Role of orbitofrontal cortex in incubation of oxycodone craving ......neural mechanisms underlying incubation of craving to oxycodone, a commonly abused prescription opioid, and we

OR I G I N A L A R T I C L E

Role of orbitofrontal cortex in incubation of oxycodone cravingin male rats

Rachel D. Altshuler | Eddy S. Yang | KristineT. Garcia | Ian R. Davis |

Adedayo Olaniran | Meron Haile | Syrus Razavi | Xuan Li

Department of Psychology, University of

Maryland College Park, College Park, MD, USA

Correspondence

Xuan Li, Department of Psychology, University

of Maryland College Park. 4,094 Campus Dr.,

College Park, MD 20742, USA.

Email: [email protected]

Funding information

University of Maryland Department of

Psychology Startup Funds (XL)

Abstract

One of the main challenges in treating opioid-use disorders is relapse during absti-

nence, triggered by re-exposure to drug-associated cues. Previous studies have

demonstrated that drug-seeking in rats progressively increases over time during

withdrawal (incubation of drug craving). Here, we used male rats and examined

neural mechanisms underlying incubation of craving to oxycodone, a commonly

abused prescription opioid, and we focused on orbitofrontal cortex (OFC), a brain

region previously implicated in incubation of heroin craving. We first used neuronal

activity marker Fos and measured neuronal activation in OFC (ventral and lateral

OFC) associated with day-1 and day-15 relapse tests. Next, we determined the effect

of pharmacological reversible inactivation of OFC on incubated oxycodone seeking

on withdrawal day 15. Finally, we determined the effect of reversible inactivation

of OFC on nonincubated oxycodone seeking on withdrawal day 1. We found that

lever presses during relapse tests were higher on withdrawal day 15 than on with-

drawal day 1 (incubation of oxycodone craving). Incubation of oxycodone craving

is accompanied with a time-dependent increase of Fos protein expression in both

ventral and lateral OFC. Lastly, OFC inactivation decreased oxycodone seeking on

withdrawal day 15 but had no effect on withdrawal day 1. Together with the previ-

ous heroin study, results here show that OFC plays a critical role in incubation of

opioid craving.

K E YWORD S

Fos, incubation of craving, orbitofrontal cortex, oxycodone, relapse, self-administration

1 | INTRODUCTION

Relapse to drug use is a major barrier to addressing the ongoing pre-

scription opioid epidemic in the United States.1–3 One of the common

factors for triggering relapse is exposure to drug associated-cues.4

Previous studies have shown that drug seeking, including cocaine,5,6

heroin,7 nicotine,8 alcohol,9 and methamphetamine,10 progressively

increases after withdrawal in rats with a history of drug self-adminis-

tration, a phenomenon termed “incubation of drug craving”.6 Subse-

quent studies identified several neural mechanisms underlying this

incubation, primarily focusing on cocaine.11 Recent studies also inves-

tigated neural substrates involved in incubation of heroin12–14 and

methamphetamine craving.15–25 However, only three recently publi-

shed studies26–28 began exploring neural mechanisms underlying incu-

bation of oxycodone craving. At the molecular level, Blackwood

Rachel D. Altshuler and Eddy S. Yang have contributed equally to this work.

The data that support the findings of this study are available from the corresponding author

upon request.

Received: 20 February 2020 Revised: 6 April 2020 Accepted: 14 May 2020

DOI: 10.1111/adb.12927

Addiction Biology. 2020;e12927. wileyonlinelibrary.com/journal/adb © 2020 Society for the Study of Addiction 1 of 11

https://doi.org/10.1111/adb.12927

https://orcid.org/0000-0002-1771-9287https://orcid.org/0000-0003-2559-1102https://orcid.org/0000-0002-4048-6856https://orcid.org/0000-0002-6737-1223mailto:[email protected]://doi.org/10.1111/adb.12927http://wileyonlinelibrary.com/journal/adbhttps://doi.org/10.1111/adb.12927

et al,26,27 demonstrated time-dependent changes of gene expression

(e.g., opioid receptors or fibroblast growth factors) associated with

incubation of oxycodone craving in male rats' striatum and hippocam-

pus. At the functional level, Fredriksson et al,28 showed that systemic

administration of a dopamine stabilizer decreased incubation of oxy-

codone craving in male rats after either forced abstinence or electric

barrier-based abstinence.

