Localization of Orphanin FQ (Nociceptin) Peptide and

Localization of Orphanin FQ (Nociceptin)Peptide and Messenger RNA in theCentral Nervous System of the Rat

CHARLES R. NEAL, JR.,1,2* ALFRED MANSOUR,3 RAINER REINSCHEID,4

HANS-PETER NOTHACKER,5 OLIVIER CIVELLI,5 AND STANLEY J. WATSON, JR.1,6

1Mental Health Research Institute, University of Michigan Medical Center,Ann Arbor, Michigan 48109-0720

2Department of Pediatrics, University of Michigan Medical Center,Ann Arbor, Michigan 48109-0720

3Pharmaco Genesis Corporation, West Bloomfield, Michigan 483224University Hospital Eppendorf, Hamburg, Germany

5Department of Pharmacology, College of Medicine, University of California-Irvine,Irvine, California 92697-4625

6Department of Psychiatry, University of Michigan Medical Center,Ann Arbor, Michigan 48109-0720

ABSTRACTOrphanin FQ (OFQ) is the endogenous agonist of the opioid receptor-like receptor

(ORL-1). It and its precursor, prepro-OFQ, exhibit structural features suggestive of the opioidpeptides. A cDNA encoding the OFQ precursor sequence in the rat recently has been cloned,and the authors recently generated a polyclonal antibody directed against the OFQ peptide. Inthe present study, the authors used in situ hybridization and immunohistochemistry toexamine the distribution of OFQ peptide and mRNA in the central nervous system of the adultrat. OFQ immunoreactivity and prepro-OFQ mRNA expression correlated virtually in allbrain areas studied. In the forebrain, OFQ peptide and mRNA were prominent in theneocortex endopiriform nucleus, claustrum, lateral septum, ventral forebrain, hypothalamus,mammillary bodies, central and medial nuclei of the amygdala, hippocampal formation,paratenial and reticular nuclei of the thalamus, medial habenula, and zona incerta. No OFQwas observed in the pineal or pituitary glands. In the brainstem, OFQ was prominent in theventral tegmental area, substantia nigra, nucleus of the posterior commissure, central gray,nucleus of Darkschewitsch, peripeduncular nucleus, interpeduncular nucleus, tegmentalnuclei, locus coeruleus, raphe complex, lateral parabrachial nucleus, inferior olivary complex,vestibular nuclear complex, prepositus hypoglossus, solitary nucleus, nucleus ambiguous,caudal spinal trigeminal nucleus, and reticular formation. In the spinal cord, OFQ wasobserved throughout the dorsal and ventral horns. The wide distribution of this peptideprovides support for its role in a multitude of functions, including not only nociception but alsomotor and balance control, special sensory processing, and various autonomic and physiologicprocesses. J. Comp. Neurol. 406:503–547, 1999. r 1999 Wiley-Liss, Inc.

Indexing terms: immunohistochemistry; in situ hybridization; mapping; mRNA; polyclonal antibody

Following the cloning of the mu (µ), delta (d), and kappa(k) receptors, a novel clone also was isolated (Marchese etal., 1994). This cDNA encoded a putative membrane receptorwith homology to the µ, d, and k opioid receptors (Bunzowet al., 1994; Wick et al., 1994). Early studies of this newclone demonstrated that it is homologous to opioid recep-tors in rat, mouse, and human but is distinct in itsstructure and distribution (Chen et al., 1994; Fukuda etal., 1994; Lachowicz et al., 1994). This orphan clone hasbeen isolated and named opioid receptor-like (ORL1) recep-

tor to emphasize its relationship to the known opioidreceptors (Mollereau et al., 1994). Splice variants and

Grant sponsor: National Institute of Mental Health; Grant number:5 T32 MH15794; Grant sponsor: National Institute of Drug Abuse; Grantnumber: RO1 DA08920.

*Correspondence to: Charles R. Neal, Jr., M.D., Ph.D., Mental HealthResearch Institute, 205 Zina Pitcher Place, Ann Arbor, MI 48109-0720.E-mail: [email protected]

Received 15April 1998; Revised 16 October 1998;Accepted 23 October 1998.

THE JOURNAL OF COMPARATIVE NEUROLOGY 406:503–547 (1999)

r 1999 WILEY-LISS, INC.

Abbreviations

3V third ventricle4V fourth ventricle6 abducens nucleus6n root of the abducens nerve7 facial nucleus7n facial nerve8n vestibulocochlear nerve9n glossopharyngeal nerve10 dorsal motor nucleus of the vagal nerve12 hypoglossal nucleus12n root of the hypoglossal nerveA5 A5 noradrenaline cellsA7 A7 noradrenaline cellsAAA anterior amygdaloid areaac anterior commissureAcb accumbens nucleusAcbC accumbens nucleus, coreAcbSh accumbens nucleus, shellACo anterior cortical amygdaloid nucleusAD anterodorsal thalamic nucleusAHA anterior hypothalamic areaAHiA amygdalohippocampal areaAI agranular insular cortexAL anterior lobe, pituitary glandalv alveusAM anteromedial thalamic nucleusAmb nucleus ambiguousAMPO anterior medial preoptic nucleusAOD anterior olfactory nucleus, dorsalAOL anterior olfactory nucleus, lateralAOM anterior olfactory nucleus, medialAOP anterior olfactory nucleus, posteriorAOV anterior olfactory nucleus, ventralAPir amygdalopiriform transition areaAPT anterior pretectal nucleusAPTD anterior pretectal nucleus, dorsal partAPTV anterior pretectal nucleus, ventral partAq aqueductArc arcuate nucleusATg anterior tegmental nucleusAV anteroventral thalamic nucleusAVPO anteroventral preoptic nucleusB basal nucleus of MeynertBAOT bed nucleus of the accessory olfactory tractbas basilar arterybic brachium of the inferior colliculusBIC nucleus of the brachium of the inferior colliculusBL basolateral amygdaloid nucleusBM basomedial amygdaloid nucleusBSTIA bed nucleus of the stria terminalis, intraamygdaloid divisionBSTl bed nucleus of the stria terminalis, lateral divisionBSTld bed nucleus of the stria terminalis, lateral division, dorsal partBSTlj bed nucleus of the stria terminalis, lateral juxtacapsular partBSTlp bed nucleus of the stria terminalis, lateral posterior partBSTlv bed nucleus of the stria terminalis, lateral ventral partBSTma bed nucleus of the stria terminalis, medial anterior partBSTmpl bed nucleus of the stria terminalis, medial posterolateral partBSTmpm bed nucleus of the stria terminalis, medial posteromedial partBSTmv bed nucleus of the stria terminalis, medial ventral partC1 C1 adrenaline cellsC2 C2 adrenaline cellsC3 C3 adrenaline cellsCA3sl field CA3 of Ammon’s horn, stratum lucidumCA1–3so fields CA1–CA3 of Ammon’s horn, stratum oriensCA1–3sp fields CA1–CA3 of Ammon’s horn, stratum pyramidalCA1–3sr fields CA1–CA3 of Ammon’s horn, stratum radiatumcc corpus callosumCC central canalCe central amygdaloid nucleusCeL central amygdaloid nucleus, lateralCeM central amygdaloid nucleus, medialCer cerebellumCg cingulate gyrusCG central grayCGD central gray, dorsalCGPn pontine central grayCI caudal interstitial nucleus, medial longitudinal fasciculuscic commissure of the inferior colliculusCIC central nucleus of the inferior colliculus

Cl claustrumCL centrolateral thalamic nucleusCLi (B8) caudal linear nucleus of the rapheCM central medial thalamic nucleusCnF cuneiform nucleuscp cerebral peduncleCPO caudal periolivary nucleusCPu caudate putamen (striatum)csc commissure of the superior colliculusctg central tegmental tractcu cuneate fasciculusCu cuneate nucleusDA dorsal hypothalamic areaDC dorsal cochlear nucleusDCIC dorsal cortex of the inferior colliculusdcs dorsal corticospinal tractDEn dorsal endopiriform nucleusdf dorsal fornixDG dentate gyrusDGgr dentate gyrus, granule cell layerDGhi dentate gyrus, hilumDGmo dentate gyrus, molecular layerDGpo dentate gyrus, polymorph layerDH dorsal horn of the spinal cordDk nucleus of Darkschewitschdlf dorsal longitudinal fasciculusDLL dorsal nucleus of the lateral lemniscusDM dorsomedial hypothalamic nucleusDMSp5 dorsomedial spinal trigeminal nucleusDMTg dorsomedial tegmental areaDP dorsal peduncular cortexDPGi dorsal paragigantocellular nucleusDpMe deep mesencephalic nucleusDPO dorsal periolivary nucleusDR (B6, B7) dorsal rapheDRG dorsal root ganglionDTg dorsal tegmental nucleusECIC external cortex of the inferior colliculusECu external cuneate nucleusEnt entorhinal cortexEP entopeduncular nucleusEW Edinger-Westphal nucleusf fornixF nucleus of the fields of Forelfr fasciculus retroflexusFr frontal cortexFStr fundus striatiGi gigantocellular reticular nucleusGI granular insular cortexGiA gigantocellular reticular nucleus, alphaGiV gigantocellular reticular nucleus, ventralGP globus pallidusgr gracile fasciculusGr gracile nucleusHDB nucleus of the horizontal limb of the diagonal band of Brocahi hilum of the dentate gyrusI–X laminae (I–X) of the spinal cordI intercalated nuclei of the amygdalaIAD interanterodorsal thalamic nucleusICj islands of Callejaicp inferior cerebellar peduncleIG indusium griseumIl intermediate lobe, pituitary glandIL infralimbic cortexILL intermediate nucleus of the lateral lemniscusIML intermediolateral cell columnIMLF interstitial nucleus of the medial longitudinal fasciculusIMM intermediomedial cell columnInCo intercollicular nucleusIntA interposed cerebellar nucleus, anterior partIntDL interposed cerebellar nucleus, dorsolateral partIntDM interposed cerebellar nucleus, dorsomedial partIntP interposed cerebellar nucleus, posterior partIO inferior oliveIOA inferior olive, medial subnucleus AIOB inferior olive, medial subnucleus BIOD inferior olive, dorsalIODM inferior olive, dorsomedialIOM inferior olive, medial

504 C.R. NEAL, JR. ET AL.

Abbreviations

IOPr inferior olive, principalIPC interpeduncular nucleus, caudalIPD interpeduncular nucleus, dorsalIPL interpeduncular nucleus, lateralIPN interpeduncular nucleusIPR interpeduncular nucleus, rostralIPRL interpenduncular nucleus, rostrolateralIRt intermediate reticular nucleusKF Kolliker-Fuse nucleusLa lateral amygdaloid nucleusLA lateroanterior hypothalamic nucleusLat lateral (dentate) cerebellar nucleusLatC lateral cervical nucleusLatPC lateral cerebellar nucleus, parvocellular partLC locus coeruleusLd lambdoid septal zoneLD laterodorsal thalamic nucleusLDTg laterodorsal tegmental nucleuslfp longitudinal fasciculus of the ponslfu lateral funiculus of the spinal cordLGN lateral geniculate nucleusLH lateral hypothalamic areaLHb lateral habenulall lateral lemniscusLM lateral mammillary nucleuslo lateral olfactory tractLO lateral orbital cortexLOT nucleus of the lateral olfactory tractLP lateral posterior thalamic nucleusLPB lateral parabrachial nucleusLPGi lateral paragigantocellular nucleusLPO lateral preoptic areaLRt lateral reticular nucleusLS lateral septal nucleusLSD lateral septal nucleus, dorsalLSI lateral septal nucleus, intermediateLSO lateral superior oliveLSp lateral spinal nucleusLSV lateral septal nucleus, ventralLV lateral ventricleLVe lateral vestibular nucleusLVPO lateroventral periolivary nucleusMA3 medial accessory oculomotor nucleusMCPC magnocellular nucleus of the posterior commissureMCPO magnocellular preoptic nucleusMD mediodorsal thalamic nucleusMdD medullary reticular nucleus, dorsalMdV medullary reticular nucleus, ventralME medial eminenceMe5 mesencephalic trigeminal nucleusMeAD medial amygdaloid nucleus, anterodorsalMeAV medial amygdaloid nucleus, anteroventralMed medial (fastigial) cerebellar nucleusMedDL medial cerebellar nucleus, dorsolateral protuberanceMePD medial amygdaloid nucleus, posterodorsalMePV medial amygdaloid nucleus, posteroventralMGN medial geniculate nucleusMHb medial habenulaMiTg microcellular tegmental nucleusml medial lemniscusML medial mammillary nucleus, lateral partmlf medial longitudinal fasciculusMM medial mammillary nucleus, medial partMMn medial mammillary nucleus, median partMnR (B5) median raphe nucleusMO medial orbital cortexmo molecular layer of the dentate gyrusMo5 motor trigeminal nucleusmp mammillary peduncleMPA medial preoptic areaMPB medial parabrachial nucleusMPO medial preoptic nucleusMS medial septal nucleusMSO medial superior olivemt mammillothalamic tractMTu medial tuberal nucleusMVe medial vestibular nucleusMVeV medial vestibular nucleus, ventralMVPO medioventral periolivary nucleus

Oc occipital cortexopt optic tractOPT olivary pretectal nucleusOT nucleus of the optic tractox optic chiasmPa4 paratrochlear nucleusPa5 paratrigeminal nucleusPar parietal cortexPaS parasubiculumPBG parabigeminal nucleuspc posterior commissurePC paracentral thalamic nucleusPCom nucleus of the posterior commissurePCRt parvocellular reticular nucleusPDTg posterodorsal tegmental nucleusPe periventricular hypothalamic nucleusPeF perifornical nucleusPH posterior hypothalamusPin pineal glandPir piriform cortexPit pituitary glandPL paralemniscal nucleusPLCo posterolateral cortical amygdaloid nucleusPLd paralambdoid septal nucleusPLi posterior limitans thalamic nucleusPMCo posteromedial cortical amygdaloid nucleusPMD premammillary nucleus, dorsalPMR paramedian raphe nucleusPMV premammillary nucleus, ventralPn pontine nucleiPN paranigral nucleusPnC pontine reticular nucleus, caudalPnO pontine reticular nucleus, oralPnR pontine raphe nucleusPnV pontine reticular nucleus, ventralPo posterior thalamic nucleus groupPP peripeduncular nucleusPPT posterior pretectal nucleusPPTg pedunculopontine tegmental nucleusPR prerubral fieldPr5 principal sensory trigeminal nucleusPrC precommissural nucleusPrH prepositus hypoglossal nucleusPrS presubiculumPT paratenial nucleusPVA paraventricular thalamic nucleus, anteriorPVNm paraventricular hypothalamic nucleus, magnocellularPVNp paraventricular hypothalamic nucleus, parvocellularPVP paraventricular thalamic nucleus, posteriorpy pyramidal tractRAmb retroambiguous nucleusRbd rhabdoid nucleusRCh retrochiasmatic areaRe reunions thalamic nucleusRh rhomboid thalamic nucleusRI rostral interstitial nucleus, medial longitudinal fasciculusRLi rostral linear nucleus of the rapheRMC red nucleus, magnocellularRMg (B3) raphe magnus nucleusROb (B2) raphe obscurus nucleusRPa (B1) raphe pallidus nucleusRPC red nucleus, parvocellularRPO rostral periolivary regionRR retrorubral nucleusRRF retrorubral fieldrs rubrospinal tractRSA retrosplenial agranular cortexRSG retrosplenial granular cortexRt reticular thalamic nucleusRtTg reticulotegmental nucleus of the ponsS subiculums5 sensory root of the trigeminal nerveSC superior colliculusSC(DpG) superior colliculus, deep gray layerSC(DpWh) superior colliculus, deep white layerSC(InG) superior colliculus, intermediate gray layerSC(InWh) superior colliculus, intermediate white layerSC(Op) superior colliculus, optic nerve layerSC(SuG) superior colliculus, superficial gray layer

ORPHANIN FQ DISTRIBUTION IN THE RAT CNS 505

possible brain heterogeneity of this receptor have beenreported (Wang et al., 1994; Mathis et al., 1997) as well asits presence outside of the central nervous system (CNS;Halford et al., 1995). Functional studies of this receptorhave demonstrated that, similar to opioid receptors, itsactivation stimulates GTPgS binding and inhibits adenyl-ate cyclase (Wu et al., 1997). However, the expressed ORL1receptor has a poor affinity for the known opioid ligands(Meng et al., 1996; Sim et al., 1996; Ma et al., 1997).Binding and mutation analyses have established thatORL1 has features that specifically exclude known opioidsand promote the high-affinity binding of its own endog-enous ligand (Standifer et al., 1994; Meng et al., 1996;Mollereau et al., 1996a).

