Opioid activation of toll-like receptor 4 contributes to drug reinforcement.

Abstract

Opioid action was thought to exert reinforcing effects solely via the initial agonism of opioid receptors. Here, we present evidence for an additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its MyD88-dependent signaling. Blockade of TLR4/MD2 by administration of the nonopioid, unnatural isomer of naloxone, (+)-naloxone (rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned place preference. (+)-Naloxone also reduced opioid (remifentanil) self-administration (rats), another commonly used behavioral measure of drug reward. Moreover, pharmacological blockade of morphine-TLR4/MD2 activity potently reduced morphine-induced elevations of extracellular dopamine in rat nucleus accumbens, a region critical for opioid reinforcement. Importantly, opioid-TLR4 actions are not a unidirectional influence on opioid pharmacodynamics, since TLR4(-/-) mice had reduced oxycodone-induced p38 and JNK phosphorylation, while displaying potentiated analgesia. Similar to our recent reports of morphine-TLR4/MD2 binding, here we provide a combination of in silico and biophysical data to support (+)-naloxone and remifentanil binding to TLR4/MD2. Collectively, these data indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions, and provides further evidence supporting a role for central proinflammatory immune signaling in drug reward.

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Cellular/Molecular
OpioidActivationofToll-LikeReceptor4Contributesto
DrugReinforcement
M.R.Hutchinson,1,4*A.L.Northcutt,1*T.Hiranita,7X.Wang,1,3S.S.Lewis,1J.Thomas,5K.vanSteeg,4,6T.A.Kopajtic,7
L.C.Loram,1C.Sfregola,1E.Galer,1N.E.Miles,1S.T.Bland,8J.Amat,1R.R.Rozeske,1T.Maslanik,2T.R.Chapman,1
K.A.Strand,1M.Fleshner,2R.K.Bachtell,1A.A.Somogyi,5H.Yin,3J.L.Katz,7K.C.Rice,9S.F.Maier,1
andL.R.Watkins1
Departmentsof1PsychologyandNeuroscience,2IntegrativePhysiology,CenterforNeuroscience,and3ChemistryandBiochemistry,CenterforNeuroscienceand
theBiofrontiersInstitute,UniversityofColorado-Boulder,Boulder,Colorado80309,Disciplinesof4Physiologyand5Pharmacology,SchoolofMedicalSciences,
UniversityofAdelaide,Adelaide,SouthAustralia5005,Australia,6DivisionofPharmacology,FacultyofScience,UtrechtUniversity,3805TDUtrecht,theNetherlands,
7DepartmentofHealthandHumanServices,NationalInstituteonDrugAbuse,NationalInstitutesofHealth,IntramuralResearchProgram,BiomedicalResearchCenter
MDRB,Baltimore,Maryland21224,8DepartmentofPsychology,UniversityofColorado-Denver,Denver,Colorado80217,and9ChemicalBiologyResearchBranch,National
InstituteonDrugAbuseandNationalInstituteonAlcoholAbuseandAlcoholism,NationalInstitutesofHealth,Rockville,Maryland20892
Opioidactionwasthoughttoexertreinforcingeffectssolelyviatheinitialagonismofopioidreceptors.Here,wepresentevidenceforan
additional novel contributor to opioid reward: the innate immune pattern-recognition receptor, toll-like receptor 4 (TLR4), and its
MyD88-dependentsignaling.BlockadeofTLR4/MD2byadministrationofthenonopioid,unnaturalisomerofnaloxone,(?)-naloxone
(rats), or two independent genetic knock-outs of MyD88-TLR4-dependent signaling (mice), suppressed opioid-induced conditioned
placepreference.(?)-Naloxonealsoreducedopioid(remifentanil)self-administration(rats),anothercommonlyusedbehavioralmea-
sureofdrugreward.Moreover,pharmacologicalblockadeofmorphine-TLR4/MD2activitypotentlyreducedmorphine-inducedeleva-
tionsofextracellulardopamineinratnucleusaccumbens,aregioncriticalforopioidreinforcement.Importantly,opioid-TLR4actions
are not a unidirectional influence on opioid pharmacodynamics, since TLR4?/?mice had reduced oxycodone-induced p38 and JNK
phosphorylation,whiledisplayingpotentiatedanalgesia.Similartoourrecentreportsofmorphine-TLR4/MD2binding,hereweprovide
acombinationofinsilicoandbiophysicaldatatosupport(?)-naloxoneandremifentanilbindingtoTLR4/MD2.Collectively,thesedata
indicate that the actions of opioids at classical opioid receptors, together with their newly identified TLR4/MD2 actions, affect the
mesolimbic dopamine system that amplifies opioid-induced elevations in extracellular dopamine levels, therefore possibly explaining
altered opioid reward behaviors. Thus, the discovery of TLR4/MD2 recognition of opioids as foreign xenobiotic substances adds to the
existing hypothesized neuronal reinforcement mechanisms, identifies a new drug target in TLR4/MD2 for the treatment of addictions,
andprovidesfurtherevidencesupportingaroleforcentralproinflammatoryimmunesignalingindrugreward.
Introduction
The reinforcing/rewarding effects of opioids contribute to their
widespread abuse (Compton and Volkow, 2006; Manchikanti,
2006). Acute opioid reinforcement is traditionally thought to be
mediated through the activation of mesolimbic dopamine neu-
rons projecting from the ventral tegmental area to the nucleus
accumbens (NAc) shell (Ikemoto, 2007). However, additional
complexities to this system have also been postulated (Laviolette
et al., 2002; Vargas-Perez et al., 2009).
While there has long been a focus on classical opioid recep-
tors in exploring opioid actions, reconsideration is necessary.
The seminal research of Takagi et al. (1960) demonstrated the
pharmacodynamic relevance of nonclassical nonstereoselec-
tive opioid actions, and Goldstein et al. (1971) found a 30-fold
greater abundance of nonstereoselective but saturable opioid
binding sites compared with saturable stereoselective opioid
binding. However, until recently there has been little research
on these aspects of opioid pharmacology (Hutchinson et al.,
2011). Reevaluation of these data implies additional sites of
opioid action, which are capable of recognizing structurally
ReceivedFeb.11,2012;revisedJune14,2012;acceptedJune25,2012.
Authorcontributions:M.R.H.,A.L.N.,T.H.,X.W.,S.S.L.,J.T.,K.v.S.,T.A.K.,L.C.L.,S.T.B.,J.A.,R.R.R.,A.A.S.,H.Y.,J.L.K.,
K.C.R.,S.F.M.,andL.R.W.designedresearch;M.R.H.,A.L.N.,T.H.,X.W.,S.S.L.,J.T.,K.v.S.,T.A.K.,L.C.L.,C.S.,E.G.,N.E.M.,
S.T.B.,J.A.,R.R.R.,T.M.,T.R.C.,K.A.S.,M.F.,K.C.R.,andL.R.W.performedresearch;M.R.H.,A.L.N.,T.H.,X.W.,S.S.L.,J.T.,
K.v.S.,T.A.K.,L.C.L.,C.S.,E.G.,N.E.M.,S.T.B.,J.A.,R.R.R.,K.A.S.,A.A.S.,H.Y.,J.L.K.,K.C.R.,S.F.M.,andL.R.W.analyzeddata;
M.R.H.,A.L.N.,T.H.,X.W.,S.S.L.,J.T.,K.v.S.,T.A.K.,R.K.B.,A.A.S.,H.Y.,J.L.K.,K.C.R.,S.F.M.,andL.R.W.wrotethepaper.
This research was supported by NIH Grants DA024044, DE017782, DA023132, DA025740, NS067425,
DA027977, and DA026950 and by the NIH Intramural Research Programs of the National Institute on Drug
AbuseandtheNationalInstituteonAlcoholAbuseandAlcoholism.SupportwasalsoprovidedbyanInterna-
tional Association for the Study of Pain international collaborative research grant, an American Australian
Association Merck Company Foundation Fellowship, a National Health and Medical Research Council CJ
Martin Fellowship (ID 465423; M.R.H. 2007–2010), Australian Research Council Research Fellowship
(DP110100297;M.R.H.2011),andAustralianPostgraduateAward(J.T.)andDanoneResearchforSpecialised
Nutrition (The Netherlands) (K.vS). We thank Dr. David White and the NIDA Addiction Treatment Discovery
Program(NIDAATDP)fortheNovaScreendatageneratedthroughacontractwithCaliperLifeSciencesaswell
as for the biogenic amine transporter data generated through a contract with Research Service (R&D-22),
DepartmentofVeteransAffairsMedicalCenter,Portland,OR.
*M.R.H.andA.L.N.contributedequallytothiswork.
Theauthorsdeclarenocompetingfinancialinterests.
CorrespondenceshouldbeaddressedtoLindaR.Watkins,DepartmentofPsychologyandNeuroscience,Campus
Box345,UniversityofColoradoatBoulder,Boulder,CO80309-0345.E-mail:linda.watkins@colorado.edu.
DOI:10.1523/JNEUROSCI.0684-12.2012
Copyright©2012theauthors 0270-6474/12/3211187-14$15.00/0
TheJournalofNeuroscience,August15,2012 • 32(33):11187–11200 • 11187

Page 2

diverse ligands beyond the more well studied opioid active
(?)-isomers.
Structurally diverse opioids (including stereochemistries)
could also be viewed as xenobiotics, akin to detection of chemi-
cals by the liver’s pregnane X receptor (Matic et al., 2007), thus
recognized as substances “foreign” to the CNS. Within the CNS,
pattern recognition receptors, such as toll-like receptors (TLRs),
can serve this sentinel role identifying “molecular patterns” as
“nonself” or “danger” signals (Buchanan et al., 2010). TLR4 has
recently received increasing attention as it responds to highly
diversemolecularpatternsassociatedwithGram-negativebacte-
ria and endogenous substances released from stressed/damaged
host cells. Such ligands activate the TLR4 complex, inducing the
productionandreleaseofproinflammatory,neuroexcitatoryme-
diators via MyD88-dependent intracellular pathways (Yirmiya
and Goshen, 2011).
We have previously examined the xenobiotic-mediated TLR4
actionsofopioidsusingacollectionofinvivo,invitro,molecular,
and in silico strategies, demonstrating that various opioids, in-
cluding morphine, activate TLR4 signaling (Hutchinson et al.,
2010a; Wang et al., 2012b) through binding to an accessory pro-
tein of TLR4, myeloid differentiation protein 2 (MD2), thereby
inducing TLR4 oligomerization and triggering proinflammation
(Wang et al., 2012b). The potential importance of activation of
TLR4 signaling by opioids, in addition to opioid activation of
classical neuronal opioid receptors, for opioid reinforcement is
unknown, and is explored here for the first time.