In the current study, we aimed to identify neural substrates

underlying incubation of oxycodone craving by focusing on

orbitofrontal cortex (OFC). We chose the OFC based on heroin stud-

ies using both brain imaging in humans and neurobiological

approaches in rats. Human imaging studies showed that OFC activity

in heroin-dependent individuals enhances in response to heroin-

related cues, which positively correlates with subjective ratings of

craving.29 Moreover, the resting-state OFC function increases in

heroin-dependent individuals who relapsed compared with those who

did not relapse under methadone maintenance treatment.30 In rats,

cue-induced reinstatement of heroin seeking and incubation of heroin

craving is associated with increased ania-3 (an immediate early gene)

mRNA expression31 and Fos (an immediate early gene and commonly

used neuronal activity marker32) protein expression33 in OFC, respec-

tively. At the functional level, reversible inactivation of lateral OFC

(LOFC; but not ventral OFC [VOFC]) decreases heroin seeking after

15-day, but not 1-day withdrawal from heroin self-administration,

indicating a critical role of OFC in incubation of heroin craving.33

However, whether the role of OFC in incubation of craving general-

izes to oxycodone is unknown. In this regard, previous studies have

demonstrated dissociable roles of OFC in mediating cocaine versus

methamphetamine relapse: reversible inactivation of rat OFC

decreases cue- and context-induced reinstatement of cocaine

seeking,34,35 but has no effect on incubation of methamphetamine

craving.18

Here, we first assessed neuronal activation of VOFC and LOFC

associated with oxycodone seeking after 1- and 15-day withdrawal

using Fos immunohistochemistry. We found Fos expression progres-

sively increased in both VOFC and LOFC during incubation of oxyco-

done craving. Based on this finding, we reversibly inactivated the

entire OFC with muscimol + baclofen (GABAA and GABAB agonists36)

to determine the causal role of the OFC in incubated oxycodone seek-

ing on withdrawal day 15. Finally, we followed up the positive findings

and examined the effect of reversible inactivation of OFC on non-

incubated oxycodone seeking on withdrawal day 1.

2 | METHODS AND MATERIALS

2.1 | Subjects

We used male Sprague–Dawley rats (Charles River, total n = 64),

weighing 275–320 g prior to surgery and 300–325 g at the start of

the drug self-administration procedure; we maintained the rats under

a reverse 12:12-h light/dark cycle with food and water freely avail-

able. We kept the rats three to four per cage prior to surgery and then

housed them individually after surgery. We performed the experi-

ments under the protocols approved by the University of Maryland

College Park Animal Care and Use Committee and in accordance with

the Guide for the Care and Use of Laboratory Animals (National Insti-

tute of Health). We excluded 15 rats due to failure of catheter

patency (n = 2), health-related issues (n = 2), failure to acquire stable

oxycodone self-administration (n = 3), or cannula misplacement or

cannula-related necrosis (n = 8). The number of rats reported herein

refers to rats included in the statistical analysis.

2.2 | Intravenous surgery

We anesthetized the rats with isoflurane (5% induction, 2%–3%

maintenance) or ketamine and xylazine (80 and 10 mg/kg, i.p., respec-

tively) and inserted silastic catheters into the rats' jugular vein as pre-

viously described.17,37 We injected the rats with ketoprofen

(2.5 mg/kg, s.c.) after surgery to relieve pain and inflammation; we

allowed them to recover 5–7 days before oxycodone self-

administration training. During the recovery and training phases, we

flushed the catheters every 24–48 h with gentamicin (Hospira;

5 mg/ml) dissolved in sterile saline.

2.3 | Cannula implantation

Immediately after intravenous surgery, we implanted bilateral guide

cannulas (23 gauge; Plastics One) 1.0 mm above OFC. We set the

nose bar at −3.3 mm and used the following coordinates from Bregma

based on our previous study18: AP, +3.1 mm; ML, ±3.5 mm (10�angle);

DV, −5.0 mm or −4.7 mm. We anchored the cannulas to the skull with

jeweler's screws and dental cement. Note that we used two different

DVs in two cohorts of animals because we found a significant number

of misplacements when we set the DV at −5.0 mm under our current

experimental conditions. For final statistical analysis and data presen-

tation, we only included animals with the correct cannula placement

(Figures 3 and 4).

2.4 | Apparatus

We trained the rats in self-administration chambers located inside

sound-attenuating cabinets and controlled by a Med Associates

(Georgia, VT) system. Each chamber has two levers located 8–9 cm

above the floor. During self-administration training, presses on the

retractable (active) lever activated the infusion pump (which

delivered an oxycodone infusion); presses on the stationary (inactive)

lever were not reinforced with the drug. For oxycodone intravenous

infusions, we connected each rat's catheter to a liquid swivel

(Instech) via polyethylene-50 tubing, protected by a metal spring.

We then attached the liquid swivel to a 20-ml syringe via

polyethylene-50 tubing and to a 22-gauge modified needle (Plastics

One, VA).

2 of 11 ALTSHULER ET AL.

2.5 | Oxycodone self-administration training

We used a training procedure previously described by Li et al.18 We

trained the rats to self-administer oxycodone for 6 h per day, under a

fixed-ratio-1 (FR1) with 20-s timeout reinforcement schedule. We dis-

solved oxycodone (kindly provided by National Institute on Drug

Abuse Drug Supply Program) in saline, and the rats self-administered

oxycodone at a dose of 0.1 mg/kg/infusion over 3.5 s

(0.10 ml/infusion). We trained the rats for 10 sessions over an 11-day

period (one off day between fifth and sixth day). We used Brevital

(3 to 4 mg/kg) to check catheter potency for low responders during

the training.