In the pursuit to identify a neuropeptide that activatesthe ORL1 receptor, orphanin FQ (OFQ), a novel heptadeca-peptide also referred to as nociceptin, recently was discov-ered. OFQ exhibits structural features suggestive of endog-enous opioid peptides (Civelli et al., 1997) and has beenshown to be an endogenous agonist of the ORL1 receptor(Meunier et al., 1995; Reinscheid et al., 1995; Saito et al.,1995, 1996, 1997). This novel peptide demonstrates spe-cific binding characteristics with the ORL1 receptor (Dooleyand Houghten, 1996; Reinscheid et al., 1996; Shimohiga-shi et al., 1996; Ardati et al., 1997; Butour et al., 1997;Guerrini et al., 1997). In addition, high-affinity binding ofOFQ peptide fragments with the ORL1 receptor recentlyhas been reported (Dooley et al., 1997). Orphanin FQ hasbeen shown to depend on peptidase metabolism (Montiel etal., 1997; Noble and Roques, 1997), to stimulate ORL1-induced Ca21 and K1 conductance changes (Connor et al.,1996, 1997; Knoflach et al., 1996; Nicol et al., 1996;Vaughan and Christie, 1996; Abdulla and Smith, 1997;

Ikeda et al., 1997; Vaughan et al., 1997; Yu et al., 1997;Wagner et al., 1998) and protein kinase C activation (Louet al., 1997), and to effect function of other neurotransmit-ter systems (Faber et al., 1996; Giuliani and Maggi, 1996;Murphy et al., 1996; Wang et al., 1996; Gintzler et al, 1997;Liebel et al., 1997). Unlike other opioid peptides, OFQ failsto produce cross tolerance with morphine (Hao et al., 1997)or conditioned place preference (Devine et al., 1996b).However, it has been implicated subsequently in manyother physiologic and behavioral processes, including car-diac and vascular control (Champion and Kadowitz,1997a,b; Champion et al., 1997; Gumusel et al., 1997),diuresis (Kapusta et al., 1997), feeding (Pomonis et al.,1996), learning (Sandin et al., 1997), locomotion (Devine etal., 1996a; Florin et al., 1996, 1997a), and nociception(Grisel et al., 1996; Mogil et al., 1996a,b; Rossi et al., 1996,1997; Stanfa et al., 1996; Xu et al., 1996; Dawson-Basoaand Gintzler, 1997; Heinricher et al., 1997; King et al.,1997; Liebel et al., 1997; Morgan et al, 1997; Nishi et al.,1997; Tian et al., 1997a,b; Yamamoto et al., 1997; Zhu etal., 1997). Its possible role as an anxiolytic also has beenreported (Moreau et al., 1997).

OFQ is derived from a larger precursor, prepro-OFQ,which shares structural homology to the opioid peptideprecursors, prodynorphin and preproenkephalin, and ithas been suggested recently that a coordinated mecha-nism of evolution has separated the OFQ system from theopioid system (Reinscheid et al., 1998). The primarystructure of the rat and human OFQ precursor has re-cently been reported, as has tissue distribution of preproor-phanin mRNA in rat (Mollereau et al., 1996b; Nothacker etal., 1996) and mouse (Houtani et al., 1996; Pan et al.,1996). Antisera to OFQ have been produced, and detection

Abbreviations

SC(Zo) superior colliculus, zonal layerSCh suprachiasmatic areascp superior cerebellar peduncleSd subiculum, dorsalSFO, SF subfornical organSG suprageniculate nucleusSGe supragenual nucleusSHi septohippocampal nucleusSHy septohypothalamic nucleusSI substantia innominatasm stria medullaris of the thalamusSNC substantia nigra, pars compactaSNL substantia nigra, pars lateralisSNR substantia nigra, pars reticulataso stratum oriensSO supraoptic nucleussol solitary tractSol nucleus of the solitary tractSolC nucleus of the solitary tract, commissuralSolL nucleus of the solitary tract, lateralSolM nucleus of the solitary tract, medialSOR supraoptic nucleus, retrochiasmatic (diffuse)sp stratum pyramidalsp5 spinal trigeminal tractSp5C spinal trigeminal nucleus, caudalSp5I spinal trigeminal nucleus, interpolarSp5O spinal trigeminal nucleus, oralSPFPC subparafascicular thalamic nucleus, parvocellularSPO superior paraolivary nucleusSPTg subpeduncular tegmental nucleusSpVe spinal vestibular nucleussr stratum radiatumst stria terminalisSTh subthalamic nucleus

Su3 supraoculomotor central graySubC subcoeruleus nucleusSubG subgeniculate nucleusSuM supramammillary nucleussumx supramammillary decussationSuVe superior vestibular nucleusT temporal cortexTC tuber cinereumTe terete hypothalamic nucleusTM tuberomammillary nucleusTMC tuberal magnocellular nucleusTT tenia tectaTu olfactory tubercletz trapezoid bodyTz nucleus of the trapezoid bodyVCA ventral cochlear nucleus, anteriorVCP ventral cochlear nucleus, posteriorVDB nucleus of the vertical limb of the diagonal band of BrocaVEn ventral endopiriform nucleusvfu ventral funiculus of the spinal cordVH ventral horn of the spinal cordVL ventrolateral thalamic nucleusVLL ventral nucleus of the lateral lemniscusVLTg ventrolateral tegmental nucleusVM ventromedial thalamic nucleusVMH ventromedial hypothalamic nucleusVO ventral orbital cortexVP ventral pallidumVPL ventral posterolateral thalamic nucleusVPM ventral posteromedial thalamic nucleusVTA ventral tegmental areaVTg ventral tegmental nucleusZI zona incerta


of OFQ-like immunoreactivity has been reported in thespinal cord (Riedl et al., 1996; Lai et al., 1997; Schuligoi etal., 1997) and other structures within pain-modulatoryregions in the rat (Schulz et al., 1996), and the hypothala-mus of the rodent and monkey (Quigley et al., 1998).

Autoradiographic analysis of 3H-OFQ receptor bindingand immunohistochemical analysis of the ORL1 receptorhave been reported in rat, mouse, and human (Anton et al.,1996; Florin et al., 1997b; Makman et al., 1997), as hasagonist-stimulated binding of 35S-labeled GTPgS to theORL1 receptor in the guinea pig brain (Sim and Childers,1997). No such critical anatomic analysis of OFQ has beenreported to date. In this study, by using antisera that weregenerated against the orphanin heptadecapeptide, and35S-labeled cRNA corresponding to the 58 portion of theprepro-OFQ precursor sequence, we performed in situhybridization and immunohistochemistry to analyze thedistribution of prepro-OFQ mRNA expression and OFQpeptide immunoreactivity in the adult rat forebrain, brain-stem, and spinal cord.

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats (Charles River, Por-tage, MI; 250–300 g) were used for all in situ and immuno-cytochemistry studies. Handling and use of all animalsstrictly conformed to National Institutes of Health guide-lines. In addition, protocols for animal use in this studywere approved by the University Unit for LaboratoryAnimal Medicine (ULAM) at the University of MichiganMedical Center.

Tissue preparation

For in situ hybridization, adult male Sprague-Dawleyrats were killed by decapitation, and their brains wereremoved and immediately frozen in isopentane at 230°C.In addition to whole brains, the pituitary gland and aportion of the cervical and thoracic spinal cord weredissected from each animal and similarly frozen. Brain,pituitary, and spinal cord tissue were stored at 280°Cuntil sectioning. All CNS material was sectioned coronallyon a Bright cryostat at 15 µm, thaw mounted on polylysine-subbed microscope slides, and then stored at 280°C untiluse.

For immunohistochemistry, adult male Sprague-Dawleyrats were deeply anesthetized with 75 mg/kg of sodiumpentobarbital i.p. and transcardially perfused with approxi-mately 50–75 ml of 0.9% NaCl containing 2.2% sodiumnitrite. This was followed with approximately 400 ml ofZamboni’s fixative (Zamboni and De Martino, 1967). Sevenanimals were injected with colchicine (300 µg in 7.5 µl ofsterile saline, i.c.v.) 48 hours prior to transcardiac perfu-sion. After perfusion was completed, the brain, pituitary,and portions of the thoracic and cervical spinal cord wereremoved and postfixed in Zamboni’s fixative for 1 hour.Tissue was then transferred to a solution of 10% sucrose in50 mM potassium phosphate-buffered saline, pH 7.2(KPBS), for 24–48 hours. Brains were frozen in liquidisopentane at 230°C, then stored at 280°C until section-ing. All material was sectioned coronally on a LeicaCM1800 cryostat at 30 µm and stored in cryoprotectant(sodium phosphate buffer, pH 7.4, with 0.9% saline, 30%sucrose, and 30% ethylene glycol) at 220°C until use.

cRNA probe

Hybridization of CNS tissue was performed by using35S-UTP and 35S-CTP-labeled riboprobes generated againstthe rat prepro-OFQ mRNA sequence. A polymerase chainreaction fragment corresponding to the 58 portion of theprepro-OFQ sequence was used to prepare a 35S-labeledcRNA probe. This cDNA fragment spans 580 base pairs(bases 106–686) and contains the entire open readingframe of the prepro-OFQ precursor molecule (Houtani etal., 1996; Mollereau et al., 1996b; Nothacker et al., 1996).

Antibody production

The entire OFQ heptadecapeptide (FGGFTGARK-SARKLANQ) was used for polyclonal antibody production.The purity of this peptide and its activity at the ORL-1receptor have been reported previously (Reinscheid et al.,1995). By using glutaraldehyde, OFQ was conjugated tothyroglobulin, and additional free aldehyde sites wereblocked by saturating the solution with glycine. OFQthyroglobulin conjugates were then divided into aliquotsand delivered to HRP Inc. (Denver, PA), where rabbitanti-OFQ antiserum was produced.

At HRP Inc., antisera was generated by inoculatingrabbits (New Zealand White; n 5 2) with 350 µg of thethyroglobulin-conjugated OFQ peptide suspended in com-plete Freund’s adjuvant. Rabbits were injected initiallyonce a week for 3 weeks, given 2 weeks rest, and then bledon week 6. On week 7, the animals were inoculated with190 µg of conjugated peptide suspended in incompleteFreund’s adjuvant. They were allowed to rest on week 8and were bled on week 9. This second inoculation-rest-bleed procedure was continued until a total of eight bleedswere obtained, after which the rabbits were exsangui-nated. Sera obtained from each rabbit were tested immuno-histochemically for each bleed. Based on initial results,antisera from animal 217, bleeds 5 and 6, were affinitypurified by using the OFQ heptadecapeptide coupled to asepharose-4B-cyanogen bromide column. After final analy-sis, affinity-purified antiserum from animal number 217,bleed 6, was used in this study.

Immunohistochemistry

The immunohistochemistry technique employed in thisstudy has been described previously for detection of the µand k opioid receptors in the rat CNS (Mansour et al.,1995a, 1996). Floating rat brain and spinal cord sectionswere washed in 50 mM KPBS to remove cryoprotectant.Sections were then incubated for 30 minutes in 0.3% H2O2and rinsed in 50 mM KPBS. Next, tissue was incubatedwith an avidin-D blocking agent for 15 minutes, rinsedwith 50 mM KPBS, and followed with a 15-minute incuba-tion with a biotin-blocking solution (blocking agents ob-tained from Vector Laboratories, Burlingame, CA). Prior tothe addition of primary antibody, sections were incubatedfor 60 minutes in bovine serum albumin (BSA) diluent (50mM KPBS with 0.9% NaCl, 1% BSA, 1% normal goatserum, and 0.4% Triton X-100), then transferred to asolution containing affinity-purified OFQ antibody dilutedto 1:1,500 with BSA diluent, and incubated at 24°C for36–48 hours. After primary antibody incubation, the sec-tions were washed in KPBS and incubated with biotinyl-ated goat anti-rabbit immunoglobulin G (IgG; 1:1,000) for60 minutes at room temperature, followed by an avidin-biotin complex (ABC) coupled to horseradish peroxidase


(1:1,000; Vector Elite; Vector Laboratories) for 60 minutes,also at room temperature. Immunostaining was visualizedwith a 0.04% solution of 3,38-diaminobenzidine tetrahydro-chloride (DAB) containing 2.5% nickel chloride and 0.01%H2O2 dissolved in 0.1 M sodium acetate. After 6 minutes inDAB, the reaction was terminated by two consecutivewashes in 0.9% NaCl. Sections were mounted from 0.9%NaCl onto polylysine-subbed microscope slides, dehy-drated with graded alcohols followed by xylene, and cover-slipped with Permount. Tissue sections adjacent to thosethat were stained immunohistochemically were mountedonto polylysine-subbed slides and Nissl counterstainedwith cresyl violet to aid in anatomic localization. Immuno-histochemical brightfield staining was viewed with a ZeissAxiphot microscope (Thornwood, NY) and a Leitz DM RDmicroscope (Wetzlar, Germany) with camera attachment.Representative sections were photographed, and imagesfrom negatives were generated on high-quality Kodakphotographic paper for illustrations (Rochester, NY) forillustrations. Processing of all photomicrographs was per-formed under identical darkroom conditions.

In situ hybridization

The in situ hybridization technique employed in thisstudy has been described previously for detection of opioidreceptor mRNA in the rat CNS (Mansour et al., 1993,1994). Adjacent sections of frozen brain, pituitary, andspinal cord with dorsal root ganglia were removed fromstorage (280°C) and placed into 4% paraformaldehyde for60 minutes at room temperature. Sections were next giventhree 5-minute rinses in a solution of 300 mM sodiumchloride and 30 mM sodium citrate, pH 7.2 (SSC; 2 3 SSC),followed by treatment with proteinase K [1 µg/ml in 100mM Tris and 50 mM ethylenediamine tetraacetic acid(EDTA), pH 8.0] for 10 minutes at 37°C. Sections wererinsed once in water then treated with 0.1 M triethanol-amine and acetic anhydride (400:1 volume/volume), pH8.0, for 10 minutes at room temperature. Sections wererinsed again in water, dehydrated in graded alcohols, andair dried.

Prepared tissue was hybridized with a 35S-UTP- and35S-CTP-labeled riboprobe generated to the rat OFQ pep-tide precursor as described above. The cRNA probe wasdiluted by using a hybridization buffer composed of 75%formamide, 10% dextran sulfate, 3 3 SSC, 0.1 mg/ml yeasttRNA, 1 3 Denhardt’s, and 10 mM dithiothreitol in 50 mMNa2 PO4, pH 7.4. The activity of 35S-labeled cRNA used forhybridization was in the range of 1–2 3 106 cpm/35 µl.Volumes of 35 µl of diluted probe were applied to tissuesections, and glass coverslips were placed, keeping thehybridization buffer in contact with tissue. Slides wereplaced in sealed humidifying chambers containing 50%formamide and hybridized overnight in a VRW Scientific1535 incubator (Cornelius, OR) at 55°C.

On day 2, glass coverslips were removed, and slides wererinsed twice in 2 3 SSC for 5 minutes, then treated withRNase A for 60 minutes at 37°C (200 µg/ml RNase A and0.5 M NaCl in 100 mM Tris, pH 8.0). Following RNase Atreatment, sections were washed in 2 3 SSC for 5 minutes,followed by 1 3 SSC for 5 minutes, and 0.5 3 SSC for 5minutes, all at room temperature. Low salt wash wascompleted with incubation in 0.1 3 SSC for 60 minutes at65°C. Sections were then rinsed in water, dehydratedthrough graded alcohols, and air dried.

On completion of hybridization, slide-mounted sectionswere opposed to Kodak XAR-5 x-ray film for 5 days thendipped in NTB2 film emulsion. Brain sections were thendeveloped following a 30-day exposure to NTB2 emulsion.The exposure time was chosen to maximize the detection ofin situ hybridization grains and was determined empiri-cally by periodic development of test slides of tissuesections dipped in the NTB2 emulsion. Following develop-ment of the NTB2 film emulsion, all slides were rinsed inrunning water at room temperature for 30 minutes andNissl counterstained with cresyl violet, dehydrated ingraded alcohols followed by xylene, and coverslipped withPermount. Hybridized tissue was analyzed by using a‘‘Dark-Lite’’ darkfield stage light on a Leitz DM RD micro-scope with camera attachment. Representative sectionswere photographed, and images from negatives were gen-erated on high-quality Kodak photographic paper forillustrations. Like the immunohistochemical images, allprocessing was done under identical darkroom conditions.