The present studies aimed to define whether opioid-induced
TLR4 activation contributes to opioid reinforcement and its as-
sociated elevations of NAc dopamine (Ikemoto, 2007). This was
conducted using mice deficient in TLR4 and MyD88 signaling
andbypharmacologicalblockadeofTLR4by(?)-naloxone.Im-
portantly,ineachconditiontheanalgesicpropertiesoftheopioid
agonist were assessed to determine whether the TLR4-directed
intervention had simply reduced all the pharmacodynamic ac-
tions of the opioid agonist, rather than selectively the rewarding
properties. It is apparent that an understanding of opioid-TLR4
actionswithinthemesolimbicdopaminerewardsystem,inaddi-
tion to the established opioid receptor-dependent response, will
have implications for how opioid reinforcement is viewed, and
the opportunities that await the use of pharmacological TLR4
blockade in drug reward.
MaterialsandMethods
Subjects
For studies at the University of Colorado, viral-free adult, male Sprague
Dawley rats (250–325 g; Harlan) were pair-housed in standard Plexiglas
cageswithadlibitumchoicefoodandwater.Thecolonyroomwasmain-
tained at 21°C on a 12/12 h light/dark cycle. All experiments were con-
ducted during the light phase. Upon arrival, rats were allowed 1 week of
acclimationbeforeanyprocedures.Allprocedureswereapprovedbythe
University of Colorado Institutional Animal Care and Use Committee.
Forthedrugself-administrationprocedure,performedattheNational
Institute on Drug Abuse, viral-free adult male Sprague Dawley rats
(weighing ?300 g at the start of the study), obtained from Taconic
Farms, served as subjects after acclimation to the laboratory for at least 1
week. Food (Scored Bacon Lover Treats, BIOSERV) and tap water were
available in their home cages. After acclimation, weights of rats were
maintained at ?320 g by adjusting their daily food ration. The animal
housingroomwastemperatureandhumiditycontrolledandmaintained
on a 12/12 h light/dark cycle with lights on at 07:00 A.M. All procedures
were approved by the National Institute on Drug Abuse Intramural Re-
searchProgramInstitutionalAnimalCareandUseCommittee.Allstud-
ies were performed during the light phase.
Formousestudies,conductedattheUniversityofAdelaide,pathogen-
free adult male wild-type BALB/c mice were obtained from The Univer-
sity of Adelaide Laboratory Animal Services (Adelaide, SA, Australia),
and two null mutant mouse strains, TLR4?/?and MyD88?/?, were
originally sourced from Professor Akira (Osaka University, Osaka,
Japan) via Dr. Paul Foster from University of Newcastle (Newcastle,
NSW,Australia).Micewerehousedina12/12hlight/darkcycle(lighton
at 7:00 A.M.) in temperature-controlled rooms (23 ? 3°C). Food and
water was available ad libitum. After arrival, the mice were allowed to
acclimate for at least 5 d and were handled at least 3 d before testing
commenced. The mice were always tested during the light phase of the
light/dark cycle. All procedures were approved by the Animal Ethics
Committee of the University of Adelaide. All studies were performed
during the light phase.
Drugs
(?)-Morphine sulfate was gifted by Mallinckrodt. (?)-Naloxone was
synthesized by Dr. Kenner Rice (Chemical Biology Research Branch,
National Institute on Drug Abuse and National Institute on Alcohol
Abuse and Alcoholism, National Institutes of Health, Bethesda, MD).
Oxycodoneand(?)-naloxonewaspurchasedfromSigma.Remifentanil
(brand name Ultiva) was purchased from Mylan Institutional. Glycine
vehicle was purchased from Sigma. Drugs were confirmed to be
endotoxin-free by the limulus amebocyte lysate assay (Lonza) con-
ducted per the manufacturer’s instructions. Drugs doses are reported
as free base.
Experiment1:Ratconditionedplace preference
Apparatus
The Plexiglas place preference apparatus measured 72 [length (L)] ? 30
[width (W)] ? 30 [height (H)] cm and comprised two distinct condi-
tioning environments with a neutral space in-between. Each condition-
ing environment measured 30 (L) ? 30 (W) ? 30 (H) cm. One
environment had a floor consisting of 5 mm metal bars spaced 1.5 cm
apart (edge-to-edge) and walls with alternating 2 cm wide black and
white stripes. The floor of the second environment was a black anodized
aluminum plate, perforated across the surface with evenly spaced 5 mm
holes, and the walls were black with evenly spaced 50 mm white polka
dots. The neutral area measured 12 ? 30 ? 30 cm, with sanded, black
Plexiglas flooring. During the conditioning phase, Plexiglas partitions
matching their respective environments were inserted to restrict the rats
to their specific, designated environment.
The activity of each rat was recorded using Logitech Quickcam Pro
5000 webcams mounted 1.0 m above the center of the conditioned place
preference apparatus. The cameras were connected to a computer run-
ningAnyMaze(Stoelting),totrackandrecordthetimearatspentineach
of the three compartments.
Procedure to assess the effect of (?)-naloxone on morphine
conditioned place preference
An unbiased conditioned place preference protocol was used. Rats were
handledandweighedthedaybeforethestartofanexperiment.Onday1,
all rats were placed individually in the conditioned place preference ap-
paratus and allowed to freely explore the entire apparatus for 20 min, to
assess baseline preferences or biases to either environment. Any rat that
spent ?20% or ?80% of the entire time in either environment was
removed from the study. Each rat was then randomly assigned to treat-
ment group and conditioning environment in a counterbalanced fash-
ion, so that half the rats in each treatment group were assigned to the
environment they preferred, and half were assigned to the environment
theylesspreferred.Ratsreceivedeither(1)salinesubcutaneouslyinjected
immediatelywith(?)-morphine(5mg/kg,s.c.)orsalinesubcutaneously
onalternatingdays;(2)salinesubcutaneouslyinjectedimmediatelywith
saline subcutaneously every day; (3) (?)-naloxone (1 mg/kg, s.c.) in-
jected immediately with (?)-morphine (5 mg/kg, s.c.) or with saline
subcutaneously on alternating days; or (4) (?)-naloxone (1 mg/kg, s.c.)
injected immediately with saline subcutaneously every day. Upon com-
pletionofinjections,subjectswereplacedintothedesignatedcondition-
ingenvironmentfor45min.Placepreferencetestingoccurredonday10
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(preexposure day 1, days 2–9 conditioning, day 10 preference test) and
was run identically to baseline testing on day 1. That is, rats were placed
in the place preference apparatus, in a drug-free state, and allowed to
explore the entire apparatus for 20 min. The time spent in each environ-
ment was recorded and conditioning was calculated as a difference be-
tween the time spent in the drug-paired environment before and after
conditioning. All testing was performed using blinded procedures with
respect to group assignments.
Procedure to assess the possible aversive effect of (?)-naloxone
Thesameparadigmwasusedasabove,exceptthatratsreceivedeither(1)
(?)-naloxone(1mg/kg,s.c.)versussalinesubcutaneouslyonalternating
days, or (2) saline subcutaneously every day. Place preference was then
assessed as above.
Experiment2:Rat self-administration
Apparatus
Experimental sessions were conducted with subjects placed in operant-
conditioning chambers (modified ENV-008CT, Med Associates) that
measured 25.5 ? 32.0 ? 25.0 cm, and were enclosed within sound-
attenuating cubicles equipped with a fan for ventilation and white noise
to mask extraneous sounds. On the front wall of each chamber were two
responselevers,5.0cmfromthemidlineand4.0cmabovethegridfloor.
A downward displacement of a lever with a force approximating 20 g
defined a response, which always activated a relay mounted behind the
front wall of the chamber producing an audible “feedback” click. Three
light-emitting diodes (LEDs) were located in a row above each lever. A
receptacleforthedeliveryoffoodpelletswasmountedbehinda5.0?5.0
cm opening in the front wall midline between the two levers and 2.0 cm
above the floor. A pellet dispenser (ENV-203, Med Associates) could
deliver 45 mg food pellets to the receptacle. A syringe driver (Model 22,
Harvard Apparatus) placed above each chamber delivered injections of
specified volumes and durations from a 10 ml syringe. The syringe was
connected by Tygon tubing to a single-channel fluid swivel (375 Series
SingleChannelSwivels),whichwasmountedonabalancearmabovethe
chamber. Tygon tubing from the swivel to the subject’s catheter was
protected by a surrounding metal spring and completed the connection
to the subject.
Procedures
Subjectswereplacedinchambersduringexperimentalsessionsthatwere
conducted daily, 7 d per week. During sessions, subjects were trained
with food reinforcement (45 mg of food pellets, BIOSERV) to press the
right lever, and were subsequently trained under a fixed-ratio (FR)
5-response schedule of reinforcement (each fifth response produced a
foodpellet).Fooddeliverieswerefollowedbya20stimeout(TO)period
during which all lights were off and responses had no scheduled conse-
quences other than the feedback click. During this training, sessions
lasted for 20 min or until 30 food pellets were delivered.
After subjects were responding at a rate sufficiently high that they
obtained 30 food pellets within each of three consecutive sessions, they
were surgically implanted in the right or left external jugular vein with a
chronic indwelling catheter that exited at the mid-scapular region of the
animal’s back. Catheter implantation was performed under anesthesia
(ketamine 60 mg/kg, i.p. and xylazine 12 mg/kg, i.p.). Catheters were
infused daily with 0.1 ml of a sterile saline solution containing heparin
(30 IU/ml) and penicillin G potassium (250,000 IU/ml) to minimize the
likelihoodofinfectionandtheformationofclotsorfibroids.Allanimals
were allowed to recover from surgery for ?7 d before cocaine self-
administration studies were initiated.
Rats were trained to self-administer cocaine first, a standard training
paradigm as previously described (Hiranita et al., 2011). Cocaine self-
administration sessions were conducted in 2 h daily sessions until the
responseratesandpatternsofrespondingshowednosubstantialsession-
to-session trends. During these sessions, the LEDs above the right lever
were illuminated when cocaine injections were available. Completion of
five responses turned off the LEDs and activated the infusion pump,
delivering a dose of 0.89 mg/kg. A 20 s TO, during which LEDs were off
and responses produced no consequences, started with the injection.
