostriatal circuits that are implicated in the maintenance of
compulsive drug seeking.
56
As such, future studies designed to dis-
sect the relative contributions of nicotine on these circuits on the
enhancement of compulsive drug taking are warranted.
ACKNOWLEDGMENTS
Research reported in this manuscript was supported by a grant from
the National Institute on Alcohol Abuse and Alcoholism (T32:
AA007583) to SCH and a grant from the National Institute on Drug
Abuse (DA048336) to GCL.
CONFLICT OF INTERESTS
The authors have no conflicts to declare.
AUTHOR CONTRIBUTIONS
GCL and SCH were responsible for the study concept and design.
SCH, MSP, RDC and AM collected the data. GCL and SCH were
responsible for data analyses and interpretation of findings. SCH and
GCL drafted the manuscript. MSP, RDC, and AM assisted in editing
the manuscript. All have approved the final version for publication.
There are no conflicts of interest to declare.
DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the
corresponding author upon reasonable request.
ORCID
Gregory C. Loney https://orcid.org/0000-0003-3936-1885
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SUPPORTING INFORMATION
Additional supporting information may be found in the online version
of the article at the publisher's website.
How to cite this article: Honeycutt SC, Paladino MS,
Camadine RD, Mukherjee A, Loney GC. Acute nicotine
treatment enhances compulsive-like remifentanil self-
administration that persists despite contextual punishment.
Addiction Biology. 2022;27(3):e13170. doi:10.1111/adb.13170
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