Controls

For immunohistochemical controls, the OFQ antibodywas preabsorbed by incubating the primary antiserumwith 25 µM of the OFQ heptadecapeptide overnight priorto the addition of floating tissue sections. Preabsorptioncontrols and normal immunohistochemical studies wereperformed on adjacent tissues from both normal andcolchicine-treated animals.

The specificity of the 58 portion of the prepro-OFQsequence used for in situ hybridization was determinedwith two separate control conditions. After the 60-minuteincubation in 4% paraformaldehyde, sections from repre-sentative brain regions were incubated in RNase A for 60minutes at 37°C (200 µg/ml RNase A and 0.5 M NaCl in100 mM Tris, pH 8.0). They were then run through theentire hybridization procedure with 35S-labeled cRNA, asdescribed above. A separate set of adjacent, representativebrain regions was run through the entire hybridizationprocedure as described above, with the exception that a35S-labeled mRNA (sense strand) was used for the hybrid-ization. Other than the alterations described above (RNaseA and sense controls), all control tissue was treatedidentically and ran along side adjacent sections undernormal conditions for comparison.

RESULTS

Colchicine treatment

Colchicine injection into the lateral ventricle markedlyaltered OFQ immunohistochemical staining in all animalsstudied. Brains of untreated animals contained morerobust fiber staining than what was observed in colchicine-treated animals, but they also demonstrated very fewimmunolabeled cell bodies. Areas of heaviest immunolabel-ing (e.g., lateral septum, bed nucleus of the stria termina-lis, central nucleus of the amygdala, arcuate nucleus,central gray, nucleus of Darkschewitsch, solitary nucleus)did contain some labeled perikarya in untreated animals.Untreated (normal) animals provided an added source ofinformation concerning the distribution of OFQ fiber andterminal immunoreactivity, complimenting fiber and cellstaining observed in colchicine-treated animals. Descrip-tions presented here are a synthesis of immunolabelingobserved in both colchicine-treated and normal animals,with descriptions of cell labeling derived primarily from


colchicine-treated animals, and fiber/terminal labelingderived primarily from untreated animals (Fig. 1).

Controls

No mRNA expression was detected in tissues pretreatedwith RNase A prior to in situ hybridization using 35S-labeled cRNA directed to the 58 portion of the prepro-OFQcDNA sequence (antisense). Messenger RNA levels inthese tissues were negligible in all levels of the forebrain,brainstem, and spinal cord. In addition, no mRNA-expressing cells were detected in tissues hybridized with a35S-labeled mRNA directed to the 58 portion of the prepro-OFQ cDNA sequence (sense strand). Like the RNase Atreatment, these tissues contained no mRNA-expressingcells at all levels studied (Fig. 2).

Unanticipated, nonspecific background staining notedduring initial immunohistochemical trials was found to bedue to impurities in the ABC-horseradish peroxidasereaction kit obtained from Vector Laboratories (VectorElite kit). Nonspecific background staining was removedcompletely by using an avidin-D and biotin-blocking solu-tion, also provided by Vector Laboratories. Therefore,these blocking agents were used in all immunohistochemi-cal studies reported below.

Preabsorption of the primary OFQ antibody with OFQheptadecapeptide virtually eliminated all immunolabeling(Fig. 3). However, there were two notable exceptions. Adensely stained group of cell bodies was observed clusteredlateral to the paraventricular nucleus of the hypothalamusin a perifornical distribution dorsal to the anterior hypotha-lamic area. Within this prominently immunolabeled cellgroup, a very small population of these neurons was notblocked completely in preabsorption controls. Unblockedperikarya were visible but were labeled very lightly.Within the forebrain, the neurons within this cell groupwere the only perikarya that were not blocked completelyin preabsorption controls. Preabsorption of the OFQ anti-body with the OFQ peptide completely blocked immunore-activity in every other forebrain region. In the cerebellum,dense staining of perikarya was observed throughout thePurkinje cell layer in preabsorption controls. These immu-nolabeled cells, which were confined to the cerebellum,were considered to be nonspecific because 1) they werepresent equally in both colchicine-treated and untreatedanimals, 2) this cell population was confined to the Pur-kinje layer and remained completely unblocked in allpreabsorption control studies, and 3) no mRNA expressionwas observed in this cell layer at any brainstem level.Therefore, in the cerebellum, the Purkinje cell layer wasconsidered to be void of specific immunolabeling. In con-trast, immunolabeling in the granular layer and deepcerebellar nuclei is treated as specific, because cerebellarlabeling in these areas was blocked completely in preab-sorption control studies.

Immunohistochemical and in situ hybridization controlsdemonstrated high specificity of the OFQ antiserum andthe 35S-labeled prepro-OFQ riboprobe used in this study. Adetailed description of the distribution of prepro-OFQmRNA and OFQ peptide is presented below. Anatomicdescriptions and nomenclature are based on those de-scribed for the rat CNS by Paxinos and Watson (1986). Asummary of OFQ mRNA and peptide distribution is pro-vided in Table 1. An overview of OFQ mRNA expression inthe brain and spinal cord is presented in Figures 4 and 5.

Cortex

In situ hybridization. Prepro-OFQ mRNA expres-sion is found throughout the rostral-to-caudal extent of theneocortex. Cells containing OFQ mRNA are scatteredprimarily in layers II, III, V, and VI, with the strongestexpression in layer VI. Prepro-OFQ mRNA-expressingcells are most numerous in the frontal cortex (Fig. 2A),

Fig. 1. Brightfield photomicrographs demonstrating orphanin FQ(OFQ)-containing fibers (arrows) and terminal-like structures (arrow-heads) in noncolchicine-treated tissue. A: High-power image of thestriatum demonstrating the fine immunoreactive fibers found scat-tered throughout this structure. B: High-power photomicrograph ofdense OFQ-containing fibers in the median eminence. C: High-powerphotomicrograph of OFQ immunoreactivity in the posteroventral partof the medial nucleus of the amygdala demonstrating immunolabeledfibers and terminal-like structures in this region. For abbreviations,see list. Scale bar 5 50 µm.


Fig. 2. Darkfield autoradiograms of in situ hybridization controls.A: mRNA expression obtained after hybridization with a 35S-labeledcRNA (antisense strand) generated against the 58 portion of thepreproorphanin sequence in the frontal cortex (AS). B: mRNA expres-sion is absent in an adjacent section hybridized with a 35S-labeledmRNA (sense strand) generated against the same region of thepreproorphanin sequence (S). C: Photomicrograph of mRNA-express-ing neurons in the rostral claustrum. D: Labeling is absent in an

adjacent section treated with RNase A prior to hybridization (R).E: Antisense signal generated in the peripeduncular nucleus of themesencephalon. F: Labeling is absent in an adjacent section followinghybridization with sense strand. G: mRNA-expressing neurons inlaminae VIII and IX of the ventral horn of the cervical spinal cord. InH, labeling is absent in an adjacent section that was treated withRNase A prior to hybridization. Scale bar 5 200 µm in A–F, 100 µm inG,H.

with mRNA expression in the temporal cortex weakest ofall regions. No mRNA expression is observed within thecorpus callosum.

Messenger RNA expression is also strong in other corti-cal regions. Rostrally, moderate mRNA expression is seenin medial, lateral, and ventral orbital cortices, primarily inlayer II. Expression levels are strongest in the rostral poleof the orbital cortex, tapering off at caudal levels. In therostral agranular insular cortex, mRNA-containing neu-rons are numerous and are found mostly in layers II, III,and V. This mRNA expression becomes most intense incaudal insular regions, with adjacent granular cortex

maintaining a more moderate distribution of prepro-OFQ-containing neurons. In the rostral forebrain, mRNAexpres-sion is strong in the prefrontal cortical regions. MessengerRNA-containing neurons are numerous in the infralimbicand dorsal peduncular cortical regions ventrally and in therostral cingulate cortex dorsally, mostly in layers II, III,and V. Rostral cingulate expression is strongest in area 1,but mRNA expression intensity is strongest in area 2caudally, into the retrosplenial cortices. Messenger RNAlevels throughout the retrosplenial cortex remain heavy inthe agranular part, primarily in layers II and V rostral,and in layers II and III caudal. In the rostral tenia tecta,

Fig. 3. Brightfield photomicrographs demonstrating immunohisto-chemistry controls in colchicine-treated animals: Immunolabeling inarea CA1 of Ammon’s horn in the hippocampal formation afterincubation with primary OFQ antiserum (A) is absent in the sameregion when incubated in primary antiserum after preabsorption with

25 mM OFQ peptide (B). Densely immunolabeled neurons within thesubstantia nigra, pars reticulata (C) are unlabeled in adjacent controltissue (D). Cell and fiber immunolabeling within the dorsal horn of thespinal cord (E) also is blocked completely in adjacent control tissue (F).For abbreviations, see list. Scale bar 5 75 µm in A,B, 200 µm in C–F.


TABLE 1. Distribution of Orphanin FQ Peptide and mRNAin the Rat Central Nervous System1

CNS region

Orphanin FQ peptide PreproorphaninFQ mRNAexpressionCells F/T

NeocortexFrontal

Layer I 2 2 2Layer II 1 1 111Layer III 11 1 111Layer IV 2 2 2Layer V 1 1 11Layer VI 11 1 111

ParietalLayer I 2 2 2Layer II 1 2 111Layer III 1 2 11Layer IV 2 2 2Layer V 1 1 1Layer VI 11 1 111

TemporalLayer I 2 2 2Layer II 2 1 1Layer III 2 1 1Layer IV 2 2 2Layer V 2 1 1Layer VI 1 1 11

OccipitalLayer I 2 2 2Layer II 2 2 11Layer III 2 2 11Layer IV 2 2 2Layer V 1 1 1Layer VI 11 1 11

Other cortical regionsAI 1 2 111cc 2 2 2Cg 11 1 111DP 2 2 1Ent 2 2 111GI 1 2 11IL 1 2 11LO 1 2 111MO 2 2 11Pir 1 11 11RSA 11 1 111RSG 11 1 11TT 11 2 11VO 11 2 111

Ventral forebrainac 2 2 2AcbC 1 1111 11AcbSh 1 11 1AOD 2 2 2AOL 2 2 1AOM 2 2 11AOP 11 1 1AOV 1 2 11FStr 2 111 2HDB 1111 11 1111ICj 2 2 2SI 111 11 111Tu 1 1 11VDB 1 11 111VP 1 111 11

Septumdf 2 2 2Ld 2 2 2LSD 111 11 11111LSI 11 11 1111LSV 1 111 111MS 1 2 1PLd 2 2 2SHi 11 11 11SHy 111 111 1111

Basal gangliaB 111 11 11Cl 11 2 111CPu 1 11 1Den 11 1 111EP 11 11 11GP 111 1 111SNC 11 11 111SNL 11 1 11SNR 111 1 111STh 1 1 1VEn 11 2 11

Basal telencephalonAMPO 1 1111 1AVPO 111 111 1111BSTl 11 1111 11

TABLE 1. (continued)

CNS region


Basal telencephalon(continued)

BSTld 111 111 1111BSTlj 1 11 1BSTlp 111 111 1111BSTlv 1 1111 111BSTma 111 1111 11111BSTmpl 1111 11 11111BSTmpm 1111 11 11111BSTmv 111 1111 1111f 2 2 2LOT 11 1 11LPO 11 111 111MCPO 111 1 111MPA 11 1111 1111MPO 111 1111 1111Pe 1 111 1st 1 11 2

HypothalamusAHA 11 1 111Arc 111 111 111DA 1 11 11DM 11 111 111LA 11 1 1LH 111 1 11LM 1111 11 11ME 2 1111 2ML 2 2 2MM 2 2 2MMn 11 111 1mp 2 2 2MTu 11 2 11Pe 1 111 1PeF 1 2 1PH 11 2 111Pin 2Pit 2 2 2PMD 11 111 111PMV 11 111 11PVNm 1 1 1PVNp 11 11 11RCh 111 1 1SCh 2 2 2SO 2 11 2SOR 2 111 2SuM 11 11 111sumx 2 2 2TC 11 11 11Te 11 2 2TM 11 1 11TMC 1 2 1VMH 1 111 2/1

AmygdalaAAA 2 1 1ACo 1 1 1AHiA 2 2 1APir 2 2 2BAOT 1 11 1BL 1 1 11BM 1 2 1BSTIA 1 11 11CeL 1 111 11CeM 111 1111 1111I 1 11 2La 2 2 2MeAD 11 111 11MeAV 2 1 1MePD 1111 1111 11111MePV 1 111 111opt 2 2 2PLCo 2 2 2PMCo 11 1 11

Hippocampal formationCA1so 1 11 1CA1sp 111 1 111CA1sr 11 11 1CA2so 1 2 2CA2sp 11 2 11CA2sr 2 2 1CA3sl 111 11 111CA3so 2 1 2/1CA3sp 11 1 11CA3sr 2 1 1DGgr 11 1 111DGhi 2 2 2DGmo 1 11 1



CNS region


Hippocampal formation(continued)

DGpo 1 1 1IG 111 1 111PaS 1 2 11PrS 1 2 1S 111 1 111SFO 1 11 11SHi 11 11 11

ThalamusAD 2 11 2AM 2 2 2AV 2 1 11CL 2 2 2CM 2 2 2F 2 2 2IAD 2 111 2LD 2 2 1LGN 2 2 1LHb 2 11 11LP 2 2 2MD 2 2 1MGN 2 2 2MHb 11 111 1mt 2 2 2PC 2 11 2PLi 1 2 11Po 1 2 11PR 2 2 1PrC 11 111 11PT 2 111 111PVA 1 111 11PVP 2 1 11Re 2 1 1Rh 2 1 2RI 2 2 1Rt 1111 11 11111SG 111 2 11SPFPC 11 111 111SubG 2 2 11VL 2 2 2VM 2 2 2VPL 2 2 2VPM 2 2 2ZI 11 11 111

MesencephalonAPT 111 11 11APTD 11 11 11APTV 111 1 111ATg 1 1 2bic 2 2 2BIC 2 11 2CG 1111 111 1111CGD 1 1 11cic 2 2 2CIC 1 2 2CLi (B8) 11 1 11CnF 2 2 2cp 2 2 2csc 2 2 2ctg 2 2 2DCIC 1 1 1Dk 1111 11 11111dlf 2 2 2DLL 2 2 2DpMe 1 1 11DR (B7) 11 11 11DTg 111 1 1111ECIC 11 1 111EW 2 2 2fr 2 2 2lfp 2 2 2ILL 2 2 2IMLF 11 1 111InCo 2 2 2IPC 1 1 11IPD 11 111 1111IPL 2 1 111IPR 111 1 111LC 11 11 111LDTg 111 11 111LGN 2 2 1ll 2 2 2MA3 2 2 2MCPC 1 2 11Me5 2 2 2MGN 2 2 2


CNS region


Mesencephalon(continued)

MiTg 2 2 2ml 2 11 2MnR (B8) 111 1 11mp 2 2 2OPT 11 2 1OT 11 1 11Pa4 2 2 2PBG 2 2 2pc 2 2 2PCom 111 2 111PL 1 2 2PLi 1 2 11PMR 11 2 1Pn 2 1 2PN 111 1 1111PnO 1 11 11PP 111 11 1111PPT 11 2 11PPTg 11 1 111PR 2 2 2Rbd 2 2 2RLi 11 1 11RMC 1 1 11RPC 2 1 1RPO 1 2 2RR 11 2 1RRF 11 1 11rs 2 2 2RtTg 2 2 2SC(DpG) 2 2 1SC(DpWh) 2 2 2SC(InG) 111 1 111SC(InWh) 2 2 2SC(Op) 11 1 11SC(SuG) 1 1 1SC(Zo) 2 1 2scp 2 2 2SG 11 2 111SNC 11 1 111SNL 11 1 11SNR 111 11 111sp5 2 2 2SPTg 2 2 2Su3 11 1 11tz 2 2 2Tz 11 2 111VLL 2 2 2VLTg 2 2 2VTA 111 11 111VTg 1 1 11ZI 11 11 111

CerebellumLobules 1 2 1IntA 2 2 2IntDL 2 2 2IntDM 2 2 2IntP 2 2 2Lat 2 2 2LatPC 2 2 2Med 111 11 11MedDL 11 11 11

Metencephalon6 2 2 26n 2 2 27 2 2 27n 2 2 28n 2 2 2A5 11 2 11A7 2 2 2CGPn 11 1 11CPO 2 1 2DC 11 1 1DMTg 1 2 11DPO 1 2 11DR (B6) 11 11 11DTg 11 2 11Gi 1 2 11GiA 11 2 11GiV 111 2 111Gr 2 2 2icp 2 2 2KF 2 2 2LC 11 11 111LDTg 1 2 11LPB 11 1 111


scattered neurons expressing OFQ mRNA emerge in layerIII at the level of the rostral accumbens. Caudally, moreintense labeling is observed in its dorsal part in layers IIand III. The piriform cortex maintains persistent celllabeling throughout its rostral-to-caudal extent. Messen-ger RNA levels in this cortical region are light to moderate,primarily in layer III, with occasional scattered neurons inlayer II. In the entorhinal cortex, mRNA expression ismoderate throughout but is observed mostly in layers IIand III.

