Opioid Receptors: Methods and Protocols

268 Pages·5.82 MB·English

by  Santi M. Spampinato

Methods in
Molecular Biology 2201
Santi M. Spampinato Editor
Opioid
Receptors
Methods and Protocols
Second Edition
M M B
ETHODS IN OLECULAR IO LO GY
SeriesEditor
JohnM.Walker
School of Lifeand MedicalSciences
University ofHertfordshire
Hatfield, Hertfordshire, UK
Forfurther volumes:
http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
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Opioid Receptors
Methods and Protocols
Second Edition
Edited by
Santi M. Spampinato
Department of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy
Editor
SantiM.Spampinato
DepartmentofPharmacy
andBiotechnology
UniversityofBologna
Bologna,Italy
ISSN1064-3745 ISSN1940-6029 (electronic)
MethodsinMolecularBiology
ISBN978-1-0716-0883-8 ISBN978-1-0716-0884-5 (eBook)
https://doi.org/10.1007/978-1-0716-0884-5
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Dedication
Tomywife,Anna,forherlove,patienceandencouragement
TomygrandchildrenAuroraandSanti
v
Preface
Opioidreceptors,beyondtheirinvolvementinpaintransmission,playanumberofrelevant
physiologicalrolesinthecentralnervoussystemandinperipheralorgans.Opioidreceptors
canbeconsideredacrossroadswhereendogenousopioidpeptidesandforeignopioidsand
opiatesmeetthecellandtransmittheirmessagestoanother vastarrayofstimulus-response
mechanism.Inrecentyears,studiesontheiremergingroleshavebeenfavoredbynumerous
andfruitfultechniquesthathaveopenednewavenuesofpreclinicalandclinicalresearchthat
demandsmultidisciplinaryapproaches.
The post-genomic era has opened up novel opportunities for the exploitation of these
novel technologies. As an increasing number of investigators seek to harness the fruits of
knowledge in these emerging fields, it is essential that well-tested protocols are made
availabletoresearchers.Withthisinmind,itisappositetoprovideacollectionofprotocols
tofavorinnovativestudiesonopioidreceptorswrittenbyexpertswhoareroutinelyemploy-
ing thesetechniques in their laboratories. Thisbook presentsthe protocolsin the stepwise
“cookbook”style of thiswell-known bookseries and summarizes ofstate-of-the art meth-
ods that have been utilized for understanding opioid receptor functionality at molecular,
cellular,structural,andorganismlevels.
Opioid Receptors: Methods and Protocols—Second Edition is, hence, an invaluable guide
for researchers in the fields of neuroscience, biochemistry, pharmacology, and molecular
biology.
PartIofthisbook(Chapters1–8)focusesonprocedurestoevaluategeneticsofopioid
receptors as well as their transcriptional and posttranscriptional regulation. An overview
of genetic analysis of opioid receptors with sequencing methods is included (Chapter 1).
A technique for the posttranscriptional analysis of opioid receptor genes is presented
(Chapter2).Furthermore,aprotocoldedicatedtoco-localizationandcellulardistribution
ofopioidreceptorsisdescribed(Chapter3),andtheuseofbioluminescenceenergytransfer
in living cells to investigate protein-protein interactions of opioid receptors is included
(Chapters 4 and 5). Imaging of opioid receptors is investigated adopting an ultrasensitive
technique based on bioconjugation of nanoruby (Chapter 6), and these receptors are
detected in peripheral tissues by immunohistochemistry (Chapter 7) and in immune cells
byreal-timereversetranscriptionPCR(Chapter8).
PartII(Chapters9–16)illustratesmethodsfortheanalysisofopioidreceptorsactivated
by selective agonists. A technique aimed to monitor internalization and interaction of mu
opioid receptors in the spinal cord of morphine-tolerant mice is reported (Chapter 9).