After the time out, the LEDs were illuminated and responding again had
scheduled consequences. Once rates of responding maintained by co-
cainewerestableacrosssessions,thesessionwasdividedintofive20min
components, each preceded by a 2 min TO. This arrangement allowed
the assessment of a different cocaine dose within each component. By
adjustinginfusionvolumesanddurations,thecocainedoseperinjection
wasincrementedinthefivesequentialcomponentsinanascendingorder
as follows: no injection (also referred to as extinction, or EXT, because
responses had no scheduled consequences other than turning off the
LEDs for 20 s), 0.026, 0.089, 0.29, and 0.89 mg/kg/inj. Infusion volumes
and durations were respectively 0, 5.6, 18, 56, 180 ?l and 0, 0.32, 1, 3.2
10 s, based on a body weight of 0.32 kg. A response-independent “sam-
ple” injection of cocaine at the corresponding dose was administered
immediately before each component.
Training continued until: (1) at least 4.5 mg/kg of cocaine was self-
administered within a session with ?20% variation in the total number
ofcocaineinjectionscomparedwiththeprevioussession;(2)thedoseof
cocaine that maintained maximal response rates varied by no more than
one-half log unit over two consecutive test sessions; and (3) maximum
response rates were at least five-fold higher than response rates main-
tained during EXT.
Onceperformanceswerestableacrosssuccessivesessions,theeffectof
substitutions for cocaine of remifentanil (dose range 0.09–2.9 micro-
gram/kg/inj, i.v.) was assessed, with a minimum of 72 h between treat-
ments. Subsequently, the effects of presession intraperitoneal injections
of (?)-naloxone on the response rates maintained by remifentanil
injection were assessed. The opioid remifentanil was chosen because
its ultra-short half-life increases rapidity and stability of bar-pressing
for drug (Panlilio et al., 2003). Due to high rates of remifentanil
self-administration, infusion durations were reduced to 0, 0.24, 0.75,
2.4, 7.5 s to avoid excessive fluid intake and emptying of the syringe.
Experiment 3: Rat in vivo microdialysis
Surgery
A single microdialysis guide cannula was stereotaxically implanted per
rat under isoflurane anesthesia (MWI Veterinary Supply). Each sterile
CMA 12 gauge guide cannula (CMA Microdialysis) was aseptically im-
planted and aimed at the right or left NAc shell (stereotaxic coordinates
relative to bregma: anterior/posterior ? ?1.7 mm; medial/lateral ?
?0.8 mm; relative to dura: dorsal/ventral ? ?5.6 mm, bite bar ? 0;
(Paxinos and Watson, 1998) in a counterbalanced fashion. The guide
cannula and a tether screw (CMA microdialysis) were attached to the
skull using three jeweler’s screws and dental cement. After surgery (iso-
fluraneanesthesia),ratswereindividuallyhousedandallowedtorecover
for at least 1 week.
In vivo microdialysis procedures
The microdialysis study was undertaken with a minimum of 1 week
recovery from anesthesia and guide cannula insertion. The afternoon
before the microdialysis experiment, the rats were transferred to the
dialysis room that was on the same light/dark cycle as the colony room.
TheratswereplacedinseparatePlexiglasbowlswithadlibitumfoodand
water. Microdialysis probes (CMA 12, MW cutoff 20,000 Da, 2 mm
active membrane) were inserted through each guide cannula and artifi-
cial CSF (145 mM NaCl, 2.7 mM KCl, 1.2 mM CaCl, 1.0 mM KCl) was
perfused through the probes using a CMA infusion pump at a rate of 0.2
?l/min overnight. The next morning, the flow rate was increased to 1.5
?l/min where it remained for the rest of the experiment. The rats were
given 2 h to acclimatize to the experimental flow rate before any samples
were taken. All dialysates were collected in tubes prefilled with 3 ?l of
0.02% EDTA (antioxidant) in 1% ethanol. The sample tubes were man-
ually changed every 20 min for a total of 3 h (9 samples total). Three
baselinesampleswerecollectedforthefirsthouroftheexperiment.After
thefourthsampletubewasinserted,ratsbeganreceivinginjections.Each
ratreceivedtwosubcutaneousinjections,oneimmediatelyfollowingthe
other. The first injection was either (?)-naloxone (1 mg/kg) or saline
and the second injection was either morphine (6 mg/kg) or saline. The
morphineand(?)-naloxonedoseswerebasedonpilotstudies.Thenthe
(?)-naloxone dose was similarly based on pilot studies. Following drug
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administration, samples were taken every 20 min across a 100 min time
course.
Microdialysis probe placement verification
After completion of the microdialysis study, rats were killed with intra-
peritoneal 65 mg/kg sodium pentobarbital (Abbott Laboratories) before
brain extraction. The brains were frozen in chilled isopentane and cryo-
stat sectioned (30 ?m) at ?20°C. Brain sections containing each rat’s
cannula track were mounted on gelatin-treated slides and stained with
cresylviolet,coverslipped,andviewedunderalightmicroscopetodefine
thesiteofmicrodialysissampling.Tobeincludedindataanalysis,atleast
75%oftheprobehadtobewithintheNAcshell.Dialysatesamplesfrom
rats fulfilling this requirement for dopamine were analyzed using high
performance liquid chromatography (HPLC) along with electrochemi-
cal detection using a method previously described (Bland et al., 2009).
Experiment4:Mouseconditionedplace preference
Apparatus
ThePlexiglasplacepreferenceapparatusmeasured50(L)?25(W)?35
(H) cm and comprised two distinct conditioning environments (22 ?
19 ? 35 cm divided into 4) with a neutral passage way in-between
(16.6 ? 4.8 ? 35 cm). Each conditioning environment measured 10.9
(L) ? 9.3 (W) ? 35 (H) cm. One environment had a floor consisting of
5 mm plastic black bars spaced 5 mm apart (edge-to-edge). The floor of
the second environment was black plastic perforated across the surface
with evenly spaced 5 mm holes. The walls of each environment were
black or white (balanced randomized assignment). The neutral passage
waymeasured16.6?4.8?35cm,withsanded,blackPlexiglasflooring.
During the conditioning phase, Plexiglas partitions matching their re-
spective environments were inserted to restrict the mice to their specific,
designated environment.
TheactivityofeachmousewasrecordedusingLogitechQuickcamPro
5000 webcams mounted 1.0 m above the center of the conditioned place
preference apparatus. The cameras were connected to a computer run-
ningAnyMaze(Stoelting),totrackandrecordthetimeamousespentin
each of the three compartments.
Procedure
An unbiased conditioned place preference protocol was used. On day 1,
all mice were placed individually in the conditioned place preference
apparatus and allowed to freely explore the entire apparatus for 20 min,
to assess baseline preferences or biases to either environment. Each
mouse was then randomly assigned to treatments group and condition-
ing environment in a counterbalanced fashion, so that half the mice in
each treatment group was assigned to the environment they preferred,
and half was assigned to the environment they did not prefer. Mice
receivedeither(1)oxycodone(20mg/kgi.p.)versussalineonalternating
days,or(2)saline(vehicleofoxycodone)everyday.Oxycodonewasused
here as it produced robust, consistent, and reliable conditioned place
preference in wild-type BALB/c mice, while morphine did not. Upon
completion of injections, mice were placed into the designated condi-
tioning environment for 30 min (total of four conditioning sessions,
conditioningsessions1and2separatedfromconditioningsessions3and
4 by a 2 d weekend). Place preference testing occurred on day 8 and was
run identically to baseline testing on day 1. That is, mice were placed in
the place preference apparatus, in a drug-free state, and allowed to ex-
plore the entire apparatus for 20 min. The time spent in each environ-
mentwasrecordedandconditioningwascalculatedasadifferenceofthe
time spent in the drug-paired environment before and after condition-
ing. All testing was performed using blinded procedures with respect to
group assignments.
Phase1controlexperimentstoassesstheselectivityofthe
observedopioiddrugresponsefollowingpharmacological
andgeneticmodificationtoTLR4
1A: Rat Hargreaves test for thermal sensitivity
Acute indwelling lumbosacral catheters. Catheter implantations via the
L5/L6intervertebralapproachanddrugmicroinjectionswereperformed
based on Milligan et al. (1999). Rats were briefly anesthetized under
isofluraneanesthesiaandan18-gaugeneedlewasplacedbetweenL5and
L6 into the intrathecal space to serve as a guide. Polyethylene-10 tubing
was threaded rostrally through the guide and terminated over the lum-
bosacral enlargement. The 18-gauge needle was removed after catheter
placement and the tubing secured to the superficial musculature of the
lower back with 3-0 silk suture. The tubing was then threaded subcuta-
neously to exit the nape of the neck and the skin incision closed. Cathe-
terswere75cminlength,preloadedwithdrugsattheintrathecalendand
theremainderfilledwithsterilesaline,asusedpreviously(Hutchinsonet
al., 2008b). This allowed remote injection of drug during behavioral
testing without disturbing the animal, and the injection of a small void
volume that ensured delivery of drugs. Behavioral testing began 2 h after
intrathecal catheter placement.
Procedure. Thermal testing measured withdrawal latency to radiant
heatappliedseparatelytothetailandtheplantarsurfaceofeachhindpaw
inamodifiedHargreavestest(Hargreavesetal.,1988).Baselinelatencies
before drug administration were calculated as the average of three laten-
cies,measured5minapart.Followingfirstdrugadministration,latencies
to withdrawal from each paw and the tail were measured at 3 min inter-
vals for 45 min. The intensity of the heat source was adjusted such that
predrug latencies to withdrawal were 3–4 s, with a 10 s cutoff to avoid
tissue damage. This allowed both analgesia and hyperalgesia to be mea-
sured. All testing was blind with respect to group assignment.
1B: Mouse hotplate test for thermal sensitivity
Mice received at least three 5 min habituations to the test environment
before behavioral testing. Latencies for behavioral responses to the 50°C
hotplate were assessed. All testing was conducted blind to group assign-
ment.Acutofftimeof60swasimposedtoavoidtissuedamage.Baseline
latencies for the hotplate response ranged from 24 to 32 s. Baseline re-
sponse latencies were recorded before drug administration. Data are ex-
pressed as percentage maximum potential effect (% MPE). For the
construction of the dose-responses to oxycodone, mice (wild-type and
TLR4?/?)hotplatelatencieswereassessedbeforeand20minafterchal-
lenge doses of oxycodone (0.01, 0.1, 1, 2, and 5 mg/kg). Each oxycodone
challenge dose involved 8 mice. Mice, which displayed freezing symp-
toms or urinated during hotplate latency testing, were excluded from
datasets because of inherent errors in data collection under these condi-
tions. Mouse behavioral responses that were two SDs or greater than the
mean were also excluded (max n ? 2 per group, and replaced).
1C: Morphine tissue concentration quantification
Experimentalprocedure.(?)-Naloxone(5mg/kg)orequivolumevehicle
(1 ml/kg saline) was administered subcutaneously 10 min before mor-
phine (10 mg/kg, s.c.). The (?)-naloxone and morphine doses and tim-
ingswerechosentomatchthoseusedinstudiesofreinforcement,below.