The daily training sessions started at the onset of the dark cycle

and began with the extension of the active lever and the illumination

of the red house light. The house light remained on for the duration of

the 6-h session. During training, active lever presses led to the deliv-

ery of an oxycodone infusion and a compound 5-s tone-light cue (the

tone and light modules were located above the active lever). During

the 20-s timeout, we recorded the nonreinforced lever presses. We

set 90 infusions as the maximum for each 6-h session to prevent over-

dose. The red house light was turned off and the active lever retracted

after the rats received the maximum infusions or at the end of the 6-h

session.

2.6 | Withdrawal phase

During the withdrawal phase, we housed the rats individually in the

animal facility and handled them two to three times per week.

2.7 | Relapse test

We conducted all relapse tests (2 h) immediately after the onset of

the dark cycle. The sessions began with the extension of the active

lever and the illumination of the red house light, which remained on

for the duration of the session. Active lever presses during testing

(the operational measure of drug seeking in incubation of craving

studies12,39,40) resulted in contingent presentations of the tone-light

cue, previously paired with oxycodone infusions but did not result in

drug infusions. Inactive lever presses were used as a measure of non-

specific activity and/or response generalization.39,40

2.8 | Food self-administration

We trained rats to self-administer food pellets (Test Diet, #1811155)

for 1 h per day during the middle of their dark cycle, under an FR1

20-s timeout reinforcement schedule; pellet delivery was paired with

5-s light cue. To increase rats' motivation to press for food pellets, we

restricted their diet in home cage to 20 g/day and fed them after they

finished the food self-administration session. Additionally, to facilitate

the acquisition of food self-administration, we gave 1-h magazine

training before the operant training during the first two training days.

During magazine training, we presented the food pellets to the rats

every 5 min; pellet delivery was paired with 5-s light cue.

2.9 | Intracranial injection

We dissolved muscimol + baclofen (Tocris) in sterile saline and

injected the drugs 15 min before the relapse test sessions. The dose-

s of muscimol + baclofen (50 + 50 ng/0.5 μl/side) were based on pre-

vious studies.41,42 The injectors extended 1.0 mm below the tips of

the guide cannulas for OFC. We injected vehicle (saline) or drug at a

rate of 0.5 μl/min and left the injectors in place for an additional

minute to allow diffusion. We connected the syringe pump (Harvard

Apparatus) to 10 μl Hamilton syringes and attached the Hamilton

syringes to the 30-gauge injectors via polyethylene-50 tubing. After

testing, we extracted the rats' brains and stored them in 4% parafor-

maldehyde (PFA) for 48 h at 4�C. We sectioned the rat brains (50 μm

sections) using a Leica cryostat and stained the sections with cresyl

violet. Finally, we verified cannula placements under a light

microscope.

2.10 | Fos immunohistochemistry

Immediately after the relapse tests on withdrawal day 1 and 15, we

anesthetized the rats with isoflurane and perfused them transcardially

with �100 ml of 0.1 M sodium phosphate (PBS) followed by 400 mlof 4% PFA in PBS. We extracted the brains and postfixed them in 4%

PFA for 2 h, then transferred them to 30% sucrose in PBS for 48 h at

4�C. We froze the brains on dry ice and kept them at −80�C until sec-

tioning. We cut serial coronal sections (40 μm) using a Leica Micro-

systems cryostat and preserved the sections in cryoprotectant (20%

glycerol and 2% DMSO in PBS, pH 7.4).

For Fos immunohistochemistry, we processed 1-in-5 series of

sections from OFC of each rat for immunochemical detection of Fos.

We repeatedly rinsed free-floating sections in PBS (3 × 10 min

washes) and incubated them with 3% normal goat serum (NGS) in PBS

with 0.25% Triton X-100 (PBST) for 1 h at room temperature. Next,

we incubated the sections with anti-c-Fos primary antibody (1:5000,

Cat #5348, Cell Signaling, RRID:AB_10557109) diluted in 3% NGS in

PBST overnight at 4�C. We washed the sections with PBS (3 × 10 min

washes) and incubated the sections with biotinylated goat anti-rabbit

secondary antibody (1:600, Vector Laboratories, Cat# BA-1000,

RRID:AB_2313606) diluted in 1% NGS in PBST for 2 h at room tem-

perature. Next, we washed the sections with PBS (3 × 10 min washes)

and incubated the sections with avidin-biotin-peroxidase complex

(ABC, ABC Elite Kit, #PK6100, Vector Laboratories) for 1 h at room

temperature. We then washed the sections with PBS (3 × 10 min

washes) and developed the sections in 3,30-Diaminobenzidine (DAB)

for 100 s. We washed the section in PBS (4 × 5 min) and mounted the

section on glass slides (Fisherbrand™ Superfrost™ Plus Microscope

Slides, Cat #12-550-15). Once dried, the slides were dehydrated in a

ALTSHULER ET AL. 3 of 11

series of ethanol (30%, 60%, 90%, 95%, 100%, and 100%) and cleaned

with Citrisolv (Fisher Scientific). We then cover-slipped slides with

Permount (Fisher Scientific).