Strategiesforanalysisofsignalingeventsmodulatedbyopioidreceptorsactivatedbyagonist
ligands are described. Following opioid receptor activation, GTP will replace GDP on the
α-subunit of the G-protein, leading to a dissociation of the βγ-subunit. The [35S]GTPγS
autoradiographyassay,describedinChapter10,isusefultomonitoropioidreceptoractiva-
tionindiscretebrainareas.Thereafterisdescribedatechniquetoevaluateopioid-mediated
adenylylcyclaseinhibitioninlivingcellsbyabioluminescence-basedassay(Chapter11).The
developmentofawhole-cellpatchclamptoinvestigatetheactivationofinwardlyrectifying
potassium channel opioid receptor agonists in peripheral sensory neurons following nerve
injuryisreported(Chapter12).
vii
viii Preface
Clinical studies indicate that opioids may exacerbate the symptoms of HIV-associated
neurocognitive disorders (HAND). Interestingly, morphine increases neuronal iron and
ferritin levels, suggesting that opioids may also modulate neuronal iron homeostasis.
Understanding how opioids regulate neuronal iron metabolism may help to identify novel
drug targets in HAND with potential relevance to other neurocognitive disorders. New
molecularapproachesthatcanbeusedtounderstandhowopioidsmodulateneuronaliron
aredescribedinChapter13.
Procedures to investigate two innovative research areas involving opioid receptors in
modulatingimmunecelladhesion(Chapter14)andbraincorticalcellviability(Chapter15)
arereported.
Finally, biased agonists at opioid receptors have attracted increasing interest in the last
decadeastheyhaveemergedasmoreeffectiveandsafercandidateanalgesics.InChapter16,
promises, pitfalls, and future perspective of biased agonists at MOR and KOR opioid
receptors are discussed. Methodological insights are provided with regard to the most
appropriate experimental settings to be employed aiming at developing novel biased KOR
agonists.
Part III (Chapters 17–19) reports experimental techniques to investigate opioid
receptor-mediated functions at organismal level in a physiological or pathological context.
In Chapter 17 is described the analysis of cutaneous stimulation-induced sensory input by
vonFreyhairs.Theimmunosuppressiveeffectsmediatedbyopioidsarecentraltotheinvivo
activation of opioid receptors, and Chapters 18 and 19 explain strategies toward attaining
thisobjective.
Part IV (Chapters 20–24) showcases protocols for the analysis of behavioral effects
induced by opioids. Opioid addiction represents an important health concern, and animal
models have been crucial in understanding the neurobiology and pathophysiology of this
complex disease. Although animal models of addiction do not fully reproduce the human
condition, they do permit investigation of specific elements of this process as well as
identification of potential therapeutic targets. In Chapter 20, a step-by-step description of
morphine-conditionedplacepreferencemodelisreportedthatrepresentsausefulpreclinical
model extensively used to study the rewarding/aversive effect of drugs. In Chapter 21, a
procedure to evaluate heroin-seeking reinstatement in the rat is described that is useful to
studythemechanismsunderlyingrelapsetoheroinandvulnerabilityfactorsthatenhancethe
resumption of heroin-seeking behavior. In Chapter 22 is presented a procedure to investi-
gatetheroleofopioidreceptorsinalcoholismbyadoptingamodelthatcombineschronic
ethanolexposureprocedureswithvoluntaryethanoldrinkinginrodents.
Chapter 23 considers an animal model to study the pathophysiological alterations
connected to hypothalamic-pituitary-adrenal axis hyperactivity. This alteration is induced
byanearly-lifestressfulprocedureinvolvingtheendogenousopioidsystem.Behavioraltests
designedtoevaluatetheactivityandinvolvementofopioidreceptorsinpupsaredescribedin
Chapter24.
Isincerelyhopethattheseprotocolswillhelpbothexperiencedandnewentrantsinthis
fieldtocarryouttheirexperimentssuccessfully.Finally,Iwouldliketothankalltheauthors
for their outstanding contributions and the series editor, Professor John M. Walker, for
valuableeditorialhelp.