Aseriesofbloodsamples(?0.5ml/sample)werecollectedvialateraltail
vein nick into heparinized tubes. Sampling occurred just before mor-
phine(time0)and5,15,and30minaftermorphine.Heparinizedblood
samples were kept on ice until centrifugation, collection of plasma, and
storage of plasma at ?80°C before extraction for analysis of morphine
content. Half the rats of each group were killed immediately after the 5
min sample collection. The rest were killed immediately after the 30 min
sample collection. Upon death by unanesthetized decapitation, brain
samples(hippocampus,cerebellum)wererapidlyisolated,flashfrozenin
liquid nitrogen, and stored at ?80°C before extraction for analysis of
morphine content.
Morphine extractions and analyses. Tissue morphine concentrations
were quantified by a modification of a HPLC electrochemical detection
method previously described (Van Crugten et al., 1997; Doverty et al.,
2001). The system consists of an ESA 5600A Coularray detector with an
ESA 5014B analytical cell and an ESA 5020 guard cell. The column was
anESAMD-150(C-18,3?m,150?3.2mm),andthemobilephasewas
ESA buffer MD-TM. The analytical cell potentials are kept at ?100 mV
and ?250 mV and the guard cell at ?300 mV. Tissue was weighed and
then sonicated in 1 ml of deionized water, while plasma samples were
dilutedinwaterforatotalvolumeof1ml.Sampleswerethenalkalinized
with500?lofsodiumbicarbonatebuffer(500mM;pH9.6)andextracted
withchloroform(6ml)byvortexingfor120sfollowedbycentrifugation
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(1700 ? g; 10 min). The upper aqueous layer is aspirated to waste fol-
lowed by a further addition of the sodium bicarbonate buffer. Samples
were then vortexed (10 s) and centrifuged (1700 ? g; 10 min). After
aspiratingtheaqueouslayertowaste,morphinewasbackextractedfrom
5 ml of chloroform into 300 ?l of NaH2PO4(50 mM; pH 2) by vortexing
for 120 s. After centrifugation, an aliquot (100 ?l) of the aqueous phase
was injected onto the system. Calibration standards ranged from 0.25
ng/ml to 400 ng/ml, and samples above this were diluted with water.
High(300ng/ml)andlow(1ng/ml)qualitycontrolsampleswereassayed
with each assay and were expected to be within 10% of the nominal
concentrations. The lower limit of quantification was 0.25 ng/ml.
Phase2controlexperimenttodefinespeedof(?)-naloxone
effectsonmitogen-activatedproteinkinasephosphorylation
inmicefollowingoxycodone
Immediately following hotplate behavioral testing, mice were overdosed
with sodium pentobarbital (400 mg/kg), perfused with 0.9% isotonic
saline, and then dissected to obtain spinal cord. The spinal cord tissue
was homogenized in 1 ml of 1? denaturing buffer and then heated to
100°C in a heating block for 6 min to prevent protein aggregation and
degradation. Spinal tissue was stored at ?80°C until use. Spinal lysates
from wild-type and TLR4?/?mice receiving 0.01, 1, and 5 mg/kg oxy-
codone were tested for levels of p38, JNK, and ERK phosphorylation.
Total p38 was not measured because of time limitations for transcrip-
tion/translation. Before analysis, spinal cord samples were diluted 1:4
using assay diluent and protein concentrations determined using BCA
analysiskit(BDBioscience).CelllysateswerepreparedasperBDCytometric
Bead Array kit to label phosphorylated proteins. Following washing, the
samples were analyzed using a BD fluorescence-activated cell-sorting ma-
chineandanalyzedinablindedfashion.
Phase3controlexperimentstodefinebiophysicaland in
silicointeractionsofopioidsand(?)-naloxoneatTLR4/MD2
TLR4/MD2 in silico docking
To prioritize the docking calculations and to provide a possible mecha-
nistic framework for the in silico docking simulations, it was a priori
hypothesized that the TLR4 and MD2 could exist in a range of possible
conformational states ranging from a preactivation state of individual
membrane bound TLR4 and soluble extracellular MD2 through to a
complete signaling heterodimer of TLR4 and MD2.
ToexaminetheinsilicodockingofligandstotheTLR4/MD2complex,
the crystal structure of the human TLR4-human MD2-E.coli LPS Ra
complex program database (pdb) file was obtained from RCSB Protein
Data Bank (PDBID: 3FXI) as published by Park et al. (2009). All ligands,
water, and cofactors were removed from the file via Molegro Molecular
Viewer, thus eliminating exogenous water molecules and artifacts from
crystallization from future docking simulations. The modified pdb files
werefurtherpreparedusingMGLTools1.5.6.RC2(http://mgltools.scripps.
edu/) with polar hydrogen atoms added. Ligands for docking were gath-
ered using PubChem isomeric SMILES, then converted to .pdb using a
structure file generator (http://cactus.nci.nih.gov/services/translate/),
and validated by visual inspection.
The four macromolecules in the MD2-TLR4 heterodimer pdb file
wereseparatedintofourseparatepdbfiles,resultinginTLR4-A,TLR4-B,
MD2-C, and MD2-D facilitating the creation of the range of possible
conformational states using Molegro Molecular Viewer. Docking simu-
lations were conducted for all ligands (agonists and antagonists) to each
of these conformational states. Docking was conducted using Vina [ver-
sion 1.1.2 (Trott and Olson, 2010)] within PyRx [version 0.8 (Wolf,
2009)]. An exhaustiveness factor of 8 was used for all simulations, with
theVinasearchspacedimensionsandcenterdefinedforeachmacromol-
ecule using the auto-maximize function.
Fluorescence titrations
An insect expression human MD2-pAcGP67A vector was provided by
Dr. Jie-Oh Lee (Korea Advanced Institute of Science and Technology,
Daejeon,RepublicofKorea).MD2baculoviruswaspreparedbycotrans-
fection of SF-9 insect cells with MD2-pAcGP67A vector and bright lin-
earized baculovirus DNA as described by the manufacturer’s protocol
(BD Bioscience). After 2–3 rounds of amplification, the MD2 baculovi-
russuspensionreachedatiterof?108/mlvirusparticlesandwasusedto
transfect high 5 insect cells to express MD2. MD2 was secreted into the
medium. After 3–4 d transfection, the medium was harvested and MD2
protein was purified by IgG Sepharose affinity purification.
Fluorescence measurements were performed on a Fluorolog-3 spec-
trofluorimeter (Horiba Jobin Yvon). All measurements were performed
under room temperature using a 2°—10 mm quartz cell (Starna Cells).
Thewavelengthof280nmwaschosentoexcitetheTyrandTrpresidues
in MD2 fluorescence and measurement at emission of 300–450 nm was
conducted. For the fluorescent probe Bis-ANS26, 385 nm was chosen as
the excitation wavelength and emission at 420–550 nm was recorded.
Appropriate baseline signals were subtracted from spectra obtained. Fluo-
rescence was also corrected by the relation, Fcorr? Fobsanti-log (ODex?
ODem/2) for the inner filter effect when necessary, where ODexand ODem
aretheopticaldensitiesatexcitationandemissionwavelengths,respectively
(Lakowicz,2006).
For naloxone and remifentanil fluorescence quenching assays, 0.5 ??
MD2wastitratedwithdifferentconcentrationsofnaloxoneorremifentanil,
and the fluorescence emission at 337 nm was plotted against naloxone or
remifentanil concentration. The raw data were fitted by nonlinear
least squaremethodusingtheequation:F?0.5?(2?F0?FPL?(KD?
[LT]?[PT]?((KD?[LT]?[PT])2?4?[LT]?[PT])0.5)),where[F],the
observed fluorescence; F0, initial fluorescence of protein in the absence of
ligand;FRL,adjustableparameterforprotein–ligandcomplexmolarfluores-
cence;KD,dissociationconstant;[LT],totalconcentrationoftheligand;[PT],
total protein concentration. The data were also plotted according to the
equation: lg (F0/F ? 1) ? ?lgKD? n ? lg ([ligand]) (Lakowicz, 2006),
where KD, dissociation constant; n, stoichiometry. (?)-Naloxone and
remifentanilshownofluorescencesignalatthetestedconditions.Roxithro-
mycin,whichhasbeenreportedtoshownoapparentbindingtoMD2(Res-
manetal.,2008),servedasanegativecontrolcompound.ProteinAwasused
asanegativecontrolproteintoeliminatethepossibilityofbindingtoProtein
Atag.
For the displacement assay, different concentrations of (?)-
naloxone were titrated into MD2 (0.5 ?M) and Bis-ANS (0.5 ?M)
mixture. After overnight equilibrium at room temperature, the Bis-
ANS fluorescence intensity was measured. The fluorescence emission
at 478 nm was plotted against (?)-naloxone concentration. Kiof
(?)-naloxone was determined using the equation: Ki? Kapp/(1 ?
[Bis?ANS]/KD(Bis?ANS?MD2)).
Statistics
Two-way repeated-measures ANOVAs with Bonferroni post hoc tests
whenappropriatewereusedtodeterminestatisticalsignificancebetween
groupsforboththeCPPandinvivomicrodialysismeasures.Forthedrug
self-administrationprocedure,responseratesweredeterminedbydivid-
ing responses by elapsed time in each component, excluding the time
outs that followed injections. Average values across six subjects (with
SEM) are presented below. To determine whether there was a difference
in effects of cocaine compared with remifentanil self-administration, a
two-way,repeated-measuresANOVAwasused(factorswerecomponent
and substance injected: cocaine or saline). A one-way, repeated-
measures ANOVA was used to assess the effects of successive compo-
nents in the substitution for cocaine of remifentanil. A two-way
repeated-measures ANOVA was used to assess the effects of presession
treatments of (?)-naloxone on remifentanil self-administration. For
studiesofpriordrugtreatmentsonself-administrationofremifentanil,a
post hoc Bonferroni t test was used for pairwise comparisons.
Forresponsethresholdstoradiantheat(Hargreavestest)andhotplate
tests,one-wayANOVAswithappropriateBonferroniposthocswereused
to confirm that there were no baseline differences on behavioral mea-
sures. Two-way repeated-measures ANOVAs with Bonferroni post hoc
tests when appropriate were used to determine statistical significance
between groups for thermal response threshold measures. p ? 0.05 was
considered significant. Hotplate latencies across the oxycodone dose
range were analyzed using Prism, GraphPad 5.0 software with a four
parameter dose–response model.
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For all analyses, p ? 0.05 was considered significant.