2.11 | Image acquisition and neuronal quantification

For each rat, we digitally captured bright-field images of Fos immuno-

reactive (IR) cells in four sections (bregma coordinates: +4.20 to

+3.00 mm), using a Nikon DS-Fi3 camera attached to an inverted

Nikon Eclipse Ti2 Series microscope. We tiled images captured at 10×

magnification, and used an automatic counting method (NIS-Elements,

Nikon, 5.20.00) to quantify Fos-IR cells in VOFC, LOFC and anterior

insula (AI).

2.12 | Experiment 1: effect of oxycodone seeking onFos protein expression in OFC after 1- and 15-daywithdrawal

The goal of Experiment 1 was twofold: (1) to examine whether incu-

bation of oxycodone craving occurs under our experimental condi-

tions (2) to examine whether activation of OFC (assessed by neuronal

activity marker Fos32) is associated with incubation of oxycodone

craving. We performed intravenous surgeries on four groups of rats

(n = 25) and trained them to self-administer oxycodone for 10 days in

three independent runs, as described above. We counterbalanced all

groups based on their oxycodone intake during self-administration

training. On either withdrawal day 1 or 15, we tested one group of

rats for relapse (Day 1: n = 7; Day 15: n = 7) and the other group of

rats served as no-test groups (Day 1: n = 5; Day 15: n = 6). We anes-

thetized the rats, perfused them, and extracted the brains of the rats

from the relapse-test group immediately after the 2-h test session. At

the same time, we perfused and extracted the brains of the rats from

the no-test group, which we brought to the perfusion room directly

from their home cages. Finally, we measured Fos protein expression

in VOFC, LOFC, and AI (an adjacent cortical area dorsal and lateral to

LOFC).

2.13 | Experiment 2: effect of OFC inactivation onoxycodone seeking on withdrawal day 15

The goal of Experiment 2 was to examine whether OFC plays a causal

role in incubation of oxycodone craving. We performed intravenous

surgeries and implanted bilateral cannula 1 mm above OFC on

2 groups of rats (n = 13). We then trained them to self-administer oxy-

codone in 2 independent runs, as described above. On withdrawal

day 15, we injected the rats with vehicle (n = 6) or a mixture of

GABAA and GABAB agonists muscimol + baclofen (n = 7) into OFC,

15 min before the 2-h relapse test. We counterbalanced both groups

based on their oxycodone intake during oxycodone self-

administration.


The goal of Experiment 3 was to examine whether OFC plays a spe-

cific role in oxycodone seeking after long withdrawal (day 15) versus a

general time-independent role in oxycodone seeking. We performed

intravenous surgeries and implanted bilateral cannula 1 mm above

OFC on two groups of rats (n = 11). We then trained them to self-

administer oxycodone in two independent runs. On withdrawal day

1, we injected the rats with vehicle (n = 5) or muscimol + baclofen

(n = 6) into the OFC, 15 min before the 2-h relapse test. We

counterbalanced both groups based on their oxycodone intake during

oxycodone self-administration.

Finally, to examine whether OFC inactivation under the experi-

mental parameters causes motor deficits, we trained rats from Experi-

ment 3 to self-administer palatable food pellets (n = 5) for 5 day

(1 h/day), as described above. We then injected the rats with vehicle

or muscimol + baclofen into OFC, 15 min before the 1-h food self-

administration session on the sixth and ninth days. We re-trained rats

on the seventh and eighth day and we counterbalanced the order of

the vehicle and muscimol + baclofen injections.

2.15 | Statistical analysis

We analyzed the data with SPSS (version 24) mixed analysis of

variances (ANOVAs), one-way ANOVA, or t test, as appropriate.

We followed significant interaction or main effects with Fish-

erprotected least significant difference (PLSD) or Tukey honestly sig-

nificant difference (HSD) post hoc tests. For the repeated measures

analyses of the training data, we replaced 20 outlier values of inactive

lever presses with the group mean for a given training day. We

defined outliers as three median absolute deviations (MADs) above

the group median,43 and we only replaced one outlier (the highest

value above the threshold) for each training day. For rats that were

not well-trained (n = 4) or failed Brevital test (n = 4) during the

10 training days, we extended their training (and implanted the cathe-

ters into their left jugular vein) for additional 1–5 days, and we rep-

laced their training data with data from these additional training days.

We indicate the between- and within-subject factors of the different

analyses in the Section 3. All statistical comparisons are listed in the

Table S1.