Bologna,Italy SantiM.Spampinato
Contents
Dedication.................................................................. v
Preface ..................................................................... vii
Contributors................................................................. xiii
PART I TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL
ANALYSIS AND CELLULAR DETECTION OF OPIOID RECEPTORS
1 OverviewofGeneticAnalysisofHumanOpioidReceptors................... 3
SantiM.Spampinato
0
2 RenillaLuciferaseReporterAssaytoStudy3-UTR-Driven
PosttranscriptionalRegulationofOPRM1................................. 15
GabrieleVincelliandAndreaBedini
3 FluorescenceColocalizationAnalysisofCellularDistribution
ofMOR-1 ............................................................. 27
VittoriaBorgonettiandNicolettaGaleotti
4 MonitoringOpioidReceptorInteractioninLivingCells
byBioluminescenceResonanceEnergyTransfer(BRET) .................... 35
MonicaBaiula
5 BioluminescenceResonanceEnergyTransfer(BRET)toDetect
theInteractionsBetweenKappaOpioidReceptor
andNonvisualArrestins.................................................. 45
AndreaBedini
6 FunctionalizationandBioconjugationofNanoruby
forLong-Term,UltrasensitiveImagingofMu-OpioidReceptors ............. 59
RashmiPillai,MarkConnor,andVarunK.A.Sreenivasan
7 ImmunohistochemicalAnalysisofOpioidReceptors
inPeripheralTissues..................................................... 71
YvonneSchmidtandHalinaMachelska
8 Real-TimeQuantitativeReverseTranscriptionPCRforDetection
ofOpioidReceptorsinImmuneCells ..................................... 83
MelihO¨zgu¨rCelik,DominikaLabuz,andHalinaMachelska
PART II ANALYSIS OF SIGNALING EVENTS MODULATED
BY OPIOID RECEPTORS
9 QuantitativeAnalysisofMOR-1InternalizationinSpinalCord
ofMorphine-TolerantMice .............................................. 99
VittoriaBorgonettiandNicolettaGaleotti
10 GTPγS-AutoradiographyforStudiesofOpioidReceptor
Functionality........................................................... 109
AlfhildGr¨onbladhandMathiasHallberg
ix
x Contents
11 EvaluatingOpioid-MediatedAdenylylCyclaseInhibition
inLiveCellsUsingaBRET-BasedAssay................................... 117
PreetiManandhar,ShivaniSachdev,andMarinaSantiago
12 PatchClampAnalysisofOpioid-InducedKir3Currents
inMousePeripheralSensoryNeuronsFollowingNerveInjury............... 127
ViolaSeitz,PhilipSt¨otzner,DominikaLabuz,
andHalinaMachelska
13 OpioidModulationofNeuronalIronandPotentialContributions
toNeuroHIV .......................................................... 139
BradleyNash,ElenaIrollo,RenatoBrandimarti,
andOlimpiaMeucci
14 AninVitroAssaytoStudytheRoleofOpioidsinModulating
ImmuneCellAdhesion.................................................. 163
MonicaBaiula
15 AssessingCellViabilityEffectsofOpioidsinPrimary
CorticalCellsfromRat.................................................. 171
AlfhildGr¨onbladh,ErikNylander,SofiaZelleroth,
andMathiasHallberg
16 TheQuestforMoreEffectiveAnalgesicswithReducedAbuse
LiabilityandFewerAdverseEffects:Promises,Pitfalls,
andFuturePerspectivesofBiasedAgonistsatOpioidReceptors.............. 181
AndreaBedini
PART III MODEL SYSTEMS TO STUDYING OPIOID RECEPTOR-
MEDIATED FUNCTIONS
17 MechanicalNociceptioninMiceandRats:Measurement
withAutomatedvonFreyEquipment ..................................... 195
GabrieleCampanaandRobertoRimondini
18 EvaluationofMurineMacrophageCytokineProduction
AfterInVivoMorphineTreatment ....................................... 199
SilviaFranchi,MaraCastelli,SarahMoretti,AlbertoPanerai,
andPaolaSacerdote
19 MeasurementofMacrophageToll-LikeReceptor4Expression
AfterMorphineTreatment............................................... 209
MaraCastelli,AlbertoPanerai,PaolaSacerdote,
andSilviaFranchi
PART IV BEHAVIORAL EFFECTS MEDIATED BY OPIOID RECEPTORS
20 ConditionedPlacePreference(CPP)inRats:FromConditioning
toReinstatementTest................................................... 221
MariaScherma,LianaFattore,WalterFratta,
andPaolaFadda