Results
AssessmentoftheroleofTLR4inopioid
reward/reinforcement
Experiment 1: (?)-Naloxone suppresses morphine conditioned
place preference
Toassesswhethersystemic(?)-naloxoneinfluencesbrainmech-
anisms that underlie opioid reinforcement, the effects of (?)-
naloxone on morphine conditioned place preference (CPP; n ?
6–11/group)wereassessedusingunbiasedprocedurespreviously
described (Hutchinson et al., 2008a). In CPP, rats experience
morphine in one environment, vehicle in a different environ-
ment,andarelatertesteddrug-freetodeterminewhichenviron-
menttheyprefer.Thedifferenceintimespentinthedrug-paired
side, compared with predrug baseline, is then calculated. As ex-
pected, control rats displayed preference for the side previously
paired with morphine (5 mg/kg s.c.; saline/morphine group; Fig.
1). (?)-Naloxone (1 mg/kg, s.c.) administered just before each
conditioning trial blocked the development of morphine-
induced place preference [(?)-naloxone/morphine group]. The
saline/saline and (?)-naloxone/saline groups were not statisti-
callydifferentfromeachother,norweretheysignificantlydiffer-
ent from the (?)-naloxone/morphine group, supporting the
conclusion that (?)-naloxone effectively blocked morphine in-
duced CPP. A two-way ANOVA with Bonferroni post hoc tests
revealedasignificanteffectoftreatment(F(3,30)?4.89,p?0.01).
Two-way ANOVA revealed no effect of morphine drug condi-
tioning (F(1,30)? 0.02, p ? 0.78), no interaction (F(1,30)? 0.02,
p?0.89)oreffectof(?)-naloxonetreatment(F(1,30)?0.02,p?
0.88)onthetotaldistancetraveledduringtheplaceofpreference
test, as a measure of activity. Thus, morphine-induced TLR4 ac-
tivationmaycontributetomorphinereward,asmeasuredbythis
Pavlovian conditioning paradigm.
Alternatively, (?)-naloxone could be perceived as aversive,
ratherthansuppressingrewardperse.Toexaminethispossibility
the study was repeated without morphine, with (?)-naloxone
paired with one context, rather than both contexts as above (n ?
5–6/group).(?)-Naloxonefailedtoconditioneitheraversionor
preference (change in preference mean ? SEM for saline: ?
15.4?46.6s;for(?)-naloxone:1.5?44.35s;one-wayANOVA,
p ? 0.05). A two-tailed Student’s t test revealed no effect of (?)-
naloxone treatment (t ? 0.15, p ? 0.88) on the total distance
traveledduringtheplaceofpreferencetest,asameasureofactiv-
ity.Togetherwiththestudyabove,thesedatasupporttheconcept
that morphine-induced TLR4 signaling contributes to this mea-
sure of opioid reinforcement.
Experiment 2: (?)-Naloxone suppresses self-administration of the
opioid remifentanil
The effect of the TLR4 antagonist (?)-naloxone was tested on
remifentanil self-administration. Remifentanil was chosen for
test given its very short half-life increases rate and stability of bar
pressing. Rats trained on cocaine self-administration, a standard
training paradigm as previously described (Hiranita et al., 2011),
were tested with remifentanil that reliably maintained self-
administration at high rates. The inverted U-shaped dose-effect
curve for remifentanil is characteristic of that for other drugs of
abuse (Fig. 2) (Panlilio et al., 2003). The highest rate of respond-
ingmaintainedbycocainewasobtainedatadoseof285.6?g/kg/
inj. Remifentanil maintained responding in a manner similar to
that maintained by cocaine and other abused drugs in all impor-
tant aspects (Fig. 2). The highest rate of responding was main-
tained at a dose of 0.9 ?g/kg/inj, with lower response rates at
higher and lower doses (Fig. 2, open circles). The maximal re-
sponse rates maintained by remifentanil were two-fold higher
than those maintained by cocaine (0.52 ? 0.20 vs 0.19 ? 0.06,
respectively), but the shape of the remifentanil dose-effect curve
wassimilartothatforcocaine.Remifentanilwas?320-foldmore
potent than cocaine. Response rates were significantly (F(4,20)?
4.20,p?0.013)affectedbydose,andposthoctestsindicatedthat
rates maintained by 285.6 and 0.9 ?g/kg/inj of cocaine and
remifentanil, respectively, were significantly greater than those
whenresponsesdidnotproduceinjections(t?2.81,p?0.043).
Treatment with (?)-naloxone immediately before the self-
administration session, dose-dependently suppressed responding
maintained by remifentanil (Fig. 2). A two-way repeated-
measures ANOVA indicated a significant effect of remifentanil
dose (F(4,40)? 5.22, p ? 0.005) but a nonsignificant effect of
presession dose of (?)-naloxone (F(2,40)? 2.54, p ? 0.128). In
addition, there was a significant interaction of the two (F(8,40)?
2.34, p ? 0.036). Post hoc tests indicated that the effects of 26.2
mg/kg of (?)-naloxone significantly (p ? 0.012) decreased re-
sponse rates maintained by the 0.29 (t ? 2.99) and 0.9 (t ? 2.98)
ug/kg/inj dose of remifentanil.
Experiment 3: (?)-Naloxone suppresses morphine-induced
elevations of NAc shell dopamine
Giventhestrikingbehavioraleffectsobservedwith(?)-naloxone
intheCPPandself-administrationparadigmsabove,theeffectof
Figure 1.
mg/kg,i.p.;4pairings)injectedwithsaline(blackbar)producedsignificantplacepreference
(two-way ANOVA with Bonferroni post hoc test, p ? 0.01). (?)-Naloxone (1 mg/kg, s.c.)
injectedjustbeforemorphineblockedmorphine-inducedpreferences(graybar).Intheabsence
of morphine, (?)-naloxone and saline were without effect on place conditioning. Data are
meanspergroup?SEMs,p?0.01,n?6–11/group.
(?)-Naloxone blocks the place conditioning effects of morphine. Morphine (5
Figure2.
persecond;abscissae,remifentanilinjectiondoseinug/kg/injection.Ext;extinction.0mg/kg
(?)-naloxone(opensquares),8.2mg/kg(?)-naloxone(opencircles),and26.3mg/kg(?)-
naloxone (filled circles). (?)-Naloxone was administered intraperitoneally at 5 min before
sessions. Note that (?)-naloxone dose dependently decreased remifentanil self-
administration.Dataaremeans?SEMs;n?6/group.
(?)-Naloxonesuppressesremifentanilself-administration.Ordinates,responses
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(?)-naloxone on morphine-induced elevations of NAc shell do-
pamine, an important neurochemical correlate of opioid rein-
forcement, was tested to define whether suppression of this
measure of reinforcement would also be observed. Dopamine
concentrations were assessed by in vivo microdialysis, as previ-
ously described (Bland et al., 2009), in rats implanted with can-
nulae confirmed to terminate within the NAc shell (Fig. 3).
Before drug treatment, there were no differences in concentra-
tions of extracellular dopamine in the NAc shell recorded from
the three baseline time points sampled (p ? 0.05). As expected,
morphine (6 mg/kg, s.c.) injected with saline vehicle increased
extracellular concentrations of dopamine in the NAc shell (Fig.
4). (?)-Naloxone (1 mg/kg, s.c.) suppressed this effect. A
repeated-measures ANOVA revealed a significant difference in
percentage increase of dopamine from baseline based on drug
treatment,F(2,128)?6.77, p ? 0.01. Bonferroni post hoc tests sup-
ported that the saline/morphine treated rats showed an increase
in dopamine efflux in the NAc shell, whereas the (?)-naloxone/
morphine treated rats were not different from the saline/saline
treatment group.
Experiment 4: Mice deficient in TLR4- or MyD88-dependent
signaling display significantly reduced oxycodone conditioned
place preference
Given the above converging lines of evidence, all of which support
that a (?)-naloxone-sensitive system is involved in opioid reward/
reinforcement,itwaspertinenttoassesswhetherthisresponsecould
be mirrored using an alternative TLR4-targeted approach. There-
fore,theroleofTLR4signalinginthebehavioralreinforcingactions
ofoxycodonewasexamined.MicedeficientinTLR4orthoselacking
theTLR4accessorysignalingproteinMyD88wereassessedonoxy-
codone CPP and compared with wild-type control mice. Deficien-
cies in TLR4 or MyD88 render mice unable to signal via the TLR4-
MyD88-dependent signaling cascade. Two-way ANOVA revealed
main strain (F(2,72)? 3.21, p ? 0.046) and drug effects (F(1,72)?
3.00, p ? 0.049) with a significant interaction (F(2,72)? 4.76, p ?
0.01; Fig. 5). Post hoc analyses revealed a significant place pref-
erence induced by oxycodone in wild-type mice (t ? 3.8, p ?
0.001), but no significant effect of oxycodone in the TLR4?/?or
MyD88?/?animals(t?0.79,p?0.05;Fig.5).Two-wayANOVA
revealed a main effect of drug conditioning (F(1,72)? 26.55, p ?
0.0001)onthetotaldistancetraveled,asameasureofactivity,butno
interaction(F(2,72)?0.93,p?0.40)oreffectofstrain(F(2,72)?1.45,
p?0.25)wereobservedduringtheplaceofpreferencetest.
Experimentalcontrolstoassesstheselectivityoftheimpactof
TLR4onthepharmacodynamicactionsofopioids
The ability to draw meaningful conclusions from the first four
experiments would be significantly hampered if no further as-
sessments were made. Therefore, three additional phases of con-
trolexperimentswereconductedtoprovideadditionalvalidation
of the four primary experiments. Phase 1 control experiments
assessed the selectivity of the observed opioid drug response
following pharmacological and genetic modification to TLR4.
Figure 3.
experiment.ProbeplacementsaredepictedusingplatesadaptedfromtheatlasofPaxinosand
Watson(1998).Notallprobescanbeseenduetooverlappingplacements.
Probe placements in the nucleus accumbens shell in the in vivo microdialysis
Figure 4.
cleusaccumbensshell.Invivomicrodialysiswasusedtotesttheeffectof(?)-naloxone-(1
mg/kg,s.c.)onmorphine-(6mg/kg,s.c.)inducedincreasesinextracellulardopaminein
thenucleusaccumbensshell.Afterthreebaselinesamples,ratsreceivedsalineplussaline,
saline plus morphine, (?)-naloxone plus saline, or (?)-naloxone plus morphine.
Repeated-measures ANOVA revealed a significant effect of treatment condition (p ?
0.01).Dataaremeanspergroup?SEMs;n?4/group.
(?)-Naloxone suppresses morphine-induced dopamine release in the nu-
Figure 5.