3 | RESULTS

3.1 | Oxycodone self-administration (Exp. 1–3)

As reported in a previous study,44 rats demonstrated reliable escala-

tion of oxycodone self-administration and a strong preference for

oxycodone-associated active lever over the nonreinforced inactive

lever during the training phase (Figure 1A, B, and C). All statistical

reporting of these data is listed inTable S1.


3.2 | Experiment 1: effect of oxycodone seeking onFos protein expression in OFC after 1- and 15-daywithdrawal

The goal of Experiment 1 was to examine whether incubation of oxy-

codone craving occurs under our experimental conditions and

whether neuronal activation in OFC is associated with incubation of

oxycodone craving. To achieve this goal, we tested rats for oxycodone

seeking (relapse tests) on withdrawal day 1 and 15 and measured Fos

protein expression in OFC after the relapse tests.

3.2.1 | Relapse tests

Active lever presses during relapse tests was significantly higher after

15 withdrawal days than after 1 day, demonstrating incubation of

oxycodone craving occurred under our experimental conditions. We

analyzed the data with the between-subject factor of withdrawal day

(day 1 and 15) and the within-subject factor of lever (active and inac-

tive lever). We observed significant main effects of lever

(F1,12 = 72.023, p < 0.001) and withdrawal day (F1,12 = 31.105,

p < 0.001), and a significant interaction between these two factors

(F1,12 = 14.483, p = 0.003; Figure 2B).

3.2.2 | Fos-expressing cells in VOFC and LOFC

Number of Fos-expressing cells in both VOFC and LOFC progres-

sively increased during incubation of oxycodone craving. We analyzed

the data with the between-subject factors of Test condition (no test,

relapse test) and Withdrawal day (Day 1 and 15). We found significant

interactions between these factors for both VOFC (F1,21 = 4.741,

p = 0.041) and LOFC (F1,21 = 4.818, p = 0.040, Figure 2C), and a signif-

icant main effect of test condition (VOFC: F1,21 = 11.783, p = 0.002;

LOFC: F1,21 = 15.768, p = 0.001) but no main effect of withdrawal

day in either VOFC or LOFC (Figure 2C,D).

In summary, the data in Experiment 1 demonstrated time-

dependent increase of oxycodone seeking under our experimental

condition. In addition, the Fos data showed that this incubation of

oxycodone craving was associated with a time-dependent increase in

neuronal activation in both VOFC and LOFC. Based on these findings,

we examined the causal role of OFC in incubation of oxycodone crav-

ing in Experiments 2 and 3.


The goal of Experiment 2 was to determine whether OFC plays a

causal role in oxycodone seeking after 15-day withdrawal (incubated

oxycodone seeking). For this purpose, we examined the effect of bilat-

eral inactivation of OFC with a mixture of muscimol and baclofen on

oxycodone seeking on withdrawal day 15.


Musicmol and baclofen injections bilaterally into the OFC on with-

drawal day 15 decreased oxycodone seeking compared with vehicle

injections. We analyzed the total responding with the between-

subject factor of drug dose (muscimol + baclofen, 0, 50 + 50 ng/side)

and the within-subject factor of lever (active and inactive lever). We

observed significant main effects of lever (F1,11 = 19.939, p = 0.001),

drug dose (F1,11 = 9.747, p = 0.010), and a significant interaction

between these two factors (F1,11 = 7.743, p = 0.018, Figure 3B). We

also analyzed the time course data (30-min interval) with the

between-subjects factor of drug dose, and the within-subject factors

of lever and session minute (30, 60, 90, and 120). We observed signif-

icant main effects of drug dose (F1,11 = 9.747, p = 0.010), lever

F IGURE 1 Oxycodone self-administration training. Data aremean ± SEM number of oxycodone (0.1 mg/kg/infusion) infusions,and active and inactive lever presses during the ten 6-h daily self-administration sessions for Exp. 1 (total n = 25), Exp. 2 (total n = 14),Exp. 3 (total n = 11). During training, active lever presses werereinforced on an FR1 20-s timeout reinforcement schedule, andoxycodone infusions were paired with a 5-s tone-light cue. OFC,orbitofrontal cortex


(F1,11 = 19.939, p = 0.001), session minute (F3,33 = 22.827, p < 0.001),

and a significant triple interaction (drug dose × lever × session minute,

F3,33 = 4.979, p = 0.006, Figure 3C).

In summary, the data in Experiment 2 indicate that the bilateral

OFC inactivation decreased oxycodone seeking on withdrawal day

15, demonstrating a critical role of OFC in incubated oxycodone seek-

ing (Figure 3B,C).

3.4 | Experiment 3: effect of inactivation of the OFCon oxycodone seeking on withdrawal day 1

The goal of Experiment 3 was to determine whether the OFC plays a

specific role in incubated oxycodone seeking (withdrawal day 15) ver-

sus a general time-independent role in oxycodone seeking. To achieve

this goal, we examined the effect of bilateral inactivation of the OFC

on oxycodone seeking on withdrawal day 1.