Immunohistochemistry. Moderately immunostainedcells are scattered throughout the rostral-to-caudal extentof both the neocortex and the allocortex. Fiber labeling isminimal, with occasional fibers noted primarily in layers Vand VI in untreated animals. These fibers are seen in allcortical regions and are sparse. Fibers in layer VI main-tain a parallel orientation, whereas those in other layersmaintain a perpendicular orientation. Immunolabeling inthe frontal cortex is light to moderate to its caudal extent,with most immunolabeled neurons within layers II, III,and VI rostral and with occasional scattered neuronsnoted in layer V caudally. Rostral parietal regions demon-strate sparse immunolabeling, but OFQ-containing neu-rons are slightly more numerous and are stained moremoderately in its middle-to-caudal regions. These peri-karya are scattered mostly in layers II, III, V, and VI. Inthe temporal cortex, the weakest OFQ neocortical immuno-labeling is observed. Perikarya are observed occasionallyin layer VI and are stained moderately. Occipital immuno-labeling is confined to scattered, moderately labeled neu-rons in layers V and VI, primarily in its dorsal part.Throughout its extent, the corpus callosum is devoid ofimmunolabeling.


CNS region


Metencephalon(continued)

LPGi 11 2 1111LRt 1 2 11LSO 1 11 11LVe 11 1 111LVPO 11 1 11mlf 2 2 2MnR (B5) 111 1 11Mo5 2 2 2MPB 2 2 2MSO 2 2 2MVeV 11 1 11MVe 11 11 11MVPO 11 1 111Pa5 1 2 11PCRt 2 11 1PDTg 1 2 111PnC 11 1 11PnR 11 1 111PnV 11 1 1PPTg 111 2 111Pr5 2 2 2py 2 2 2RMg 111 11 1111RPa (B1) 11 1 11RPO 1 11 11Sp5O 2 1 1SPO 11 11 11SpVe 1 1 11SubC 2 2 2SuVe 111 1 11Tz 11 2 111VCA 2 2 2VCP 2 2 2

Mylencephalon9n 2 2 210 2 2 212 2 2 2A5 11 2 11A7 2 2 2Amb 111 11 1111C1 2 2 2C2 2 2 2C3 2 2 2CI 11 2 111Cu 2 2 2DMSp5 111 111 11111DPGi 1 2 1ECu 2 2 1Gr 2 2 2IOA 1 2 11IOB 2 2 11IOD 111 11 11IODM 2 2 2IOM 2 2 2IOPr 111 1 111IRt 11 2 111LVe 11 1 111MdD 11 2 11MdV 11 2 11mlf 2 2 2MVeV 2 2 2MVe 11 1 11Pa5 1 2 11PrH 1111 111 1111py 2 2 2RAmb 111 2 11111RMg (B3) 111 11 1111ROb (B2) 1 1 11SGe 11 111 111sol 2 2 2Sol 11 11 1111SolC 111 11 11SolL 111 111 111SolM 11 111 111Sp5C 111 1111 11111Sp5I 2 111 1SpVe 11 1 1SuVe 11 1 11Tz 111 2 111VCA 2 2 2VCP 2 2 2

Spinal cordCervical

I 2 11 2II 11 1111 1111


CNS region


Spinal cordCervical (continued)

III 11 111 111IV 2 111 2V 2 1 1VI 2 1 1VII 1 1 11VIII 1 1 1IX 11 2 11X 11 111 111

ThoracicI 2 1 2II 11 111 1111III 2 11 111IV 2 11 2V 2 11 1VII 2 11 2VIII 11 1IX 1 2 11X 11 111 11

cu 2 2dcs 2 2 2DRG 1gr 2 2 2IML 2 2 2IMM 1 2 111LatC 111 111 1lfu 2 2 2LSp 11 111 2vfu 2 2 2

Distribution of orphanin FQ immunoreactivity and preproorphanin mRNA in thecentral nervous system (CNS) of the adult male rat. Degree of immunoreactivity andmRNAexpression was graded arbitrarily based on density and intensity of immunostain-ing, and intensity of microscopic mRNA expression was assessed based on emulsion-dipped sections. F/T, fibers/terminals. Gradations used were 11111, highest signalintensity (used for mRNA expression); 1111, intense; 111, moderate; 11, light tomoderate; 1, light or sparse; 2, undetectable. For abbreviations, see list.


Fig. 4. A–J: Distribution of prepro-OFQ mRNA in the rat brain at representative coronal sectionsthrough the forebrain. For abbreviations, see list. Scale bar 5 500 µm.


Scattered, lightly labeled cells are noted in the ventralorbital cortex, with moderately labeled immunoreactivecells noted in layers I and II of the dorsal peduncular andinfralimbic cortices. Immunolabeling in the insular cortex

is confined to scattered, lightly labeled neurons in layers IIand III, mostly in its granular part. Immunolabeled neu-rons are observed throughout the tenia tecta, mostly inlayer II (Fig. 6B), and in the indusium griseum, where

Fig. 5. A–H: Distribution of prepro-OFQ mRNA in the rat brain at representative coronal sectionsthrough the brainstem and spinal cord. For abbreviations, see list. Scale bar 5 500 µm.


darkly stained cells are seen throughout its rostral-to-caudal extent. In the piriform cortex, scattered, moder-ately stained cells are dispersed throughout layer III, witha moderate network of fibers and puncta observed in layerIII in untreated animals (Fig. 6A). No immunolabeling isobserved in layer I. Lightly immunolabeled neurons andminimal fibers are observed in the amygdalopiriformtransition area, and the entorhinal and perirhinal corticesexhibit no immunolabeling. OFQ-containing cells are darkand are most prominent in layers II and III throughout thecingulate cortex, with darkly immunolabeled neuronsnoted in layer VI in its caudal extent. This pattern persistsinto the retrosplenial cortex, where darkly stained cellsare observed in its agranular and granular parts to itscaudal extent.

Ventral forebrain

In situ hybridization. Messenger RNA expression isweak in the rostral anterior olfactory nucleus, particularlyin its lateral and dorsal parts. More caudally, mRNA levelsincrease, with scattered mRNA-expressing neurons ob-served mostly in the medial, lateral, and posterior parts.Caudally, mRNA expression in the ventral division is thestrongest of the entire anterior olfactory nucleus. Noimmunolabeled neurons are observed in the rostral pole ofnucleus accumbens. As the accumbens differentiates intocore and shell components, only scattered OFQ-containingneurons are observed in the shell throughout its rostral-to-

caudal extent. Throughout the accumbens core, mRNAexpression is very light, with mRNA-containing neuronslocalized to the lateral part of the nucleus and extendinginto the substantia innominata. Medial to the accumbensshell, the major island of Calleja is devoid of mRNAexpression. Throughout the ventral forebrain, the islandsof Calleja and fundus striati contain no OFQ mRNAexpression. Orphanin-containing neurons are lightly scat-tered in the medial ventral pallidum and are more numer-ous in its lateral aspect. Messenger RNA expression in theolfactory tubercle is moderate, with neurons scatteredprimarily throughout layer III. Moderate numbers ofOFQ-containing neurons are observed in the vertical limbof the diagonal band of Broca (Fig. 9B). Heavy mRNAexpression is observed in the horizontal limb to its caudalboundary, where a cluster of prepro-OFQ-containing neu-rons is present dorsal to the supraoptic nucleus in therostral hypothalamus. No mRNA expression is present inthe anterior commissure at any level.

Immunohistochemistry. Immunolabeling in the ante-rior olfactory nucleus is confined to sparse, lightly stainedneurons ventrally and to more numerous neurons in itsposterior division, where cells are stained more moder-ately. In untreated animals, diffuse fibers and puncta areobserved in the more caudal aspect of this nucleus extend-ing into the rostral pole of the accumbens. Immunolabelingin the rostral pole of the nucleus accumbens is limited todiffuse, fine, moderately labeled fibers that are observed

Fig. 6. Brightfield photomicrographs of OFQ immunolabeling inrostral forebrain structures. A: Fiber and neuronal immunolabelingwithin layer III of the piriform cortex. B: Photomicrograph illustratingOFQ-containing neurons within layer II of the tenia tecta. C,D: Low-

and high-power images, respectively, of immunolabeled fibers in thecore of nucleus accumbens in untreated animals. For abbreviations,see list. Scale bar 5 100 µm in A–C, 50 µm in D.


best in untreated animals. As the shell emerges, it con-tains scattered fibers and puncta with occasional darklystained cells. Fine, immunoreactive fibers extend mediallyinto the vertical limb of the diagonal band of Broca andcaudally as a continuum with the ventral division of thebed nucleus of the stria terminalis. The accumbens corecontains only occasional lightly stained neurons, whereasdiffuse fibers are spread throughout this nucleus. Fiberimmunolabeling intensifies caudally in its dorsal part,adjacent to the striatum (Fig. 6C,D), and extends ventrallywith less intensity into the ventral pallidum. In theventral pallidum, lightly immunolabeled neurons are scat-tered throughout the region, with a prominent fiber plexusobserved to its caudal extent. A similar network of densefibers and puncta is observed in the fundus striati with noimmunoreactive neurons. This pattern of fiber immunola-beling, which is observed best in untreated animals, issimilar to that seen in the ventral pallidum.

Several moderately stained neurons are found in layersII and III of the olfactory tubercle, with dark fibers andpuncta distributed primarily in layer II. No immunostain-

ing is observed in the islands of Calleja. The vertical limbof the diagonal band of Broca contains moderately immuno-labeled fibers and puncta but only occasional lightlyimmunolabeled neurons. At the emergence of the horizon-tal limb, darkly labeled OFQ-containing neurons appearand remain to its caudal pole as a cluster of denselystained neurons ventrolateral to the preoptic area. Adja-cent to the fundus striati caudally, several large, darklystained, OFQ-containing neurons and fibers are observedin the substantia innominata. Scattered fibers present inthe substantia innominata extend dorsally into the basalnucleus of Meynert, where large, moderately stained neu-rons are observed also in its caudal region. The anteriorcommissure is devoid of immunolabeling.

Septum

In situ hybridization. OFQ mRNA expression in thisentire region is very strong (Figs. 7A, 9A). Initially, lightOFQ expression is observed in the most rostral pole of thelateral septum in its ventral and intermediate parts. Atthis level, the septohippocampal nucleus has moderate

Fig. 7. Photomicrographs of mRNA expression and immunolabel-ing in the lateral septal nucleus. A: Low-power photomicrograph ofmRNA expression in the dorsal, intermediate, and ventral divisions ofthe lateral septum. B: High-power photomicrograph of the dorsal

lateral septum demonstrating numerous mRNA-containing neuronswithin this region. C: Brightfield photomicrograph of immunolabelingin the lateral septum in a colchicine-treated animal. For abbrevia-tions, see list. Scale bar 5 800 µm in A, 200 µm in B, 100 µm in C.


mRNA expression, which persists to its caudal extent. Inthe lateral septal nucleus, light OFQ expression is seeninitially in the anterior portion of its ventral part. At theemergence of the dorsal lateral septum, mRNA expressionbecomes very intense, filling this division throughout itsextent (Fig. 7B). The intermediate lateral septum also islabeled heavily but not as intensely as the dorsal part. Theventral part contains moderate mRNA expression. Scat-tered OFQ mRNA-containing neurons in the ventral lat-eral septum are observed primarily along the dorsal aspectof the accumbens shell, persisting caudally to the bednucleus of the stria terminalis. At this level, intensemRNA expression is observed intensifying and stretchingout from the lateral septal region into the septohypotha-lamic nucleus (Fig. 9A). The medial septal nucleus hasscattered OFQ-containing cells. No mRNA expression isobserved in the lambdoid zone, the paralambdoid septalnucleus, or the dorsal fornix.

Immunohistochemistry. In contrast to the intensemRNA expression observed in this region, immunolabel-ing in the lateral septum is modest (Fig. 7C). A plexus ofscattered fibers and terminals and numerous darklystained cells resides in the dorsolateral portion of thisnucleus, remaining consistently immunolabeled through-out. Moderately labeled OFQ-containing neurons in theintermediate lateral septal nucleus reside mostly along itslateral boundary, with more numerous immunolabeledneurons in the ventral part. In untreated animals, fiberand terminal staining in the intermediate division ismoderate to intense, and the ventral lateral septum con-tains scattered, light neurons among a moderate fiber andterminal plexus located primarily along the ventrolateralborder. Scattered, lightly labeled neurons are observed inthe medial septum, where fiber labeling is negligible. Noimmunolabeling is noted in the lambdoid septal zone orthe paralambdoid septal nucleus. The septohippocampalregion contains a moderate number of densely stainedneurons and fibers. Immunolabeling in the septohypotha-lamic nucleus is prominent, with moderately stained fibersand dark cells extending ventral to the bed nucleus of thestria terminalis. The dorsal fornix is devoid of immunola-beling.

Basal ganglia

In situ hybridization. In the rostral forebrain, promi-nent numbers of OFQ-containing neurons are observed inthe claustrum scattered throughout the nucleus (Fig. 2C).At the emergence of the striatum, mRNA-containing neu-rons in the claustrum become much more numerous, withlight-to-moderate mRNA expression extending ventrallyinto the dorsal and ventral endopiriform nuclei. Thestriatum contains little prepro-OFQ mRNA expression.Only scattered OFQ-containing neurons are identifiedthroughout this nucleus, with mRNA-containing neuronsmore numerous in its mediodorsal part. Messenger RNAexpression is intense throughout the globus pallidus.Neurons within this structure are not dense in number,but intensity of mRNA expression is strong, and labeledneurons are large. Near the caudal striatum, the basalnucleus of Meynert contains a moderate number of neu-rons expressing OFQ mRNA. The adjacent entopeduncu-lar nucleus has numerous large, mRNA-expressing neu-rons, mostly in its ventromedial aspect. At the caudalextent of the entopeduncular nucleus, the subthalamicnucleus contains scattered, light mRNA expression.

Caudal to the subthalamic nucleus, preproorphaninmRNA is very prominent in the substantia nigra. In itsrostral pole, scattered neurons are present in the parsreticulata. From its midrostral to midcaudal region, thepars reticulata contains heavy mRNA expression (Fig. 8C),falling off somewhat caudally, with scattered OFQ-containing neurons mostly in its dorsal part. The parscompacta contains lightly scattered, large, mRNA-contain-ing neurons in its rostral portion, with mRNA expressionbecoming quite intense into the caudal half of this region(Fig. 8D) and persisting into the pars lateralis.

Immunohistochemistry. In the rostral forebrain, mod-erately labeled neurons and occasional scattered fibers arenoted in the most rostral extent of the claustrum. Thispattern persists throughout the nucleus. The dorsal endop-iriform nucleus contains scattered, moderately labeledneurons and fibers, with most neurons residing medially.In the ventral endopiriform nucleus, immunolabeling con-sists of scattered neurons with no fibers. The striatumcontains sparse, darkly stained fibers noted throughout itsrostral-to-caudal extent. These diffuse fibers are observedequally in both colchicine-treated and untreated animals(Fig. 1A). Although these fibers can be seen in all regions ofthe striatum, there is a predominance in its ventromedialaspect. Occasional small, lightly immunolabeled neuronsare observed but are very few in number. At the emergenceof the globus pallidus, darkly immunolabeled neuronsappear and remain throughout its extent (Fig. 8A). Theseneurons, although they are not numerous, are large andintensely stained. Medial to the globus pallidus at morecaudal levels, large, darkly labeled neurons are observedthroughout the basal nucleus of Meynert (Fig. 8B). Thereis moderate fiber staining in this structure, with lightlylabeled puncta extending into the substantia innominata.The entopeduncular nucleus contains scattered, darklyimmunolabeled neurons with sparse fibers and terminals.