MyD88?/?comparedwithwild-typemice.DeficienciesinTLR4orMyD88rendermicewith
significantlyreducedoxycodoneconditionedplacepreference;mainstrain(F(2,72)?3.21,p?
0.046)anddrugeffects(F(1,72)?3.00,p?0.049)withasignificantinteraction(F(2,72)?4.76,
p?0.01).Posthocanalysisrevealedasignificantplacepreferenceinducedbyoxycodonein
wild-typemice(t?3.8,p?0.001),butnosignificanteffectofoxycodoneintheTLR4?/?or
MyD88?/?animals(t?0.79,p?0.05).n?11–17pergroup.Dataaremeans?SEMs.
Oxycodone conditioned place preference is significantly reduced in TLR4 or
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That is, it was first assessed if the previ-
ous experiments could simply be ex-
plained bya pan-pharmacodynamic
opioid attenuation response, as would be
expected if an opioid receptor antagonist
had been administered. In fact, based on
our previous publications, attenuation of
the TLR4-dependent central neuroexcit-
atory immune signaling, which has been
demonstrated to oppose opioid analgesia,
should lead to an acute potentiation of
opioid analgesia (Hutchinson et al.,
2010b). Phase 1 control experiments also
assessed if the coadministration of (?)-
naloxone may have altered the access of
morphine to the brain, and thus its active
sites for producing opioid reward.
Phase 2 control experiments assessed
if the opioid-induced TLR4-dependent
changesinbehaviorweretemporallyasso-
ciated with established TLR4-dependent
downstream signaling activation, thereby
providing evidence for the speed of the
TLR4 response. Finally, Phase 3 control
experiments aimed to determine if these
opioid-induced, TLR4-dependent
sponses were via direct opioid activation
of the TLR4 signaling complex or via
some other indirect means. Therefore, in
silico and in vitro biophysical studies were
conducted to address each of these
concerns.
re-
Phase 1 control experiments: Does
pharmacological or genetic modification of
TLR4 decrease opioid analgesia parallel to TLR4 blockade
reducing opioid reward?
1A: (?)-Naloxone potentiates acute remifentanil analgesia, in
keepingwithremifentanilasaTLR4agonist.Ifremifentanilactsas
aTLR4agonist,then(?)-naloxoneshouldpotentiateintrathecal
remifentanil analgesia just as (?)-naloxone has previously been
documented to potentiate morphine analgesia (Hutchinson et
al., 2010b). There were no baseline latency differences between
groupsofanimals(F?1.67,p?0.05).Verysimilarpatternswere
observed on both the left and the right hindpaw withdrawal la-
tencies, and thus results are presented with the two hindpaws
averaged. There were significant differences between the drug
treatment groups in the tail (F ? 44.8, p ? 0.05) and hindpaw
(F?23.29,p?0.05)responses.Bonferroniposthoctestsshowed
thatintrathecalremifentanilaloneproducedsignificantandbrief
analgesia on the tail (at 3, 6, 18, 21, and 33 min following first
remifentanil injection) and hindpaws (at 3 and 33 min following
firstremifentanilinjection),whencomparedwithglycinevehicle
alone(Fig.6).Theremifentanilanalgesiaonboththetail(3,6,9,
21, and 36 min following first remifentanil injection) and hind-
paws (3, 6, 18, 21, 33, and 36 min following first remifentanil
injection)waspotentiatedbyadministrationof(?)-naloxone10
minbeforethefirstremifentanilinjection.Thus,ashypothesized,
remifentanil acted as a TLR4 agonist causing TLR4-dependent
opposition to opioid analgesia, and hence remifentanil analgesia
waspotentiatedby(?)-naloxoneactingasaTLR4antagonist.As
such, while (?)-naloxone reduced remifentanil reward (Experi-
ment 2) it increased analgesia.
1B: Oxycodone is a more potent analgesic in TLR4?/?mice
comparedwithwild-typemice.TLR4?/?micewereusedtoassess
the involvement of TLR4 in acute oxycodone analgesia over a
range of doses. Pain responsivity was compared for wild-type
versus TLR4?/?mice dosed by intraperitoneal injection of oxy-
codone ranging from no analgesia to maximal analgesia on the
hotplatetest.Asexpected,increasingdosesofoxycodoneresulted
in significant increases in hotplate latencies. Further analysis of
theoxycodonedose–responsecurvesrevealedamainTLR4effect
where TLR4?/?mice achieved significantly longer hotplate la-
tencies (p ? 0.01). There was a fivefold leftward shift in the
TLR4?/?oxycodoneanalgesicdose–responsecurve(ED50wild-
type 1.36 mg/kg vs TLR4?/?0.26 mg/kg; F(2,71)? 24.1; p ?
0.0001) compared with wild-type mice (Fig. 7). In addition,
TLR4?/?mice had a significantly altered slope of the dose–re-
sponse curves (wild-type: 2.89 vs TLR4?/?: 0.85; F(2,71)? 24.1;
p ? 0.0001). Once again, as hypothesized, oxycodone acted as a
TLR4 agonist causing TLR4-dependent opposition of opioid an-
algesia, and hence oxycodone analgesia was potentiated in the
absence of TLR4. As such, while TLR4?/?mice had reduced
oxycodone reward (Experiment 4), they had increased analgesia.
1C: (?)-Naloxone does not prevent systemic morphine from
reaching the brain. While the CPP, self-administration, and in
vivo microdialysis studies above suggest that (?)-naloxone in-
hibits those centrally mediated opioid-induced effects, an alter-
nativeexplanationisthat(?)-naloxonepreventsmorphinefrom
reaching the brain, that it is a pharmacokinetic, and not TLR4-
based, opioid-reward mechanism. Thus, brain morphine con-
Figure 6.
TLR4agonist.Followingbaseline(BL)latencyassessmentswithradiantheattothetailandhindpaws,(?)-naloxone(75mg/kg)
or vehicle (saline) was subcutaneously administered, 10 min before the first intrathecal dosing. A total of three intrathecal
injectionsofremifentanilorvehicle(glycine)wereadministered15minapart,giventhebriefhalf-lifeofremifentanil.Remifentanil
producedasignificantandbriefanalgesiainboththetail(top;F?44.8,p?0.05)andhindpaws(bottom;F?23.29,p?0.05)
whencomparedwithglycinevehicle.Remifentanilanalgesiawasrobustlypotentiatedby(?)-naloxone(F?44.8;p?0.05).
Arrowsindicatetimingoftheremifentanilorglycinevehicleintrathecalinjections.Dataaremeans?SEMs;n?6/group.
Remifentanil analgesia is potentiated by coadministration of (?)-naloxone, as predicted if remifentanil acts as a
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centrations were quantified, as previously described (Van
Crugten et al., 1997; Doverty et al., 2001), following coadminis-
trationwith(?)-naloxone.(?)-Naloxonehadnoeffectonmor-
phineconcentrationsinbraintissueatthetimepointsmeasured,
compared with rats receiving morphine coadministered with vehi-
cle (saline) (p ? 0.05; Fig. 8). Together with the CPP, self-
administrationandtheinvivomicrodialysisdataabove,theseresults
support the conclusion that opioid-induced TLR4 signaling sub-
stantially contributes to this neurochemical change considered to
playaroleinopioidrewardindependentofpan-pharmacodynamic
changesorpharmacokineticalterations.
Phase 2 control experiments: Are the opioid-induced
TLR4-dependent changes in behavior temporally associated with
TLR4-dependent activation of mitogen-activated protein kinases?
It was first necessary to clarify whether opioid-induced TLR4-
dependentsignalingeventsoccurredfastenoughtobeassociated
with the altered pharmacodynamic responses. We have previ-
ouslypublishedthatoxycodoneiscapableofinvitroactivationof
TLR4-dependent NF-?B signaling using a stably transfected cell
lineand24hcellculture(Hutchinsonetal.,2010b).However,to
date there is no evidence for in vivo TLR4-dependent oxycodone
signaling. Thus, using wild-type and TLR4?/?mice, quantifica-
tion of mitogen-activated protein kinase (MAPK) phosphoryla-
tion following in vivo oxycodone administration was evaluated.
Immediately following analgesia testing in Phase 1 control
study 1B (20 min after drug), wild-type and TLR4?/?mice spi-
nalcordsampleswerecollectedtoenablequantificationofinvivo
signalingviatheTLR4cascade.Sampleswereanalyzedfordown-
stream TLR4 MAPK phosphorylation. Oxycodone administra-
tion caused significant dose-dependent elevations in the
phosphorylation of p38 (Fig. 9A) and JNK (Fig. 9B) in wild-type
(t?3.053,p?0.01)butnotTLR4?/?mice(t?0.98,p?0.05).
Forthephosphorylationofp38,atwo-wayANOVAresultedina
main strain (F(3,53)? 11.11, p ? 0.0016) and oxycodone dose
effect (F(1,53)? 6.94, p ? 0.0005) with a significant interaction
(F(3,53)?3.71,p?0.017).Posthocanalysisrevealedthatoxycodone
significantly increased p38 phosphorylation in wild-type over
TLR4?/?mice at 5 mg/kg (t ? 3.8, p ? 0.01; Fig. 9A). Analysis of
JNK phosphorylation revealed a main strain (F(3,53)? 9.13, p ?
0.0001)anddoseeffect(F(1,53)?60.79,p?0.0001)andsignificant
interaction(F(1,53)?14.73,p?0.001;Fig.9B). In contrast, there
werenodose-dependentelevationsinERKphosphorylationandno
TLR4-dependent differences in the response (p ? 0.05; Fig. 9C).
Therefore, acute opioid-induced TLR4-dependent p38 and JNK
Figure 7.
typemice.TLR4?/?micedemonstratesignificantlylongerhotplatelatencieswhenadminis-
tered with oxycodone compared with wild-type controls. ED50wild-type 1.36 mg/kg versus
TLR4?/?0.26mg/kg;F(2,71)?24.1;p?0.0001.n?7–8animals/dose.
Oxycodone is a more potent analgesic in TLR4?/?mice compared with wild-
Figure 9.
TLR4?/?mice had significantly reduced oxycodone-induced MAP kinase signaling. Phos-
phorylation of MAPK proteins (p38, JNK, and ERK) were measured from wild-type and
TLR4?/?spinalcordsfollowingacuteoxycodoneadministration.Oxycodonecausedsignifi-
cantdosedependentelevationsinthephosphorylationofp38(A)andJNK(B)inwild-typebut
notTLR4?/?mice.AnoxycodoneeffectwasnotobservedinERKphosphorylation(C).n?
7–8animalsperdose.PosthocWTversusTLR4?/?,*p?0.05,**p?0.01,***p?0.001.