Muscimol and baclofen injections into the OFC had no effect on oxy-

codone seeking on withdrawal day 1. We analyzed the total

responding with the between-subject factor of drug dose

(muscimol + baclofen, 0, 50 + 50 ng/side) and the within-subject fac-

tor of lever (active and inactive lever). We observed a significant main

effect of lever (F1,9 = 24.101, p = 0.001) but no effect of drug dose

(F1,9 = 0.266, p = 0.618) or an interaction between these two factors

(F1,9 = 0.519, p = 0.490, Figure 4B). We also analyzed the time course

data with the between-subjects factor of drug dose and the within-

subject factors of lever and session minute. We observed significant

main effects of session minute (F3,27 = 20.235, p < 0.001) and lever

(F1,9 = 24.101, p = 0.001) but not drug doses(F1,9= 0.266, p = 0.618)

or interactions between these three factors (F3,27 = 0.596, p = 0.623;

Figure 4B,C).

Finally, to rule out the possibility that the effect of OFC inactiva-

tion by muscimol + baclofen on oxycodone seeking in Experiment

2 was caused by motor deficits, we trained five rats from Experiment

3 to self-administer palatable food pellets and examined the effect of

vehicle or muscimol + baclofen OFC injections on ongoing food self-

administration. We found that bilateral muscimol + baclofen injections

into the OFC had no effect on food-reinforced responding (p > 0.05,

Figure S2).

In summary, the data from Experiment 3 showed that bilateral

OFC inactivation had no effect on oxycodone seeking on withdrawal

day 1 or ongoing food self-administration. Taken together with the

findings in Experiment 2, these data demonstrate a time-dependent

role of the OFC in incubation of oxycodone craving.

4 | DISCUSSION

We used immunohistochemistry in combination with pharmacological

reversible inactivation and studied the role of OFC in incubation of

oxycodone craving in male rats. We report two main findings. First,

oxycodone seeking during incubation of oxycodone craving was asso-

ciated with a time-dependent increase of neuronal activation

(assessed by Fos) in both VOFC and LOFC. Second, muscimol + baclo-

fen injections into OFC decreased oxycodone seeking on withdrawal

day 15 but had no effect on withdrawal day 1. Together, these find-

ings demonstrated a critical role of OFC in incubation of oxycodone

craving in male rats.

F IGURE 2 Incubated oxycodone seeking is associated withneuronal activation in OFC (Exp. 1). (A) Timeline of the experiment.(B) Relapse test after 1 or 15 withdrawal days. During testing, leverpresses led to contingent presentations of the tone-light cuepreviously paired with oxycodone infusions during training but notdrug infusions. Data are mean ± SEM of lever presses on thepreviously active lever and on the inactive lever during the relapse

test sessions. *Different from Day 1, p < 0.05, n = 5–7 per group.(C) Fos-expressing cells: data are mean ± SEM of Fos-expressing cellsper mm2 in VOFC (left) and LOFC (right). *Different from Day1, p < 0.05, n = 5–7 per group. (D) Representative images of Fos-expressing cells in VOFC and LOFC. LOFC, lateral orbitofrontalcortex; VOFC, ventral orbitofrontal cortex


4.1 | Methodological considerations

One issue is that the effect of muscimol + baclofen injections into

OFC on oxycodone seeking is due to motor deficits. This is unlikely

because muscimol + baclofen injections into OFC had no effect on

high lever responding during ongoing food self-administration (see

Figure S2). Furthermore, a recent study demonstrated that OFC

inactivation with the same dose of muscimol + baclofen had no

effect on reacquisition of fentanyl self-administration, which

supports our conclusion that OFC inactivation under our experimen-

tal condition does not cause motor deficits.45 Second, the null effect

of OFC inactivation on oxycodone seeking on withdrawal day

1 (Exp. 3) may be due to a floor effect attributed by low lever

responding and therefore should be interpreted with caution. A

third issue is that all animals in the current study are male rats. A

question for future studies is whether the critical role of OFC in

incubation of oxycodone craving in male rats also generalizes to

female rats.