Immediately caudal to the entopeduncular nucleus,sparse, lightly labeled fibers and neurons are observed inthe subthalamic nucleus, confined primarily to its ventro-medial part. The pars reticulata of the substantia nigraemerges with numerous, moderately labeled perikaryaand fibers that are most numerous in its ventromedial partand that extend dorsally in caudal regions (Fig. 3C).Although the pars compacta has significantly fewer peri-karya than are seen in the pars reticulata, neurons areintensely immunolabeled. Caudally, the darkly stained,large neurons in the pars compacta extend dorsolaterallyinto the pars lateralis.

Basal telencephalon

In situ hybridization. Within the forebrain, mRNAexpression is highest within the basal telencephalon (Fig.9A). In the rostral bed nucleus of the stria terminalis,mRNA expression is observed throughout the entire me-dial and lateral parts. Heavy mRNA levels are seengenerally throughout the lateral division, with very lightmRNA expression observed in its juxtacapsular part andwith heavy expression in its dorsal and ventral parts. Themedial division has a moderate-to-strong mRNA expres-sion in its ventral part (Fig. 9B), increasing dramaticallyin its caudal aspect. Intensity of mRNA expression in theposterolateral part of the medial division is the strongestof the entire bed nucleus of the stria terminalis. IntensemRNA expression persists to the caudal pole of this


nuclear complex, surrounding the fornix in the anteriorhypothalamus. In this region, scattered, mRNA-express-ing neurons extend medially from this nucleus into theregion of the paraventricular nucleus of the hypothala-mus. No prepro-OFQ mRNA expression is observed in thefornix or the stria terminalis.

In the preoptic region, heavy mRNA expression is seenin the anteroventral preoptic nucleus, contrasting sharplywith that of the anteromedial preoptic nucleus, in whichmRNA levels are much lighter. The adjacent medial preop-tic area is filled with mRNA-containing neurons through-out its rostral-to-caudal extent (Fig. 9E), with expressionheaviest in its dorsal part rostrally and in the lateralaspect of this region more caudally. This expression pat-tern extends into the lateral preoptic area, where itremains strong. The medial preoptic nucleus contains adense accumulation of cells expressing OFQ mRNA (Fig.9E). Just caudal to the horizontal limb of the diagonalband, moderate mRNA levels are observed in neuronswithin the magnocellular preoptic nucleus. In the caudalbasal telencephalon, the nucleus of the lateral olfactorytract contains scattered neurons expressing mRNA. TheseOFQ-containing neurons are light to moderate in numberand are confined mostly to layer II, with an occasionalneuron observed in layer III. No OFQ mRNA expression isseen in the lateral olfactory tract.

Immunohistochemistry. Paralleling its mRNA ex-pression pattern, immunolabeling in this region is quite

intense. In the rostral bed nucleus of the stria terminalis,dense fibers, terminals, and immunolabeled neurons ex-tend from the septohypothalamic nucleus and fill theanterior and ventral parts of the medial division. Farthercaudally, a plexus of neurons, fibers, and puncta fill theposteromedial part of the medial division and most of theposterolateral part (Fig. 9C,D). This dense, immunola-beled plexus lies adjacent to the lateral ventricle andspreads ventrally toward the preoptic area. In the caudalposterolateral part of the medial division, a unique distri-bution pattern emerges. At the level of the anteriorhypothalamus, numerous darkly stained neurons sur-round the fornix. This cell group extends medially as acontinuum to the lateral boundary of the magnocellularpart of the paraventricular nucleus of the hypothalamus,remaining outside of its boundaries and extending into thelateral hypothalamic area. Fiber and terminal labeling inthe lateral subdivision is slightly more intense than in themedial subdivision at rostral levels, but drops off inposterior regions. The juxtacapsular part of the lateralsubdivision contains only occasional cell bodies with light-to-moderate fiber and terminal staining (Fig. 9C). Peri-karya are not observed in the posterior part of the lateraldivision, and fiber labeling drops off dramatically to itscaudal extent, where immunolabeling in the medial divi-sion is most intense. Fiber staining is prominent in thestria terminalis, particularly dorsally, adjacent to thelateral ventricle. Occasional darkly stained neurons are

Fig. 8. Photomicrographs of mRNA expression and immunolabel-ing in basal ganglia-related structures. A: Brightfield photomicro-graph of OFQ immunoreactivity in the globus pallidus demonstratingOFQ-immunoreactive fibers and cell bodies. B: Cell and fiber immuno-labeling in the basal nucleus of Meynert (colchicine treated). C,D: In

the substantia nigra, orphanin mRNA expression is heavy in midros-tral to caudal levels of the pars reticulata (C) and in the caudal part ofthe pars compacta (D). For abbreviations, see list. Scale bar 5 100 µmin A,B,D, 200 µm in C.


present in this structure as well. The fornix is devoid ofimmunolabeling.

The medial preoptic area contains scattered, darklystained neurons and moderate-to-heavy fibers throughoutits extent (Fig. 9F). Fewer immunolabeled neurons areseen in the medial preoptic nucleus and the magnocellularpreoptic nucleus, and only scattered, lightly stained cellsare seen in the anteromedial preoptic nucleus. A cluster of

darkly stained neurons is observed in the anteroventralpreoptic nucleus, continuing laterally with the caudal partof the horizontal limb of the diagonal band. Fiber andterminal labeling is very strong in the anteroventral andanteromedial nuclei and throughout the medial preopticnucleus. Immunolabeled fibers and terminals are sparse inthe magnocellular preoptic nucleus, which contains acluster of moderately immunolabeled neurons. The lateral

Fig. 9. Photomicrographs of OFQ mRNA and protein expression inthe medial preoptic area and the bed nucleus of the stria terminalis.A: Low-power image of the rostral basal telencephalon demonstratingstrong mRNA signal in the lateral septum, bed nucleus of the striaterminalis, and medial preoptic area. B: High-power photomicrographdemonstrating OFQ-containing neurons in the ventral part of themedial bed nucleus of the stria terminalis. Several neurons withrobust OFQ mRNA signal in the ventral diagonal band are alsopresent. Dashed line outlines the boundary of the anterior commis-sure. C: OFQ immunoreactivity in the dorsal part of the lateral bed

nucleus of the stria terminalis, the juxtacapsular part of the lateraldivision, and the anterior part of the medial division (colchicinetreated). D: OFQ-immunoreactive fibers and neurons in the posterolat-eral medial division of the bed nucleus (colchicine treated). E: Orpha-nin expression in the midpreoptic region demonstrates moderatemRNA signal intensity, with a cluster of intense mRNA signal in themedial preoptic nucleus. F: OFQ-containing fibers and light cells inthe medial preoptic area and the medial preoptic nucleus (untreated).For abbreviations, see list. Scale bar 5 850 µm in A, 200 µm in B,F, 100µm in C, 150 µm in D, 400 µm in E.


preoptic area contains scattered, darkly stained neuronsamong moderate fibers and light terminal labeling, mostlyin its medial aspect. Ventrally in this region, the nucleus ofthe lateral olfactory tract contains moderate numbers oflightly stained cells that are confined to layers II and III.Sparsely immunolabeled fibers and no puncta are ob-served in this nucleus.

Hypothalamus

In situ hybridization. In analyzing the nuclei of thehypothalamus by regions, based on a parcellation schemethat has been described previously by Swanson (1992), itbecomes evident that the majority of OFQ in the hypotha-lamic region lies primarily in the medial zone. In theventral hypothalamus, the suprachiasmatic and supraop-tic nuclei are devoid of mRNA expression. Farther cau-dally, scattered cells are observed in the tuber cinereum,with strong expression observed within the medial tuberalnucleus and dorsal into the magnocellular part of thelateral hypothalamus. Occasional scattered neurons con-taining OFQ mRNA are noted within the retrochiasmaticarea, just caudal to the suprachiasmatic nucleus. Orpha-nin mRNA expression is more moderate in the arcuatenucleus, persisting throughout its rostral-to-caudal ex-tent, with heaviest expression in more ventral regions. NomRNA expression is seen in the median eminence. Theventromedial hypothalamic nucleus is devoid of mRNAexpression, with only an occasional OFQ-containing neu-ron noted in the shell region.

In the dorsal region of the hypothalamus, there is adecrease in mRNA levels in the transition from the poste-rior bed nucleus of the stria terminalis into the anteriorand lateral hypothalamic areas rostrally, where expres-sion remains moderately strong. An occasional OFQ-containing neuron is observed in the lateroanterior hypo-thalamic area. A moderate cluster of mRNA-expressingcells is observed in the parvocellular part of the paraven-tricular nucleus. Although several orphanin-containingneurons are observed at the lateral border of this nucleus,the magnocellular part itself has only occasional, scatteredmRNA-expressing cells. Caudal to the paraventricularnucleus, cells expressing mRNA are lightly scattered inthe dorsal hypothalamic area, moderately scattered in thetuberal magnocellular nucleus, and numerous in the dorso-medial hypothalamic nucleus. Lateral to the dorsal hypo-thalamic area, the perifornical nucleus contains only scat-tered neurons expressing OFQ mRNA, with intensityincreasing into the posterior hypothalamus. At the mid-line, the periventricular nucleus contains scattered mRNA-expressing neurons, persisting to its caudal extent. Thereis no mRNA expression detected in the terete hypotha-lamic nucleus.

In the mammillary region, OFQ mRNA expression in theventral premammillary nucleus is moderate and increasesinto the dorsal premammillary region. The supramammil-lary (Fig. 11A) and lateral mammillary nuclei containmoderate mRNA expression throughout their rostral-to-caudal extents. In contrast, the medial mammillary nucleushas very low mRNA levels and is devoid of mRNA expres-sion in its lateral and medial divisions. The mediandivision of the medial mammillary nucleus contains onlylightly scattered mRNA-expressing cells. The mammillarypeduncle and supramammillary decussation are devoid ofmRNA expression. The pituitary (Fig. 11C) and pineal

(Fig. 11D) glands also are devoid of OFQ mRNA expres-sion.

Immunohistochemistry. A cluster of large, darklystained neurons fills a region just lateral to the paraven-tricular nucleus. These large, OFQ-containing neuronsextend laterally and surround the fornix in the posterolat-eral part of the medial division of the bed nucleus of thestria terminalis (Fig. 10C). The parvocellular part of theparaventricular nucleus contains a modest number oflightly stained neurons and fibers, mostly in its medialaspect, adjacent to the third ventricle. Scattered, darklystained fibers and occasional neurons are present alsowithin the magnocellular part of this nucleus (Fig. 10C).The lateroanterior hypothalamic nucleus, lateral hypotha-lamic area, and anterior hypothalamic area contain moder-ate numbers of large, OFQ-containing neurons. Caudally,this cell cluster extends into the tuber cinereum. In all ofthese regions, sparse, lightly stained fibers are present,except for the tuber cinereum, where moderate fiberstaining is observed in untreated animals. The supraopticand retrochiasmatic supraoptic nuclei contain no immuno-labeled neurons, but both regions contain darkly immuno-labeled fibers throughout their rostrocaudal extent. Noimmunolabeling is noted in the suprachiasmatic nucleus.Caudally, the retrochiasmatic area contains numerousdarkly labeled neurons and fibers.

Throughout the rostral half of the hypothalamus, theperiventricular nucleus contains sparse, moderatelystained neurons immediately adjacent to the third ven-tricle, with moderate fiber and terminal labeling. Thisfiber and terminal staining extends ventrally at its caudalpole into the arcuate region. The arcuate nucleus containsmoderately stained cells and fibers, with numerous punctathroughout its rostral-to-caudal extent (Fig. 10A,B). Orpha-nin-containing neurons in this nucleus are more numerousin its dorsomedial part, and fibers within the arcuateprimarily maintain a dorsal-to-ventral orientation. Themedian eminence contains an intense immunolabeledfiber plexus. Large, densely stained fibers fill the moremedial aspect of the median eminence in its rostral part.In the middle to caudal regions, median eminence fibersare labeled more lightly in its medial aspect and arelabeled intensely in the lateral aspects (Figs. 1B, 10A,B).

Lateral to the arcuate nucleus, lightly stained neuronsare present only occasionally in the ventromedial hypotha-lamic nucleus. In untreated animals, moderate fibers andterminals are localized diffusely throughout the nucleus.Farther lateral, lightly immunolabeled neurons are ob-served in the medial tuberal nucleus, where no fiber orterminal labeling is noted. Moderately stained cells andfibers also are scattered in the dorsal hypothalamic areaand the dorsomedial hypothalamic nucleus. Immunostain-ing in this region extends laterally into the perifornicalnucleus and persists caudally as lightly stained neurons inthe posterior hypothalamus. Moderately labeled neuronsare observed in the terete hypothalamic and tuberomam-millary nuclei, and lightly labeled neurons are observed inthe tuberal magnocellular nucleus.

In the mammillary region, moderate-to-heavy fiber andterminal labeling is present in the ventral and dorsalpremammillary nuclei, with only scattered, lightly stainedneurons observed. The medial mammillary nucleus con-tains light-to-moderate cell and fiber immunolabeling inits median division, whereas its medial and lateral divi-sions are devoid of staining . Neuronal labeling is most


pronounced in the lateral mammillary nucleus, whichcontains a dense plexus of darkly stained fibers and cellsthroughout (Fig. 10D). Moderate cell staining and strongfiber and terminal staining is observed in the supramam-millary nucleus (Fig. 11B). The mammillary peduncle andsupramammillary decussation are devoid of immunolabel-ing in this region.

AmygdalaIn situ hybridization. Orphanin mRNA expression

in the amygdaloid complex is relatively sparse in mostnuclei, except for the central and medial amygdaloidnuclei, which contain intense OFQ mRNA expression (Fig12A,C). Rostrally, the anterior amygdaloid area containsoccasional labeled neurons, particularly in its dorsal re-gion. At the emergence of the central nucleus, scatteredorphanin-containing neurons are observed in the lateraldivision, with the majority of mRNA expression confined tothe medial division more caudally. This mRNA expressionis moderate to heavy within the medial part of the centralnucleus to its caudal extent (Fig. 12C). Light mRNAexpression is observed in the basolateral and basomedialnuclei, increasing in the intraamygdaloid division of thebed nucleus of the stria terminalis. No mRNA expression isobserved in the intercalated or lateral amygdaloid nuclei.

The medial nucleus has moderate expression rostrally inits anterodorsal part, with minimal expression in the

anteroventral division. This sparse distribution extendsventrally into the bed nucleus of the accessory olfactorytract. In the caudal one-third of the medial nucleus, mRNAexpression increases dramatically. The posteroventral partof the nucleus contains moderate numbers of scatteredneurons expressing orphanin mRNA, whereas the pos-terodorsal nucleus is filled completely with positive neu-rons (Fig. 12A). This intensity increases into the caudalpole of the medial nucleus, where the posterodorsal divi-sion is packed with OFQ-containing neurons. Just caudalto the medial nucleus, the amygdalohippocampal areacontains only scattered neurons with light mRNA levels.Messenger mRNA levels in the anterior cortical amygda-loid nucleus are very light, and this pattern persistscaudally into the posterior cortical nucleus. The posterome-dial cortical nucleus contains light-to-moderate mRNAexpression. The posterolateral cortical amygdaloid nucleusand the amygdalopiriform transition area have no mRNAexpression.