Posthocvehicleversusdose,##p?0.01,###p?0.001.
Oxycodone causes TLR4-dependent increases in p38 and JNK phosphorylation.
Figure8.
whethertheresultsfromtheconditionedplacepreferenceorinvivomicrodialysisstudiesmay
be attributed to blockade by (?)-naloxone of morphine reaching the CNS, morphine levels
weremeasuredinhippocampus,wherethisbrainstructurewaschosensimplyduetoitssize
andeaseofisolation.Ratswereinjectedsubcutaneouslywith1mg/kg(?)-naloxoneorsaline
5minbeforeintraperitoneal6mg/kgmorphine;naiveratsservedasnegativecontrols.Either5
or 30 min after injection, rats were decapitated, hippocampi were harvested, and morphine
levelsquantifiedbyHPLC(VanCrugtenetal.,1997;Dovertyetal.,2001).Nodifferencesinbrain
levelsofmorphineweredetected,comparing(?)-naloxoneandsalinegroupsadministered
morphine(two-wayANOVA;p?0.05).Dataaremeans?SEMs;n?4/group.
(?)-Naloxonedoesnotaltermorphinelevelsinbrain(hippocampus).Todefine
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phosphorylation occurred temporally associated with TLR4-
dependentalterationsinopioidpharmacodynamics.
Phase 3 control experiments: Can opioid agonists and (?)-
naloxone directly act at TLR4 or part of its extracellular signaling
complex to induce the TLR4-dependent alterations in opioid
reward?
All the evidence presented thus far implicates an opioid-induced,
TLR4-dependent mechanism in altering opioid reward/reinforce-
ment. However, this could occur via either direct opioid action at
TLR4 or indirect activation of TLR4 signaling via an undefined
mechanism(s).Therefore,aseriesofexperimentswereconductedto
ascertainifanyevidencefordirectopioidinteractionswithTLR4or
its accessory protein MD2 could be found. Our recent publication
hasalreadyoutlinedextensiveevidence for direct morphine inter-
actions with TLR4 and its accessory protein MD2 (Wang et al.,
2012b).Similaroxycodoneinsilicodataandoxycodone-induced
TLR4-dependent NF-?B signaling data have also previously
been published (Hutchinson et al., 2010b), suggesting a simi-
lar mechanism of action to morphine. Here, we sought to
examine the direct TLR4 activity of the fully synthetic
4-anilinopiperidine, remifentanil, and the (?)-4,5-epoxy-
morphinan isomer (?)-naloxone. Two independent lines of
evidence were pursued for each compound to address this
issue: (1) in silico docking of the ligand to the TLR4/MD2
complex, and (2) biophysical assessments of competitive
binding of the ligand to MD2.
3A: Remifentanil and remifentanil acid dock in silico to the
TLR4/MD2 complex. Morphine, remifentanil, and its opioid in-
active metabolite, remifentanil acid, were
assessed for their in silico docking to vari-
ous conformational states of the TLR4/
MD2 complex using Vina and previously
published TLR4/MD2 pdb files, to assess
if any possibly relevant physicochemical
interactions occurred. The metabolite of
remifentanil was analyzed, in addition to
the parent compound, given our prior
findingthattheopioidinactivemetabolite
of morphine proved to be a TLR4 agonist
(Lewis et al., 2010). Interestingly, mor-
phine’sdockingenergydidnotdiffersub-
stantially
conformations, but remifentanil and
remifentanil acid both displayed lower
dockingenergieswhenboundtotheMD2
portion of the available conformational
states. Moreover, all three compounds
docked to the critical lipopolysaccharide
binding domain of MD2 (Fig. 10). These
data suggest
remifentanil acid displayed the physico-
chemical characteristics that may enable
them to interact with the TLR4/MD2
complex.
3B: Remifentanil binds to MD2. Fol-
lowing on from the promising in silico
data results, binding studies were con-
ducted.Toinvestigatewhetherremifenta-
betweenanyofthese
thatremifentanil and
Figure10.
remifentanil, and its opioid inactive metabolite, remifentanil acid, were determined to dock in silico to the lipopolysaccharide-
bindingdomainofMD2.Comparisonsofthedockingenergyestimates( y-axis)forremifentanilandremifentanilacidacrossthe
multipleconformationalstates(x-axis)suggestthatsuchdockingconformationspreferredtheformationoftheactivesignaling
heterodimerTLR4/MD2complexratherthantheMD2aloneconformation.Incontrast,morphinefailedtodisplayanysuchdocking
energypreferencetotheTLR4/MD2complexstates,insteaddemonstratinghighaffinityforallstates.
Remifentanil,itsmajormetaboliteremifentanilacid,andmorphineeachdocktoTLR4andMD2complex.Morphine,
Figure11.
MD2andcausesthequenchingofexpressedMD2intrinsicfluorescenceinvitro,whileroxithromycin,acompoundusedasanegativecontrol,showsnoMD2bindingactivity.KD?6.0?1.1?Mfor
theremifentanil-MD2interaction.B,ReplottingthedatafromAaccordingtotheequation:lg(F0/F?1)??lgKD?n?lg([remifentanil]),revealingaslopeof1.06?0.07andaKD?8.2?
1.1?Mfortheremifentanil-MD2interaction.C,SpecificityofremifentanilbindingisrevealedbyitsfailuretobindproteinA.Remifentanildemonstratesnegligiblebindingtoanegativecontrol
protein,proteinA.Astheexcitation,280nmwasused,andemissionat308nm(peakposition)wasplottedagainstthetitratedremifentanilconcentration.Note:thebindingconstantsderivedhere
arelikelyunderestimatingtheaffinityconstantsofremifentaniltoMD2duetothelackofcofactorsfoundinvivo.Dataaremeans?SEMs;n?2replicationspergroup.
BiophysicaldeterminationofremifentanilbindingtoMD2invitro.A,Titrationcurvesoffluorescencequenchingassaywiththeincreasingdrugconcentrations.Remifentanilbindsto
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nil can bind to MD2, a fluorescence quenching assay was
performed. As shown in Figure 11A, remifentanil caused the
quenchingofMD2intrinsicfluorescenceuponbinding,whilethe
negative control compound roxithromycin caused little quench-
ing of MD2 fluorescence, demonstrating the specific binding of
remifentanil to MD2. A dissociation constant (KD) of 6.0 ? 1.1
?M was derived using a one-site binding model and nonlinear
least-squares fit of MD2-remifentanil interaction. Figure 11B
shows the lg(F0/F?1) versus lg([remifentanil]/?M) plot. A stoi-
chiometry value of 1.06 ? 0.07 and a KDof 8.2 ? 1.1 ?M were
obtained for the binding of remifentanil to MD2, which lends
further support to the one-site binding model. To eliminate the
possibility that the observed MD2-remifentanil binding was due
toProteinAtag-remifentanilinteraction,wealsotestedthebind-
ing of remifentanil to Protein A (Fig. 11C) and no apparent
quenching of Protein A intrinsic fluorescence was observed. The
overall result excludes the possible Protein A
tag-remifentanil binding. These data suggest
that remifentanil is capable of direct binding
to TLR4/MD2.
3C: In silico evidence that (?)-naloxone
docks to the coreceptor MD2 of TLR4. Using
theapproachusedincontrolExperiment3A,
complementary in silico data using previ-
ously available crystal structures (Park et al.,
2009; Trott and Olson, 2010) show that
docking of (?)-naloxone to MD2 spatially
overlaps the docking of both morphine,
remifentanil,andremifentanilacid,support-
ing competition at this binding pocket (Fig.
12). Therefore, (?)-naloxone has the neces-
sary characteristics to allow for blockade of
TLR4/MD2 binding to morphine and
remifentanil.
3D: (?)-Naloxone binds to MD2. As
noted above, prior studies have provided
support that (?)-naloxone disrupts TLR4
signaling, but failed to identify the location
along the TLR4 signaling cascade, even
whether this involves an extracellular site
versus intracellular site following activation
of the TLR4/MD2 receptor complex. Here,
theinvitrononstereoselectivebindingofnal-
oxone to purified expressed MD2 (Hutchin-
son et al., 2011) was assessed using ligand
quenching of MD2 intrinsic fluorescence
(Fig. 13A). Both (-)-naloxone and (?)-
naloxone bound MD2 and quenched MD2
intrinsicfluorescencewithcomparableaffin-
ities (17.7 ? 3.2 ?M and 16.6 ? 4.7 ?M, re-
spectively). In comparison, the previously
reported negative control roxithromycin
(Resman et al., 2008) showed no MD2 bind-
ing activity, thus demonstrating specific
binding of (?)-naloxone to MD2. Bis-ANS,
a MD2 molecular probe, binds the lipopoly-
saccharide binding pocket of MD2 and its
fluorescence intensity is enhanced when
bound to MD2 (Mancek-Keber and Jerala,
2006). (?)-Naloxone decreased Bis-ANS-
MD2 complex fluorescence, suggesting that
(?)-naloxone replaces Bis-ANS binding to
MD2 (Fig. 13B). Collectively, these data sug-
gest (?)-naloxone binds to the critical binding domain of MD2.
Discussion
Here, we provide evidence that TLR4 is a novel contributor to
opioid reward behaviors and neurochemistry. Specifically, we
demonstrated using (?)-naloxone-TLR4 pharmacological
blockade, or MyD88-TLR4-dependent signaling genetic knock-
outs, that opioid-TLR4 signaling is a novel and important con-
tributor to: (1) opioid reward behavior, as measured by CPP and
self-administration; and (2) reward neurochemistry, through
opioid-induced, but (?)-naloxone-sensitive, NAc shell extracel-
lular dopamine elevations. Critically, these opioid-TLR4 actions
are not simply a requirement for all opioid responses, since
opioid-induced TLR4 signaling decreased opioid analgesia. We
also conclude that opioids of diverse chemical structures can
dockwiththeactivedomainofMD2,suggestingadirectmodeof
Figure12.
TLR4.Morphine,remifentanil,anditsopioidinactivemetabolite,remifentanilacid(displayedastheoverlappingsticksymbol
structures), were determined to dock in silico to the lipopolysaccharide-binding domain of MD2 (represented as a ribbon
peptide),inaconformationthatspatiallyoverlapwith(?)-naloxone(spatialcloudrepresentingpreferreddockingconfor-
mation)andmorphineinsilicodocking.