F IGURE 3 Reversible inactivation of OFC decreased oxycodone seeking on withdrawal day 15 (Exp. 2). (A) Timeline of the experiment. (B–C)Relapse test after 15 withdrawal days after bilateral injections of vehicle or muscimol + baclofen (50 + 50 ng/0.5 μl/side) into the OFC. Data aremean ± SEM of responses on the previously active lever or inactive lever during the relapse tests. *Different from vehicle, p < 0.05. n = 6–7 pergroup. (D) Approximate placement (mm from Bregma38) of injection tips (vehicle: open circles; muscimol + baclofen: closed circles), andrepresentative cannula placements. LOFC, lateral orbitofrontal cortex; VOFC, ventral orbitofrontal cortex

F IGURE 4 Reversible inactivation of OFC had no effect on oxycodone seeking on withdrawal day 1 (Exp. 3). (A) Timeline of the experiment.(B–C) Relapse test after one withdrawal day after bilateral injections of vehicle or muscimol + baclofen (50 + 50 ng/0.5 μl/side) into the OFC.Data are mean ± SEM of responses on the previously active lever or inactive lever during the relapse tests. n = 5–6 per group. (D) Approximateplacement (mm from Bregma38) of injection tips (vehicle: open circles; muscimol + baclofen: closed circles), and representative cannulaplacements. OFC, orbitofrontal cortex


A final issue is the anatomical specificity of muscimol + baclofen

injections into OFC. One possibility is that behavioral changes

observed in Exp. 2 is partially due to drug diffusion to adjacent areas

after OFC injections.46 One such area is AI, which has recently been

implicated in methamphetamine42 and fentanyl seeking47 after food-

choice based voluntary abstinence and alcohol seeking after

punishment-imposed voluntary abstinence.41 To explore this possibil-

ity, we measured Fos protein expression in AI. Although we found a

significant interaction between test condition and withdrawal day

(F1,21 = 4.480, p = 0.046, Figure S1), the number of Fos-expressing

cells associated with oxycodone seeking in AI is minimum compared

with in OFC, which suggests AI might play a less prominent role in

incubation of oxycodone craving.

In addition, based on our Fos data showing that both VOFC and

LOFC exhibited a similar time-dependent increase of Fos expression

associated with incubation of oxycodone craving, we inactivated OFC

that includes both VOFC and LOFC. However, Fanous et al, 33 previ-

ous study showed that reversible inactivation of LOFC but not VOFC

decreases incubated heroin seeking. Therefore, it is possible that

LOFC alone plays a critical role in incubation of oxycodone craving.

An earlier study also demonstrated that reversible inactivation of

LOFC, but not medial OFC (an anterior structure that lies medial to

VOFC), decreases cue-induced reinstatement of cocaine seeking.34 In

contrast, a recent study showed that blocking dopamine-1 receptor

signaling in medial OFC also decreases both cue-induced and cocaine-

primed reinstatement of cocaine seeking.48 Furthermore, a new study

demonstrated a critical role of VOFC and LOFC in fentanyl seeking

after food-choice based voluntary abstinence.45 These findings,

together with our results, raise questions for future studies to exam-

ine the causal roles of OFC subregions in incubation of oxycodone

craving.

4.2 | OFC and incubation of drug craving

The first study that implicates OFC in incubation of drug craving

focused on heroin and demonstrates that incubated heroin seeking

is associated with neuronal activation in OFC, and either pharmaco-

logical reversible inactivation or chemogenetic ablation of the OFC

neuronal ensembles decreases incubation of heroin craving.33 Based

on these findings and earlier findings on the role of OFC in cue and

context-induced reinstatement of cocaine seeking,34,35 we subse-

quently examined whether OFC plays a role in incubation of meth-

amphetamine craving. Unexpectedly, we found that reversible

inactivation of OFC has no effect on incubated methamphetamine

seeking.18 Our current findings on oxycodone extend previous incu-

bation studies and, more broadly, extend the body of literatures on

the role of OFC in cocaine,34,35,48,49 alcohol relapse50 assessed by

extinction-based animal models, and cognitive processes associated

with drug addiction.51–57 It is of note that although our inactivation

studies here are consistent with the previous heroin study,33 we

observed differences in Fos protein expression between these two

studies. Fanous et al,33 showed that Fos protein expression in OFC

(without differentiating VOFC and LOFC) increases during incubated

heroin seeking, but there is no interaction between test condition

and withdrawal day. Here, we found significant interactions

between test condition and withdrawal day when we analyzed Fos

protein expression in both VOFC and LOFC, indicating a time-

dependent neuronal activation of OFC during incubation of oxyco-

done craving.