Immunohistochemistry. OFQ peptide expression atthe emergence of the amygdaloid region is very sparse,with a scattering of fibers and puncta and with no immuno-labeled neurons in the anterior amygdaloid area. A moder-ate density of fibers and puncta reside in the rostralintercalated amygdaloid nuclei adjacent to the centralnucleus; then, immunolabeling falls off dramatically, with

Fig. 10. Brightfield photomicrographs of orphanin immunoreactiv-ity in the hypothalamus of colchicine-treated animals. A,B: Photomi-crographs of rostral and caudal levels of the arcuate nucleus andmedian eminence. Note orphanin-containing median eminence fiberslocated medially at rostral levels and laterally at caudal levels.C: Orphanin immunolabeling in the paraventricular nucleus demon-

strating labeled parvocellular neurons medially with scattered immu-nolabeled neurons in the magnocellular part and darkly labeledneurons just lateral to this nucleus. Dashed line outlines the boundaryof the paraventricular nucleus at that level. D: Immunolabeling in thelateral mammillary nucleus. For abbreviations, see list. Scale bar 5200 µm in A,B,D, 145 µm in C.


only sparse terminal staining in the more caudal nuclei.The central nucleus contains moderate-to-heavy fiber stain-ing in its rostral pole, with no immunolabeled cells pre-sent. The nucleus quickly fills with a dense plexus of fibers,terminals, and numerous immunoreactive neurons bymidcaudal levels. Heavy cell and fiber immunolabelingpredominates within the medial division, whereas thelateral division contains primarily fibers and has very fewimmunolabeled neurons (Fig. 12D). The medial amygda-loid nucleus presents a variable staining pattern from itsrostral to caudal poles. In its rostral pole, very lightlylabeled fibers are seen in the anteroventral division. At theemergence of the anterodorsal division, fiber stainingincrease significantly, and lightly stained immunolabeledcells are observed. Throughout the caudal one-third of thisnucleus, the posterodorsal division is filled with darklystained fibers, terminals, and OFQ-containing neurons(Fig. 12B). The posteroventral division contains scattered,lightly labeled cells and fibers in its core and is surroundedby a dense plexus of fibers and terminals (Figs. 1C, 12B).Lateral to this region, scattered, moderately immunola-beled cells and fibers are observed in the intercalatednuclei. The intraamygdaloid division of the bed nucleus ofthe stria terminalis contains light fiber and terminallabeling with scattered, lightly labeled cells (Fig. 12D).The bed nucleus of the lateral olfactory tract containsscattered, lightly stained neurons with moderately stained

fibers. Immunolabeling in the remaining amygdaloid nu-clei is sparse and includes lightly labeled, scattered cellsand fibers in the basomedial and basolateral nuclei (Fig.12D); light neurons and moderate fibers scattered through-out the posteromedial cortical nucleus; and light fibers inthe anteromedial cortical nucleus. The amygdalohippocam-pal and amygdalopiriform transition areas are devoid ofimmunolabeling.

Hippocampal formationand related structures

In situ hybridization. At the level of the septalnuclei, the septohippocampal nucleus contains moderatemRNA expression. Dorsal to the corpus callosum, a clusterof intense mRNA-expressing neurons resides in the indu-sium griseum, persisting throughout its rostral-to-caudalextent. Ventral to this structure, the subfornical organcontains numerous mRNA-expressing neurons. The fornixis devoid of mRNA expression. More caudally, the dentategyrus contains moderate numbers of prepro-OFQ-contain-ing neurons, primarily within the granule cell layer. Thereare occasional scattered neurons within the molecularlayer rostrally and within the polymorph layer caudally(Fig. 13A). The dentate hilus contains no mRNA expres-sion.

Fig. 11. A,B: Darkfield autoradiogram (A) and brightfield photomi-crograph (B) of the supramammillary nucleus of the hypothalamusillustrating distribution of the prepro-OFQ precursor mRNA and OFQpeptide in this nucleus. C,D: Darkfield photomicrographs demonstrat-

ing absence of OFQ mRNA expression in the anterior and intermedi-ate lobes of the pituitary (C) and pineal glands (D). For abbreviations,see list. Scale bar 5 200 µm in A,D, 100 µm in B, 400 µm in C.


In Ammon’s horn, the CA1 field contains strong mRNAexpression. Rostrally, it is primarily within the stratumpyramidal, with occasional mRNA-expressing neurons inthe stratum radiatum (Fig. 13A). There is no mRNAexpression detected in the stratum oriens. At more caudallevels, particularly in the ventral CA1 region, light mRNAexpression is also observed in the stratum oriens. Atrostral levels, little mRNA expression is detected in areaCA2. This is in sharp contrast to the heavy mRNA levelsobserved in the adjacent CA1 and CA3 regions. In thecaudal hippocampus, CA2 mRNA expression increasesslightly in the pyramidal layer. An occasional mRNA-expressing neuron is observed in the stratum radiatum ofarea CA2, but no mRNA expression is observed in thestratum oriens. Rostrally in the CA3 field of Ammon’shorn, there are numerous OFQ-containing neurons in thestratum lucidum extending into the hippocampal fieldfrom the stratum pyramidal, where scattered, mRNA-containing neurons are observed. This persists into thecaudal regions, where mRNA levels in the ventral CA3field decrease in the stratum lucidum and increase in thestratum pyramidal. Messenger RNA expression in thestratum radiatum of area CA3 is very light. In the dorsalsubiculum, moderate mRNA expression persists through-out, with most OFQ-containing neurons confined to thepyramidal layer. In the ventral subiculum, mRNA expres-sion is weak rostrally but increases in its more caudalaspect. Neurons expressing OFQ mRNA are numerous and

most dense in the pyramidal layer. Scattered neurons alsoare seen in the stratum radiatum and the stratum molecu-lar. In the caudal forebrain, mRNA expression is light inthe presubiculum and slightly heavier in the parasubicu-lum at all levels.

Immunohistochemistry. In the rostral forebrain, theindusium griseum is densely packed with dark, immunore-active fibers and cells. Ventral to this structure, stainingextends into the septohippocampal nucleus and the teniatecta (Fig. 6B). The subfornical organ contains moderatefiber labeling and scattered, lightly labeled cells. Thefornix is devoid of immunolabeling.

In the dorsal dentate gyrus, moderate, darkly stainedcells are scattered along the granule cell layer, withoccasional lightly stained cells in the polymorph andmolecular layers (Fig. 13B). These moderately sized cellslikely represent interneurons and are localized primarilyalong the inner border of the polymorph layer, with fibersextending perpendicularly into the molecular layer. In theventral dentate, neuronal immunolabeling becomes slightlymore pronounced within the polymorph layer. The alveuscontains no immunolabeling at all levels of the hippocam-pal formation. In the CA3 field of Ammon’s horn, moder-ately sized, darkly immunolabeled cells are observed inthe stratum pyramidal near the dentate hilus. In thisregion, moderately labeled fibers are seen extending intothe stratum lucidum and the stratum oriens (Fig. 13C).Approaching the transition to CA2, immunolabeled pyra-

Fig. 12. Photomicrographs of mRNA expression and immunolabel-ing in the amygdala. A: Prepro-OFQ mRNA expression in the caudalmedial nucleus. B: Brightfield photomicrograph of OFQ immunoreac-tivity in the posterodorsal and posteroventral parts of the medialnucleus (colchicine treated). C: OFQ mRNA expression in the central

nucleus. D: Immunolabeling in the medial and lateral divisions of thecentral nucleus, basolateral nucleus, and intraamygdaloid bed nucleusof the stria terminalis (colchicine treated). For abbreviations, see list.Scale bar 5 180 µm in A, 200 µm in B,C, 100 µm in D.


midal cells decrease in number, and numerous, darklylabeled neurons are observed in the stratum lucidum. Incaudal CA3, moderate numbers of intensely stained neu-rons reside in the stratum pyramidal, and slightly fewerare seen in the stratum lucidum (Fig. 13C). Neuronalimmunolabeling in the stratum radiatum is very sparsethroughout the CA3 region. Very little immunolabeling isobserved in area CA2 throughout its entire extent. Sparse,lightly labeled cells are seen in the stratum pyramidal.Moderately immunolabeled neurons are scattered scantlyin the stratum radiatum adjacent to the dentate molecularlayer. There is no fiber labeling noted in CA2. In area CA1,numerous darkly labeled neurons reside in the stratumpyramidal, with moderate fibers radiating into both thestratum oriens and the stratum radiatum near the dentatemolecular layer (Fig. 3A). In ventral CA1, there is adramatic decrease in stratum pyramidal neurons, with amoderate increase in orphanin-containing neurons in thestratum radiatum. Negligible fiber labeling is observed incaudal CA1. As CA1 transitions into the dorsal subiculum,there is a slight decrease in cell size, but the intensity ofneuronal labeling remains strong (Fig. 13D). Perikaryaare scattered throughout the dorsal region, with occa-sional scattered fibers also observed. Compared with itsdorsal part, OFQ immunolabeling is decreased in theventral subiculum. The presubiculum and parasubiculumcontain scattered, lightly labeled cells with no immunola-beled fibers. The lamina dissecans of the entorhinal cortexis devoid of labeling.

Thalamus

In situ hybridization. In the caudal basal telencepha-lon, moderate mRNA expression in the anteroventralthalamic nucleus is observed adjacent to the posterior bednucleus of the stria terminalis. At this level, strong mRNAexpression fills the paratenial nucleus and extends intothe anterior paraventricular and reuniens nuclei, wheremore moderate mRNA levels are observed. MessengerRNA expression in the paraventricular nucleus extendscaudal to the posterior division, remaining moderate intoits caudal pole. No OFQ mRNA expression is observed inthe anterodorsal, anteromedial, interanterodorsal, paracen-tral, or rhomboid thalamic nuclei. In the caudal one-thirdof the medial thalamic group, mRNA levels in the medialhabenula are very light. The lateral habenula containsslightly more mRNA expression than the medial habenula,which is most pronounced in its caudal one-third. Scat-tered neurons containing OFQ mRNA are observed in thelaterodorsal and mediodorsal thalamic nuclei. No mRNAexpression is observed in the centromedial, centrolateral,or lateroposterior thalamic nuclei, in the mammillotha-lamic tract, or in the stria medullaris.

Lateral to the midline thalamic group, the reticularnucleus contains the heaviest mRNA expression in theentire thalamic region (Fig. 14A). Messenger RNA-expressing neurons are moderate rostrally but becomevery intense in the middle one-third, filling the nucleusthroughout its rostral-to-caudal extent. Ventromedially,

Fig. 13. Photomicrographs of orphanin mRNA expression in un-treated animals and protein expression in colchicine-treated animalsin the hippocampal formation. A: Prepro-OFQ mRNA expression inthe dentate gyrus and CA1 region of Ammon’s horn. B: Brightfield

photomicrograph of OFQ immunoreactivity in the dentate gyrus.C: Higher power, detailed image of immunolabeling in CA3 area ofAmmon’s horn. D: Immunolabeling in the dorsal subiculum. Forabbreviations, see list. Scale bar 5 400 µm in A, 200 µm in B–D.


intense mRNA expression from the reticular nucleus ex-tends into the zona incerta, with high intensity in theventral zona incerta and lower intensity in its dorsaldivision. Occasional OFQ-containing neurons are observedin the subincertal nucleus and farther caudal in thesubgeniculate nucleus, just rostral to the peripeduncularnucleus in the midbrain. No mRNA expression is detectedin the fields of Forel or in the ventrolateral, ventromedial,ventral posterolateral, and ventral posteromedial tha-lamic nuclei.

In the midbrain thalamus, mRNA expression is confinedto scattered neurons in the rostral interstitial nucleus ofthe medial longitudinal fasciculus. This mRNA expressionextends laterally into the subparafascicular nucleus of thethalamus, where mRNA levels are moderate. This mRNAexpression remains strong throughout the parvocellularpart of this nucleus, with scattered mRNA-expressingneurons in the adjacent prerubral fields. Laterally, there isno mRNA expression detected in the medial geniculatenucleus, and only occasional mRNA-expressing neuronsare seen in the lateral geniculate. Adjacent to thesestructures, the suprageniculate thalamic nucleus containsa cluster of moderate mRNA-expressing cells, and theposterior thalamic group contains numerous, scattered,mRNA-containing neurons, particularly within the poste-rior limitans thalamic nucleus, where mRNA levels aremoderate. At the emergence of the mesencephalon, the

precommissural nucleus contains moderate mRNA expres-sion.

Immunohistochemistry. The anterior paraventricu-lar nucleus contains a moderate accumulation of terminalswith dispersed fibers and occasional lightly labeled neu-rons. The adjacent paratenial nucleus is filled with fineterminals and fibers and with no immunolabeled neurons.This fine fiber and terminal field spreads dorsolaterallyacross the paracentral nucleus and into the adjacentanterodorsal nucleus. Very dense terminal labeling is seenat this level in the interanterodorsal thalamic nucleus. Itpersists throughout the nucleus and does not extend intoadjacent regions. This immunolabeling pattern is observedin colchicine-treated and untreated animals. The antero-ventral nucleus contains occasional lightly labeled fiberswith no terminals. Lightly labeled fibers are scattered inthe reuniens and rhomboid nuclei at this level, but noimmunolabeled perikarya are identified. The stria medul-laris is devoid of immunolabeling. The reticular nucleuscontains numerous, intensely stained neurons in its dorsalaspect, with decreased numbers of immunoreactive neu-rons ventrally (Fig. 14B,C). There are lightly labeled,sparse fibers interspersed among the cells in the dorsalregion. This pattern of labeling persists to its caudalextent, with ventromedial extension of immunolabeledneurons into the zona incerta. In untreated animals, thezona incerta also contains moderate amounts of immunola-

Fig. 14. Photomicrographs of mRNA expression and immunolabel-ing in the thalamus. A: Low-power, darkfield autoradiogram ofprepro-OFQ mRNA expression in the reticular nucleus of the thala-mus. B: Low-power image of immunolabeling at a similar level of thereticular nucleus (colchicine treated). C: High-power, detailed view of

immunostaining in the reticular nucleus. D: Brightfield photomicro-graph from the brainstem thalamus illustrating fiber and cell stainingin the subparafascicular nucleus. For abbreviations, see list. Scalebar 5 360 µm in A, 400 µm in B, 50 µm in C, 100 µm in D.


beled fibers and puncta. No immunolabeling is observed inthe adjacent prerubral field and the nuclear fields of Forel.Dorsal to these structures, there is prominent cell, fiber,and terminal labeling in the subparafascicular thalamicnucleus (Fig. 14D).

In the caudal thalamus, a dense, immunoreactive fiberbundle courses through the medial habenula. Medial tothis fiber bundle, scattered, darkly stained neurons areobserved. Staining in the lateral habenula is confined toscattered, lightly labeled terminals. This light terminalstaining extends medioventrally into the posterior paraven-tricular nucleus, where lightly labeled terminals, fibers,and scattered immunolabeled neurons are observed to inits caudal pole. Here, a dramatic increase in terminallabeling occurs in the transition into the precommissuralnucleus, where moderate immunolabeled fibers and cellsare observed. The remainder of the thalamus is relativelydevoid of immunolabeling. The anteromedial, central me-dial, laterodorsal, mediodorsal, and centrolateral nucleiand the ventral nuclear group contain no immunolabeling.Caudally, there is light staining noted in the posteriorthalamic nuclear group, the lateral posterior nucleus, thesubgeniculate nucleus, and the lateral and medial genicu-late bodies. Adjacent to these nuclei, the suprageniculatethalamic nucleus and posterior limitans thalamic nucleuscontain lightly stained neurons, with no fiber or terminallabeling noted.

Mesencephalon

In situ hybridization. Messenger RNA expression inbrainstem thalamic and basal ganglia structures is de-scribed above. In the rostral ventral midbrain, moderatemRNA expression is observed initially within the rostrolat-eral subdivision of the interpeduncular nucleus. Messageexpression intensifies into the lateral and dorsal divisionsand remains strong (Fig. 15A). The mammillary peduncle,fasciculus retroflexus, rubrospinal tract, and medial andlateral lemniscus are devoid of mRNA expression at thislevel. The rostral ventral tegmental area contains scat-tered neurons expressing OFQ mRNA. These levels in-crease substantially in the ventral tegmental area proper,where moderate-to-heavy mRNA expression is localizedventromedially and extends into its dorsolateral part. Theparanigral nucleus contains intense expression through-out its extent (Fig. 15C). No expression is observed in theprerubral field or parabrachial pigmented nucleus.