Insilicodockingof(?)-naloxoneinthelipopolysaccharide-bindingpocketofMD2,anessentialcoreceptorof
Figure 13.
quenchingofexpressedMD2intrinsicfluorescenceinvitro,whileroxithromycin,acompoundusedasanegativecontrol,shows
no MD2 binding activity. B, (?)-Naloxone caused the decrease of Bis-ANS fluorescence from the Bis-ANS-MD2 complex,
suggestingthat(?)-naloxonereplacesBis-ANSbindingtoMD2.Note:thebindingconstantsderivedherearelikelyunder-
estimatingtheaffinityconstantsof(?)-naloxonetoMD2duetothelackofcofactorsfoundinvitro.Dataaremeans?SEMs;
n?2replications/group.
(?)-Naloxone binds to MD2 in vitro. A, Both (?)-naloxone and (?)-naloxone bind to MD2 and cause the
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Page 12

action of these agents acting as xenobiotic drugs to cause TLR4
signaling.Thus,itishypothesizedthatopioidsengageinflamma-
tory CNS processes, acting as xenobiotic-associated molecular
patterns (XAMPs) to activate TLR4, in addition to their previ-
ously characterized neuronal targets, thereby potentiating acute
dopamine changes involved in opioid reward.
Several questions arise that warrant discussion regarding the
changes in opioid reward: (1) “how” is opioid-TLR4 signaling
involved; (2) “what” cell type(s) is/are contributing; and (3)
“why” are opioid-TLR4 interactions acting in this manner? The
“how”and“what”questionsareindeliblylinked,as“how”TLR4
contributes to opioid reward is dependent on “what” cells are
expressing TLR4. As such, the two points will be discussed
simultaneously.
Which TLR4-expressing cell type(s) contributes to opioid re-
ward is unclear. Preliminary mRNA expression data using flow
cytometry-assisted cell sorting of NAc micropunches suggests
TLR4 mRNA is only found in microglia and not neurons (S.
Bilbo, personal communication). Some examples of neuronal
TLR4 expression exist (Diogenes et al., 2011; Ferraz et al., 2011),
but their functional impact and how the very low MD2 expres-
sion by this cell type (Divanovic et al., 2005; Okun et al., 2011)
impactsfunctionisunknown.Incontrast,itis clear that TLR4 is
constitutively expressed by at least microglia (Bsibsi et
al., 2002), macrophages (Fujihara et al., 2003), CNS vascular en-
dothelial cells (Bsibsi et al., 2002), and some astrocytes (Holm et
al., 2012). Thus, the highest likelihood is that the TLR4-
expressing cell type(s) contributing to opioid reward is among
these.
Narrowing the list of TLR4/XAMP cellular targets aids in the
identification of novel TLR4-dependent signaling pathways that
may contribute to opioid reward behaviors and neurochemis-
try. TLR4 activation in non-neuronal cells leads to MyD88-,
MAPK-, and NF-?B-dependent events (Okun et al., 2011). It is
now apparent all these key signals occur temporally associated
with TLR4-dependent altered opioid pharmacodynamic re-
sponses,withMyD88andp38MAPK(Zhangetal.,2012)specif-
ically implicated in opioid reward behavior. Likely molecular
mediators that result from such signaling include proinflamma-
tory cytokines, and these have been proposed as potential con-
tributors to opioid reward (Coller and Hutchinson, 2012). Such
conclusions are supported by their neuroexcitatory effects (Wat-
kinsetal.,2009)ofglutamatetransporterdownregulation(Wang
et al., 2003), GABA receptor downregulation (Stellwagen et al.,
2005), upregulation of AMPA/NMDA expression and function
(De et al., 2003), and enhancement of neurotransmitter release
(Youn et al., 2008). Proinflammatory cytokines could therefore
amplify opioid-induced neuronal activity in drug reward cir-
cuitry at multiple points, but the specifics of the underlying
mechanisms remain to be determined.
Such a TLR4-dependent proinflammatory hypothesis of opi-
oidrewardalignswithpriorliteratureimplicatingglia.Forexam-
ple, morphine increased NAc astrocyte and microglial activation
marker mRNA expression (Schwarz et al., 2011), morphine-
induced CPP concomitantly induced activation of NAc micro-
glial p38 (Zhang et al., 2012), and NAc microinjection of a
microglial or p38 inhibitor blocked both acquisition and main-
tenance of morphine-induced CPP (Zhang et al., 2012). These
regional specific studies support earlier findings that systemic
broad-spectrum glial activation inhibitors suppress morphine-
induced dopamine elevations in the NAc shell [ibudilast (Bland
et al., 2009)] and morphine-induced CPP [propentofylline, mi-
nocycline (Narita et al., 2006; Hutchinson et al., 2008a)]. Inter-
estingly, morphine reward and dependence/withdrawal may
differ regarding TLR4 involvement. Despite broad-spectrum
glial attenuators reducing proinflammation-linked opioid de-
pendence/withdrawal (Hutchinson et al., 2008a, 2009; Liu et al.,
2011), TLR4?/?mice are not protected against opioid with-
drawal,suggestiveofaTLR4-independentmechanism(Liuetal.,
2011).
The third point is “why” TLR4 is involved in opioid reward.
The evolutionary reasons for TLR4 recognizing XAMPs, includ-
ing opioids, and this central immune signal interacting with me-
solimbicdopaminerewardpathwaysareunknown.Itisclearthat
TLR4 activation by itself does not produce behavioral reward
(Narita et al., 2006). (?)-Naloxone action is not an evolutionary
controlmeasuresince(?)-naloxoneisafullysyntheticunnatural
compound. However, as we have presented here, these small
molecules dock to the same domain in MD2 as lipopolysaccha-
ride, suggesting some possible common threat-detection system.
Questions also arise as to why this opioid-TLR4/MD2 inter-
action has not previously been reported in binding or behavioral
studies. Such data have been reported but not followed up. For
example, the seminal research of Takagi et al. (1960) demon-
strated the relevance of nonclassical nonstereoselective opioid
actions,andGoldsteinetal.(1971)founda30-foldgreaterabun-
dance of nonstereoselective but saturable opioid binding sites
compared with saturable stereoselective opioid binding. A key
issue in in vitro systems, which leads to significant variability, is
thatMD2isextracellular,andcanthereforebeeasilylostfromor
not included in assays, as we have exemplified previously
(Hutchinson et al., 2010b).
An important caveat to any pharmacological studies is the
specificity of the agonist and the antagonist used. Collabora-
tively, we have screened (?)-naloxone on over 70 known
receptors, enzymes, second messengers, ion channels, and
transporters (our unpublished data), finding no significant
activity at any of these important targets. These data also ex-
tend our prior observation that (?)-naloxone blocks TLR4,
but not TLR2, signaling (Lewis et al., 2012). Other non-TLR4
actions of this agent have been reported that theoretically
could alter opioid pharmacodynamics, such as activity at Fil-
amin A (Burns and Wang, 2010) and NADPH oxidase (Wang
et al., 2012a). However, given the TLR4-MyD88 dependent
signalingrequiredtoelicitopioidCPPpresentedhere,andour
previous data showing no additional (?)-naloxone-induced
potentiation of morphine analgesia in TLR4?/?mice
(Hutchinson et al., 2010b), suggest that these documented
(?)-naloxone actions at Filamin A and NADPH oxidase are
not involved in modifying these opioid pharmacodynamic ac-
tions.Itispossiblethatothersitesof(?)-naloxoneactionmay
be downstream from the (?)-naloxone-TLR4/MD2 activity
renderingthemlessrelevanttoopioidreward,butcouldprove
important for other TLR4 ligands such as PAMPs or DAMPs.
Similarly, it is plausible that opioids may be activating other
TLRs (He et al., 2011; Zhang et al., 2011), in a fashion that is
codependent on TLR4-MyD88-dependent signaling. Such
possibilities will require further evaluation.
Additional studies are needed to understand the in vivo (?)-
naloxone and opioid TLR4/MD2 potency in these and other
models. It is apparent from the in vitro biophysical characteriza-
tion of ligand-MD2 interactions conducted to date (Wang et al.,
2012b) that this is a low affinity site. Moreover, in vivo agonist
concentrations may fall below the binding constant estimates.
Such discrepancies suggest other factors or chaperones may be
involvedthatfacilitatethisinteraction,whichareabsentinthein
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vitro systems. Other differences in (?)-naloxone requirement
were also observed, with morphine CPP and NAc dopamine ele-
vations nearly completely blocked by 1 mg/kg (?)-naloxone,
while higher doses were needed for the attenuation of remifenta-
nil self-administration. Some elevations in NAc dopamine were
anticipated to occur in the absence of opioid-TLR4 actions, sug-
gesting tonal endogenous TLR4 activity is required for elevated
mesolimbic dopamine reward function. Interestingly, Dunwid-
die et al. (1982) established that hippocampal pyramidal cell
spontaneous activity in the CA1 region was nonstereoselectively
inhibitedbynaloxone,providingfurtherimpetustoexaminethe
neuronal consequences of this TLR4-opioid response. An addi-
tionaldifferencein(?)-naloxonepotencyacrossthethreediffer-
ent models may reflect the nature of the three models and the
opioidsstudied.TheCPPandNAcdopaminemicrodialysisstud-
ies were conducted after limited opioid/XAMP exposure. How-
ever,theremifentanilself-administrationstudieswereconducted
in rats that had been repetitively trained on cocaine followed by
repeated remifentanil. Therefore, if both these treatments were
acting as XAMPs, they may have sensitized the TLR4 system
(Bowers and Kalivas, 2003), which (?)-naloxone had to over-
come via higher doses.
If activation of CNS TLR4 signaling by diverse opioid
structures reflects an innate immune system response to xe-
nobiotics (Hutchinson et al., 2011), then other drugs of abuse
maybeequallyviewedaschemicalcompoundsthatareforeign
to living organisms. Hence, multiple drugs of abuse may cause
XAMP-TLR4 signaling that could also contribute to their re-
inforcing effects. Given that drugs of abuse including alcohol
(He and Crews, 2008), cocaine (Bowers and Kalivas, 2003),
amphetamine (Thomas et al., 2004b), and methamphetamine
(Thomas et al., 2004a) are associated with glial activation, and
that systemic glial activation inhibitors have been reported to
suppress the reinforcing effects of methamphetamine (Narita
et al., 2006; Beardsley et al., 2010; Fujita et al., 2012), amphet-
amine (Sofuoglu et al., 2011), and alcohol (Agrawal et al.,
2011), examining whether XAMP-TLR4 activity contributes
to the rewarding properties of diverse drugs of abuse is war-
ranted (Coller and Hutchinson, 2012).
Notes
Supplemental material for this article is available at http://goo.gl/1f0ox;
with supplemental data for the screening of (?)-naloxone on ?70
known receptors, enzymes, second messengers, ion channels, and trans-
porters. This material has not been peer reviewed.
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