Despite that the direct evidence on the role of OFC in incubation

of cocaine craving is still lacking, results from previous18,33 and cur-

rent incubation studies suggest that the critical role of OFC in incuba-

tion of drug craving might be selective to opioids, which adds

additional evidence that distinct neural mechanisms underlie opioid

versus psychostimulant addiction.12,58 What might contribute to the

selective role of OFC in incubation of opioid craving? One possibility

is that opioids induce distinct neurological adaptations in OFC during

withdrawal. Supporting this hypothesis, previous studies showed that

spine density in OFC increases after withdrawal from morphine self-

administration59 but decreases after withdrawal from amphetamine

self-administration.60 Beside differences in structural plasticity, OFC

may also undergo molecular changes specific to incubation of opioid

craving, and one potential category of candidate molecules is opioid

receptors, the sites of action for opioids. Several studies provided

indirect evidence supporting this hypothesis. During incubation of

heroin craving, mu opioid receptor (MOR) mRNA expression in

nucleus accumbens decreases on withdrawal day 1 but returns to the

basal level on withdrawal day 15 and 30.61 Moreover, incubation of

oxycodone craving is associated with time-dependent changes of

MOR expression in dorsal striatum (increased mRNA but decreased

protein expression), and both MOR and kappa opioid receptor expres-

sion in hippocampus (decreased mRNA but increased protein expres-

sion).27 Finally, during our previous RNA-sequencing study, we found

no changes in opioid receptor expression in OFC during incubation of

methamphetamine craving.25

It is of note that the scenarios proposed above are highly specula-

tive and need validation by future experiments. Furthermore, distinct

neural mechanisms underlying incubation of craving have been dem-

onstrated within the same drug class (e.g., cocaine

vs. methamphetamine18,21). Therefore, we cannot rule out the possi-

bility that OFC, at the neurobiological levels, may play distinct roles in

incubation of craving to different opioids. Lastly, direct comparison of

the role of OFC between reinstatement and incubation studies should

be made with caution, because different self-administration proce-

dures (e.g., short-access vs. long-access) and animal models (extinc-

tion-based vs. abstinence-based) could lead to different changes at

both behavioral and neurological levels.11,14,62–65

5 | CONCLUDING REMARKS

We identified OFC as a critical neural substrate for incubation of oxy-

codone craving. Together with previous findings,18,33 our results high-

light a selective role of OFC in incubation of opioid craving. A recent

study45 examining fentanyl seeking after voluntary relapse also


suggest that the role of OFC could generalize to other forms of opioid

relapse.66 Overall, our findings set a foundation for answering ques-

tions such as whether the role of OFC in incubation of oxycodone

craving is sex-specific and/or subregion specific and what neurobio-

logical mechanisms in OFC contributes to incubation of oxycodone

craving, in future studies.

ACKNOWLEDGEMENTS

This research is supported by University of Maryland Department of

Psychology Startup Funds (X.L.). The authors declare that they do not

have any conflicts of interest (financial or otherwise) related to the

data presented in this manuscript. We thank Dr Yavin Shaham for

supporting the initiation of this project. We thank Trinity Russell for

technical support during the early phase of this project.

AUTHOR CONTRIBUTIONS

XL conceived the project, provided intellectual inputs, carried out

experiments, and wrote the paper. RDA carried out experiments, pro-

vided intellectual inputs, and wrote the paper. KTG carried out experi-

ments, analyzed the data, and wrote the paper. ESY, IRD, AO, MH,

and SR carried out experiments. All authors reviewed the content and

approved the final version for publication.

ORCID

Rachel D. Altshuler https://orcid.org/0000-0002-1771-9287

Kristine T. Garcia https://orcid.org/0000-0003-2559-1102

Ian R. Davis https://orcid.org/0000-0002-4048-6856

Xuan Li https://orcid.org/0000-0002-6737-1223

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SUPPORTING INFORMATION

Additional supporting information may be found online in the

Supporting Information section at the end of this article.

How to cite this article: Altshuler RD, Yang ES, Garcia KT,

et al. Role of orbitofrontal cortex in incubation of oxycodone

craving in male rats. Addiction Biology. 2020;e12927. https://

doi.org/10.1111/adb.12927


https://doi.org/10.1111/adb.12927https://doi.org/10.1111/adb.12927

Role of orbitofrontal cortex in incubation of oxycodone craving in male rats INTRODUCTION METHODS AND MATERIALS Subjects Intravenous surgery Cannula implantation Apparatus Oxycodone self-administration training Withdrawal phase Relapse test Food self-administration Intracranial injection Fos immunohistochemistry Image acquisition and neuronal quantification Experiment 1: effect of oxycodone seeking on Fos protein expression in OFC after 1- and 15-day withdrawal Experiment 2: effect of OFC inactivation on oxycodone seeking on withdrawal day 15 Experiment 3: effect of OFC inactivation on oxycodone seeking on withdrawal day 1 Statistical analysis

RESULTS Oxycodone self-administration (Exp. 1-3) Experiment 1: effect of oxycodone seeking on Fos protein expression in OFC after 1- and 15-day withdrawal Relapse tests Fos-expressing cells in VOFC and LOFC

Experiment 2: effect of OFC inactivation on oxycodone seeking on withdrawal day 15 Relapse tests

Experiment 3: effect of inactivation of the OFC on oxycodone seeking on withdrawal day 1 Relapse tests

DISCUSSION Methodological considerations OFC and incubation of drug craving

CONCLUDING REMARKSACKNOWLEDGEMENTS AUTHOR CONTRIBUTIONSREFERENCES

Documents

Role of orbitofrontal cortex in incubation of oxycodone craving ......neural mechanisms underlying incubation of craving to oxycodone, a commonly abused prescription opioid, and we