In the rostral dorsal midbrain, the olivary pretectalnucleus contains no mRNA expression, with moderateexpression in the ventral anterior pretectal region. Moder-ate mRNA expression is seen also in the nucleus of theoptic tract and in the posterior pretectal nucleus, and acluster of intense mRNA expression is observed in thenucleus of the posterior commissure, with lighter mRNAexpression into its magnocellular part. No mRNA-express-

Fig. 15. Photomicrographs of mRNA expression and immunolabel-ing in the ventral rostral mesencephalon. A: Prepro-OFQ mRNAexpression in the interpeduncular nucleus. B: Brightfield photomicro-graph of OFQ protein expression in the interpeduncular nucleusdemonstrates immunolabeled neurons that are concentrated mostly in

its rostral division (colchicine treated). C: High-powered view ofmRNA-expressing neurons in the ventral tegmental area at the leveldepicted in A (arrows). D: Scattered, immunolabeled neurons in theventral tegmental area (colchicine treated). For abbreviations, see list.Scale bar 5 200 µm in A, 100 µm in B–D.


ing neurons are observed in the posterior commissure or inthe commissure of the superior colliculus. In the superiorcolliculus, mRNA expression is sparse in the superficialgray and is distributed more moderately in the optic andintermediate gray layers. Other than occasional scatteredneurons within the deep gray layer, no other layer of thesuperior colliculus contains OFQ mRNA expression. In theinferior colliculus, the external cortex contains numerousmRNA-expressing neurons in layers II and III. Occasionalscattered neurons are observed in layers II and III of thedorsal cortex of the inferior colliculus, with negligiblemRNAexpression observed in the central collicular nucleus,the intercollicular nucleus, and the commissure of theinferior colliculus.

Laterally in the rostral midbrain, heavy mRNA expres-sion in the substantia nigra, pars compacta, persists intothe pars lateralis and intensifies farther away at themedial edge of the peripeduncular nucleus, which containsintense expression throughout its extent (Fig. 2E). Theretrorubral field contains moderate numbers of mRNA-containing neurons. The brachium of the inferior colliculusand its nucleus contain no mRNA expression, whereaslightly scattered neurons are present within the sub-brachial nucleus. Negligible mRNA expression is observedin the cuneiform nucleus; in the dorsal, intermediate, andventral nuclei of the lateral lemniscus; in the mesence-phalic trigeminal nucleus; in the paralemniscal nucleus;and in the retrorubral nucleus.

In the midline of the rostral midbrain, the nucleus ofDarkschewitsch is filled completely with the most intensemRNA expression in the mesencephalon. OFQ-expressingneurons fill this nucleus throughout its rostral-to-caudalextent and extend ventrolaterally into the interstitialnucleus of the medial longitudinal fasciculus, where mRNAexpression is quite strong (Fig. 16C). Dorsal to the nucleusof Darkschewitsch, the central gray also contains verystrong expression, which is more prominent in the ventralpart of the nucleus, spreading to its lateral borders (Fig.16A). There is a dramatic drop in mRNA intensity in thedorsal central gray, which contains only moderate mRNAlevels. Scattered mRNA-expressing neurons are present inits pontine division. Ventral to the central gray, theEdinger-Westphal and medial accessory oculomotor nucleiare conspicuously devoid of mRNA expression (Fig. 16C).In contrast, the supraoculomotor central gray is moder-ately labeled with OFQ-containing neurons. Farther cau-dally, the raphe nuclear complex contains prominent mRNAexpression. The rostral linear raphe nucleus containsmoderate mRNA expression. Just posterior to this region,in the caudal linear raphe nucleus, light to moderatemRNA levels are observed. The dorsal raphe emerges withmoderate mRNA expression, which persists throughout itsrostral-to-caudal extent. Numerous mRNA-containing neu-rons are observed also throughout the caudal linear rapheventrally, with extension caudally into the median raphe(Fig. 18A). The paramedian raphe contains little mRNA

Fig. 16. Messenger RNA expression and immunolabeling in theperiaqueductal region of the mesencephalon. A: mRNA-expressingneurons in the ventral central gray. B: Brightfield photomicrograph ofintensely immunolabeled neurons in the ventral division of the centralgray. C: Robust orphanin mRNA expression in the nucleus of Darks-

chewitsch and the interstitial nucleus of the medial longitudinalfasciculus. D: Intense orphanin immunolabeling in the interstitialnucleus of the medial longitudinal fasciculus and the nucleus ofDarkschewitsch (colchicine treated). For abbreviations, see list. Scalebar 5 200 µm in A,C,D, 100 µm in B.


expression (Fig. 18A), and the paratrochlear nucleus con-tains no mRNA expression.

In the remainder of the midbrain, scattered, mRNA-expressing neurons are observed within the deep mesence-phalic nucleus and in the magnocellular and parvocellularparts of the red nucleus. The central trigeminal tract,dorsal longitudinal fasciculus, rhabdoid nucleus, superiorcerebellar peduncle, microcellular, subpeduncular and ven-trolateral tegmental nuclei, sensory root of the trigeminalnerve, reticulotegmental nucleus, and rostral pontine nu-clei are devoid of mRNA expression. Moderate mRNAexpression is observed in the pontine reticular nucleus, inits oral and caudal parts, extending laterally into thepedunculopontine tegmental nucleus, where mRNA levelsincrease. Moderate mRNA expression is observed also inthe dorsal, laterodorsal, and ventral tegmental nuclei. Thenucleus of the trapezoid body contains heavy mRNAexpression at the emergence of the ventral metencephalon.

Immunohistochemistry. Immunohistochemical stain-ing in brainstem thalamic and basal ganglia structures isdescribed above. Rostrally, the interpeduncular nucleuscontains strong immunolabeling. There are numerousimmunolabeled neurons in the rostral division (Fig. 15B),and only lightly labeled, scattered neurons are found in thecaudal and lateral divisions. The dorsal division containsdarkly labeled neurons with moderate fibers and termi-

nals that extend into the ventral tegmental area. Noimmunolabeling is observed in the prerubral field, andlightly scattered OFQ-containing fibers are seen in theparvocellular part of the red nucleus. Fibers extend to themagnocellular part, where lightly stained, immunolabeledneurons are seen. The retrorubral field contains light cellimmunolabeling, with scattered fibers in its ventrolateralaspect. This pattern extends caudally and ventrolaterallyinto the pedunculopontine tegmental nucleus, where darklystained neurons are present. The rostral ventral tegmen-tal area contains numerous moderately stained neuronsand lightly stained fibers scattered throughout. This stain-ing pattern intensifies caudally into the ventral tegmentalarea proper (Fig. 15D), where scattered, darkly labeledneurons and fibers extend from the dorsolateral interpe-duncular nucleus into this region. Scattered, darkly stainedneurons spread from the lateral part of the ventral tegmen-tal area into the paranigral nucleus, where moderatelystained neurons and scattered fibers are observed. Cau-dally, immunolabeled neurons in the substantia nigra,pars lateralis, extend to the peripeduncular nucleus, wherenumerous, intensely stained neurons fill the region. Mod-erate fiber staining also fills this nucleus and is observedbest in untreated animals. Moderate fibers are observedalso in the medial lemniscus and dorsally in the region of

Fig. 17. Photomicrographs of orphanin mRNA and protein expres-sion in the dorsal metencephalon. A: mRNA-expressing neurons arenumerous in the locus coeruleus, whereas the adjacent mesencephalictrigeminal nucleus is devoid of mRNA expression. B: Brightfieldphotomicrograph of OFQ immunoreactivity in the locus coeruleus.Again, note the lack of immunolabeling in the adjacent mesencephalictrigeminal nucleus in this colchicine-treated animal. C: Lightly immu-

nolabeled neurons and fibers in the dorsal raphe with intense immuno-labeling in the dorsal tegmental and laterodorsal tegmental nuclei(colchicine treated). D: Moderate fiber and terminal labeling in thelateral parabrachial nucleus (colchicine treated). Scattered, lightlyimmunolabeled neurons also are evident. For abbreviations, see list.Scale bar 5 100 µm.


the deep mesencephalic nuclei. The cerebral and mammil-lary peduncles are without immunostaining.

In the dorsal region of the rostral midbrain, severalmoderately immunostained neurons are observed in thenucleus of the posterior commissure, the posterior pretec-tal nucleus, the olivary pretectal nucleus, and the nucleusof the optic tract. Lightly labeled, scattered neurons areobserved in the magnocellular nucleus of the posteriorcommissure. There is moderate to heavy neuronal labelingwith light fiber labeling in the dorsal and ventral divisionsof the anterior pretectal nucleus. Lightly labeled, OFQ-containing fibers also are seen in the nucleus of the optictract. The posterior commissure, the commissure of thesuperior colliculus, and the central tegmental tract aredevoid of immunolabeling. In the superior colliculus, thezonal layer contains occasional lightly labeled fibers but noimmunolabeled neurons. There are moderate numbers oflightly labeled neurons in the superficial gray layer thatincrease in number and intensity of staining in the opticnerve and intermediate gray layers, where labeled neu-rons are moderately stained. Although fibers are presentin these layers, they remain very sparse, even in untreatedanimals. No immunolabeling is observed in the intermedi-ate white, deep white, or deep gray layers of the superiorcolliculus or in the intercollicular nucleus. In the inferiorbrachium, scattered, moderately stained cells are observedprimarily in layers II and III of the external cortex. Thereare very few fibers, and no terminal labeling in this region.This pattern persists to the caudal extent of the external

cortex of the inferior colliculus. Occasional lightly labeledfibers and cells are observed in the dorsal cortex, and thecentral nucleus contains only an occasional scattered,lightly stained neuron. There is no immunolabeling in thebrachium of the inferior colliculus or in the commissure ofthe inferior colliculus. Within the nucleus of the brachiumof the inferior colliculus, moderately stained fiber pro-cesses are observed.

At the midline, numerous, intensely stained neurons,fibers, and terminals fill the ventral and lateral aspects ofthe central gray (Fig. 16B). The heaviest fiber and termi-nal immunolabeling is observed adjacent to the aqueduct,with only scattered, lightly labeled cells and fibers in thedorsal central gray. In caudal central gray regions, immu-nolabeling of neurons increases in its dorsal part. Ventro-lateral to the central gray rostrally, numerous, intenselyimmunolabeled cells with moderate fibers and terminalsfill the nucleus of Darkschewitsch (Fig. 16D). This intenseimmunolabeling persists to the caudal extent of thisnucleus. Strongly immunolabeled neurons and fibers ex-tend ventrolaterally to the interstitial nucleus of themedial longitudinal fasciculus (Fig. 16D). Lightly scat-tered cells are observed also in the deep mesencephalicnuclei of the midbrain. No immunolabeling is observed inthe Edinger-Westphal nucleus, the medial accessory oculo-motor nucleus, the supraoculomotor central gray, thedorsal longitudinal fasciculus, or the superior cerebellarpeduncle. Ventral to the central gray, several lightlyimmunolabeled neurons with scattered fibers reside in the

Fig. 18. Photomicrographs of mRNA expression and immunolabel-ing in brainstem raphe regions. A: mRNA-expressing neurons arescattered in the median and paramedian raphe nuclei. B: Immunola-beled neurons and fibers in the median and paramedian raphe nuclei.

C: mRNA expression in the raphe magnus and raphe pallidus nucleus.D: Darkly immunolabeled neurons in the raphe magnus and raphepallidum. For abbreviations, see list. Scale bar 5 100 µm in A,B, 200µm in C,D.


dorsal raphe (Fig. 17C). This labeling pattern extendsalong the midline into the median raphe. The adjacentparamedian raphe also contains scattered, darkly stainedcells (Fig. 18B). Scattered neurons with moderate stainingare observed in the rostral linear raphe. This patterncontinues into the caudal midbrain, with scattered darkneurons and lightly labeled fibers in the caudal linearraphe. Scattered fibers and cells also are observed in theanterior (Fig. 18B) and ventral tegmental nuclei. Thelaterodorsal and dorsal tegmental nuclei contain numer-ous, darkly labeled neurons and fibers (Fig. 17C). Thereticulotegmental, subpeduncular, and ventrolateral teg-mental nuclei are devoid of labeling.

In the caudal mesencephalon, the pontine reticularnucleus contains scattered, moderately stained neuronsand lightly labeled fibers in its oral part. This labelingextends laterally into the paralemniscal nucleus. No immu-nolabeling is observed in the lateral lemniscus or in theventral, dorsal, or lateral nuclei associated with it. Thecuneiform nucleus, mesencephalic trigeminal nucleus,paratrochlear nucleus, parabigeminal nucleus, longitudi-nal fasciculus, rubrospinal tract, and sensory root of thetrigeminal nerve are devoid of immunolabeling. Ventrolat-erally at this level, scattered, lightly immunolabeled fibersare present in the pontine nuclei, and caudally, moderatenumbers of modestly stained neurons lie in the nucleus ofthe trapezoid body and the in the rostral preolivary region,entering the rostral metencephalon.

Metencephalon

In situ hybridization. In the dorsal rostral pons, thelocus coeruleus contains moderate mRNA expression (Fig.17A) with light to moderate levels in the pontine centralgray and adjacent dorsal raphe. Moderate to intensemRNA expression is observed in the dorsomedial tegmen-tal area and the laterodorsal tegmental nucleus, withincreased mRNA expression caudally in the posterodorsaltegmental nucleus. The lateral parabrachial nucleus con-tains numerous, scattered, OFQ-containing neurons,whereas the medial parabrachial nucleus is devoid ofmRNA expression. Negligible expression is observed in thesubcoeruleus nucleus, principal sensory trigeminal nucleus,Kolliker-Fuse nucleus, trigeminal motor nucleus, anteriorventral cochlear nucleus, medial longitudinal fasciculus,mesencephalic trigeminal nucleus (Fig. 17A), and superiorcerebellar peduncle.

In the ventral rostral pons, the nucleus of the trapezoidbody contains intense mRNA expression, with light-to-moderate expression in the adjacent superior paraolivarynucleus (Fig. 19A). Moderate-to-strong mRNA expressionis observed in the medioventral periolivary nucleus, withscattered neurons observed in the dorsal, lateroventral,and superior periolivary nuclei and in the lateral superiorolive (Fig. 19B). No mRNA expression is observed in thecaudal periolivary nucleus or medial superior olive. Scat-tered neurons with intense mRNA levels reside in theregion of the A5 cell group (Fig. 19A), whereas no OFQmRNA is detected in the A7, C1, C2, or C3 cell groups. Thepyramidal tract and the root of the abducens nerve containno mRNA expression.

In the caudal pons, no mRNA expression is seen in thesixth, seventh, or eighth nerves or in the associatedabducens and facial nuclei. Laterally, the paratrigeminalnucleus contains a cluster of moderate mRNA-expressingneurons. In the vestibular complex, light to moderate

mRNA expression is scattered throughout the superiorand medial vestibular nuclei, primarily in their dorsalparts. Messenger RNA levels are slightly higher in thelateral vestibular nucleus, mostly in its lateral part.Moderate mRNA expression is observed in the caudal halfof the medioventral vestibular nucleus, and scatteredmRNA-expressing cells are observed in the spinal vestibu-lar nucleus and dorsal cochlear nucleus. The ventral

Fig. 19. Photomicrographs of mRNA expression and immunolabel-ing in the in the caudal metencephalon. A: mRNA expression in thesuperior paraolivary nucleus. Note scattered mRNA-expressing neu-rons within the region of the A5 cell group. B: Higher power view ofscattered mRNA-expressing neurons in the medioventral and latero-ventral periolivary nuclei. C: Brightfield photomicrograph of moder-ately immunolabeled neurons in the ventral part of the medialvestibular nucleus (colchicine treated). For abbreviations, see list.Scale bar 5 200 µm in A,B, 100 µm in C.


posterior cochlear nucleus and inferior cerebellar peduncleare devoid of mRNA expression. In the dorsal caudal pons,the abducens nucleus has no mRNA expression, but theadjacent supragenual nucleus contains moderate mRNAexpression. This persists caudally into the prepositushypoglossus, where intense mRNA expression is observedthroughout its extent (Fig. 20A). In the reticular forma-tion, scattered neurons expressing OFQ mRNA are ob-served in the parvocellular, intermediate, and dorsal para-gigantocellular reticular nuclei. Large neurons with

moderate intensity are observed in the gigantocellularnucleus and its alpha subdivision. Only sparse mRNAexpression is observed in the lateral paragigantocellularnucleus in the pons, intensifying in the medulla (Fig. 20E).

In the pontine raphe complex, modest mRNA expressionin the dorsal raphe persists caudally in its dorsal exten-sion, whereas high mRNA expression is observed in thepontine raphe nucleus. Moderate mRNA expression isobserved in the oral pontine reticular nucleus and becomesmore sparse into the caudal pontine reticular nucleus. In

Fig. 20. Photomicrographs of OFQ mRNA and protein expressionin the mylencephalon. A: Intense mRNA expression in the prepositushypoglossus. Dashed lines outline the boundary of the 4th ventricle.B: Brightfield photomicrograph of darkly immunolabeled neurons inthe prepositus hypoglossus. C: mRNA-expressing cells in the medialand lateral divisions of the midcaudal solitary nucleus. D: Immunola-beling in the medial and lateral divisions of the solitary nucleus

(colchicine treated). E: mRNA-expressing neurons in the medullarylateral paragigantocellular reticular nucleus. F: Immunolabeling inthe inferior olivary nucleus demonstrating scattered cells, fibers, andterminals in the medial, dorsal, and principal divisions (colchicinetreated). For abbreviations, see list. Scale bar 5 200 µm in A, 100 µmin B–F.


the caudal pons, intense mRNA expression is observed inthe raphe magnus and raphe pallidus (Fig. 18C), but ismuch decreased in the ventral pontine reticular nucleus.The spinal trigeminal nuclear complex has a varied mRNAexpression pattern, from rostral to caudal. Rostrally, theprincipal sensory trigeminal nucleus is devoid of mRNAexpression; however, in the caudal pons, the oral andinterpolar parts of the spinal trigeminal nucleus contain

scattered, mRNA-containing neurons. Messenger RNAexpression does not increase until the emergence of thecaudal spinal trigeminal nucleus in the medulla (Fig. 21A).

Immunohistochemistry. Immunolabeling in the dor-sal part of the rostral pons is generally sparse. Themesencephalic trigeminal nucleus and subcoeruleus con-tain no immunolabeling. The locus coeruleus containsmodest numbers of darkly stained neurons and lightly

Fig. 21. Photomicrographs of mRNA expression and immunolabel-ing in the caudal medulla and spinal cord. A: mRNA-expressingneurons in the caudal spinal trigeminal nucleus with extensionmedially into the dorsal medullary reticular nucleus and ventrallyinto the retroambiguous nucleus. B: Brightfield photomicrograph ofimmunolabeling within the caudal part of the spinal trigeminalnucleus demonstrating heavily immunolabeled fibers and terminalswith scattered, intensely labeled neurons. Note that immunolabelingis confined primarily to the lateral part of the nucleus (colchicine

treated). C: mRNA expression in the dorsal horn of the cervical spinalcord demonstrating heavy expression within laminae II and III.D: Immunolabeling in the dorsal horn demonstrating terminals andscattered neurons within laminae II and III (colchicine treated).E: mRNA-expressing neurons in lamina X and in the intermediome-dial cell column of the thoracic spinal cord. F: Immunolabeled fibersand cells within the lateral spinal nucleus (colchicine treated). Forabbreviations, see list. Scale bar 5 200 µm in A,C, 100 µm in B,E,F,50 µm in D.


labeled terminals (Fig. 17B). Scattered, lightly labeledneurons are present in the pontine central gray and in thelaterodorsal and posterodorsal tegmental nuclei. The dor-somedial tegmental nucleus contains a cluster of moder-ately immunolabeled neurons with scattered fibers. In thelateral region of the dorsal metencephalon, moderate celland light fiber immunostaining is present in the lateralparabrachial nucleus (Fig. 17D). The superior cerebellarpeduncle, medial parabrachial nucleus, A7 noradrenalinecell group, and Kolliker-Fuse nucleus are devoid of immu-nolabeling.

In the ventral rostral pons near the midline, the nucleusof the trapezoid body is filled with darkly immunolabeledneurons and few lightly labeled fibers. The lateral rostralperiolivary region contains an occasional darkly labeledneuron but is filled mostly with moderately immunola-beled fibers and terminals, which are seen well in un-treated animals. Moderately immunolabeled cells andfibers are observed in the superior periolivary nucleus,lateroventral periolivary nucleus, medioventral perioli-vary nucleus, and lateral superior olive. The medial supe-rior olive is devoid of immunolabeling. Cell immunolabel-ing is present in the lateral and dorsal superior olive but issignificantly less than that observed in the periolivarynuclei. Occasional lightly labeled fibers and no cells residein the caudal periolivary nucleus. Scattered, lightly immu-nolabeled neurons are observed in the region of the A5noradrenaline cell group. Immunolabeling was not ob-served in the C1, C2, or C3 adrenaline cell groups.

In the caudal pons, lightly immunolabeled cells andfibers are observed primarily extending through the me-dial, lateral, and superior vestibular nuclei. Fiber stainingremains light throughout, but cells become more numer-ous and darkly stained caudally in the medial nucleus andlaterodorsal part of the lateral vestibular nucleus. In themidcaudal region of the superior vestibular nucleus, acluster of intense immunoreactivity is observed just adja-cent to the inferior cerebellar peduncle. This extends aslight immunolabeling into the dorsal cochlear nucleus. Atcaudal levels, more immunolabeled neurons are observedin the superior, lateral, and ventral medial vestibularnuclei (Fig. 19C), with decreased labeling in the medialvestibular region. Scattered, darkly stained neurons areobserved in the spinal vestibular nucleus, continuing intoits caudal pole at the emergence of the solitary nucleus.The anterior and posterior ventral cochlear nuclei aredevoid of labeling.

In the pontine raphe complex, the dorsal raphe containsscattered, moderately immunolabeled cells and fibers,which diminish significantly in the region of the dorsal andlaterodorsal tegmental nuclei (Fig. 17C). Neuronal immu-nolabeling increases significantly ventral and caudal intothe pontine raphe nucleus and raphe magnus (Fig. 18D).In the reticular formation, darkly immunolabeled peri-karya are scattered throughout the gigantocellular, lateralparagigantocellular, and lateral reticular nuclei. No immu-noreactive fibers or terminals are observed in these re-gions. The parvocellular reticular nucleus contains scat-tered, lightly labeled fibers but is devoid of immunoreactivecells. Few modestly labeled neurons are present in thedorsal gigantocellular, caudal pontine, and lateral reticu-lar nuclei. Cranial nerve nuclei and fibers are relativelyunlabeled in the pontine region. The oral trigeminalnucleus does contain an occasional scattered fiber but hasno terminal or neuronal immunolabeling. The remainder

of the pontine cranial nerves and associated nuclei aredevoid of immunolabeling. Other regions with no detect-able immunolabeling include the inferior cerebellar pe-duncle, the medial longitudinal fasciculus, the pyramidaltract, the sensory root of the trigeminal nerve, the motorand principal sensory nuclei of the trigeminal nerve, andthe rostral and superior paraolivary nuclei.

MylencephalonIn situ hybridization. In the cerebellar hemispheres,

preproorphanin mRNA expression is confined to scatteredneurons in the granular layer. No mRNA expression isdetected within the molecular or Purkinje cell layers of anycerebellar lobule. Within the deep cerebellar nuclei, moder-ate numbers of neurons expressing OFQ mRNA are ob-served in the medial (fastigial) cerebellar nucleus, withslightly less intensity in its dorsolateral protuberance. NomRNA expression is detected in the lateral (dentate) orinterposed cerebellar nuclei.

Scattered OFQ-containing neurons are occasionally ob-served in the external cuneate nucleus, with the cuneatenucleus and nucleus gracilus both devoid of mRNA expres-sion. There is no OFQ mRNA expression detected in theninth or twelfth cranial nerves, in the dorsal motornucleus of the tenth nerve, or in the hypoglossal nucleus.The prepositus hypoglossus contains intense mRNAexpres-sion, which is observed initially in the caudal pons, persist-ing throughout its extent into the medulla (Fig. 20A). Thenucleus ambiguous is filled with intense mRNA expressionthroughout its extent and at its caudal boundary. Messen-ger RNA expression becomes slightly more intense into theretroambiguous nucleus in the caudal medulla (Fig. 21A).The rostral solitary nucleus emerges with moderate Mes-senger RNA expression. This persists in the medial andlateral subdivisions, with mRNA levels increasing cau-dally. In the caudal extent of the solitary nucleus, mRNAexpression is very strong in its lateral and medial divisionsand is moderate in the commissural division (Fig. 20C).Located within the solitary nuclear complex, the solitarytract contains no mRNA expression.

In the ventral medulla, the inferior olivary complexcontains weak OFQ mRNA expression at its rostral bound-ary. The medial and dorsomedial nuclei are devoid ofmRNA expression, with scattered mRNA-containing neu-rons observed in its principal and dorsal nuclei as well asin subnuclei A and B of the medial subdivision. In thereticular formation, light-to-moderate mRNA expressionis seen in the gigantocellular, gigantocellular alpha, inter-mediate, and parvocellular reticular nuclei, and moreintense expression is seen in the lateral paragigantocellu-lar nucleus (Fig. 20E). The adjacent paramedian reticularnucleus contains an occasional positive neuron but other-wise is devoid of mRNA expression. Caudally, the lateralreticular and paragigantocellular nuclei and the dorsaland ventral medullary reticular fields contain scattered,large neurons with strong mRNA expression. At the mid-line, the caudal interstitial nucleus of the medial longitudi-nal fasciculus contains intense mRNA expression, particu-larly in its ventral aspect. Orphanin mRNA expressionpersists in this nucleus to its caudal extent, extending tothe raphe obscurus. In the caudal part of the raphecomplex, the raphe magnus contains numerous intenselylabeled neurons, with a moderate number of OFQ-containing neurons in the raphe pallidus nucleus to itscaudal extent (Fig. 18C).


In the caudal mylencephalon, very strong mRNA expres-sion is noted in the spinal trigeminal nucleus. MessengerRNA expression is localized primarily in the lateral aspectof this nucleus. Very strong mRNA expression extendsdorsally into the dorsomedial spinal trigeminal nucleusand ventrally into the nucleus ambiguous and retroambigu-ous nucleus caudally (Fig. 21A). Messenger RNA levelsremain moderate to strong throughout the dorsomedialnucleus. Continuing into the transition into the cervicalspinal cord, mRNA intensity in the caudal spinal trigemi-nal nucleus is the most intense in the mylencephalon.

Immunohistochemistry. No specific orphanin immu-noreactivity was observed in the cerebellar hemispheres,as stated above. A dense plexus of cells and fibers isobserved in the fastigial (medial) nucleus, with moderateto heavy immunolabeling. This labeling drops off slightlyinto its dorsolateral protuberance but is still quite strongto its caudal extent. No immunolabeling is observed in thedentate (lateral) or interposed cerebellar nuclei or any oftheir corresponding parts.

Immunolabeling within the cranial nerve nuclei of themedulla is relatively sparse. There is no immunolabelingobserved the in ninth, tenth, and twelfth cranial nerves orin their associated nuclei. Throughout its extent, thenucleus ambiguous contains a cluster of darkly immunola-beled neurons with moderate fiber staining. This immuno-labeling remains confined to this nucleus and persists tothe retroambiguous nucleus at the level of the pyramidaldecussation. The retroambiguous nucleus contains numer-ous, densely immunolabeled, OFQ-containing neurons.Very strong neuronal immunolabeling is also observed inthe prepositus hypoglossus, with dark immunoreactivefibers visualized (Fig. 20B). At this level, the caudalinterstitial nucleus of the medial longitudinal fasciculuscontains moderate numbers of labeled neurons and fibers.

In the dorsal medulla, the nucleus of the solitary tract isstrongly immunolabeled. The inclusive solitary tract itselfis devoid of immunolabeling. A cluster of numerous, lightlyimmunolabeled cells with dark fibers and terminals is seenin the rostral solitary nucleus. At more caudal levels,immunolabeling in the solitary nucleus diverges slightly,with heavier terminal and fiber labeling in the medial andlateral parts, and with heavier cell staining in the medialand commissural parts (Fig. 20D). In this dorsal region,darkly stained neurons are observed in the supragenualnucleus medially, and the cuneate and external cuneatenuclei and nucleus gracilus are devoid of immunolabeling.Ventral to the solitary nucleus, darkly labeled neurons andfibers are scattered throughout the reticular formation.Scattered, moderately labeled neurons are observed in thecaudal gigantocellular reticular nucleus alpha and thelateral paragigantocellular nucleus. The inferior olivecontains numerous darkly immunolabeled cells and scat-tered fibers in its principal nucleus. There are also lightlystained cells and scattered fibers within the anterior,medial, and dorsal nuclei (Fig. 20F). The dorsomedialdivision is without immunolabeling. The raphe nuclei inthis ventral medullary region contain scattered, moder-ately labeled neurons into the caudal medulla. This OFQimmunoreactivity is primarily in the raphe magnus andpallidus (Fig. 18D), with scattered, lightly labeled fibersand neurons in the midline raphe obscurus.

In the lateral medulla, the interpolar spinal trigeminalnucleus contains sparse fiber labeling and no cell staining.This persists until the emergence of the caudal spinal

trigeminal nucleus, which emerges with a heavily immuno-labeled fiber and terminal field, interspersed with numer-ous darkly labeled neurons (Fig. 21B). Similar to mRNAexpression, immunolabeling extends dorsally into the dor-somedial spinal trigeminal nucleus, which contains moder-ate cell and fiber staining, and ventrally into nucleusambiguous, where intense neuronal immunolabeling isobserved. The pattern of OFQ immunoreactivity in thecaudal spinal trigeminal nucleus persists past the caudalmedulla and into the cervical spinal cord as a continuumwith the heavy fiber and terminal fields in the dorsal horn.

Spinal cord

In situ hybridization. Proximally, intense mRNAexpression in the caudal spinal trigeminal nucleus transi-tions into the rostral dorsal horn of the cervical cord,where mRNA expression is intense in lamina II and is verylight in laminae III, V, and VI (Fig. 21C). Scatteredneurons are also seen in lamina VIII dorsally. IntensemRNA expression is evident in lamina X adjacent to thecentral canal. Extending ventrally from this region, large,scattered neurons with strong mRNA expression are ob-served in the ventral horn, in the ventral aspect of laminaVII, and in what appear to be large ventral motor neuronsin lamina IX, which also are observed in immunohisto-chemical staining (Fig. 2G). The lateral cervical nucleusshows light mRNA expression. In the thoracic spinal cord,lamina I is devoid of OFQ mRNA-containing neurons, butlamina II has heavy expression. Other than occasionalneurons in laminae III and V, there is no other mRNAexpression observed in the dorsal horn. Messenger RNAlevels are moderate in lamina X of the thoracic cord, withmost neurons lying lateral in this region in the intermedio-medial cell column, where moderate numbers of OFQ-containing neurons are present (Fig. 21E). Again, thereare scattered, large, intensely labeled cells in the ventralpart of laminae IX of the ventral horn. The dorsal rootganglia contains scattered cells expressing OFQ mRNAthat are very few in number and are confined centrally inthe ganglia. No mRNA expression is observed in the lateralspinal nucleus, the intermediolateral cell column, thedorsal corticospinal tract, the gracile fasciculus, the lateralfuniculus, or the ventral funiculus in the thoracic cordregion.

Immunohistochemistry. Proximally, intense immuno-staining from the caudal spinal trigeminal nucleus per-sists into laminae II and III of the dorsal horn (Figs. 3E,21D). Lamina I contains scattered terminals and fibers,which are observed best in untreated animals, but no cells.The lateral cervical nucleus is filled with a dense plexus ofimmunolabeled fibers with moderate cell staining(Fig. 21F). Moderate puncta and fibers are seen in laminaIV, and only lightly labeled, scattered terminals andminimal fibers seen in laminae V and VI. Fiber andterminal labeling increases in the dorsal part of laminaVII, extending medially to form a moderate plexus of fibersand terminals within lamina X. Several moderately la-beled neurons also are found within this lamina. Thedorsal aspect of lamina VIII contains some lightly scat-tered fibers and no neurons. Large immunolabeled neu-rons are scattered throughout the ventral portion of laminaVIII, ventrolaterally in lamina IX, and in the ventralaspects of lamina VII proximally. No immunolabeling isobserved within the cuneate or gracile fasciculus, the


dorsal corticospinal tract, the lateral funiculus, or theventral funiculus.

In the thoracic spinal cord, there is modest fiber andterminal staining in laminae I and III, with moderatenumbers of dark neurons in lamina II. The lateral spinalnucleus contains heavy fiber and moderate cell staining,with no extension to the lateral funiculus, which is devoidof immunolabeling. Laminae IV and V have moderate fiberand terminal labeling, but no immunolabeled neurons areevident. Moderately immunolabeled fibers and puncta areseen in lamina VII and in the dorsal part of lamina VIII.Terminal and fiber labeling in lamina X is strong, withscattered immunolabeled neurons also present. Immunola-beled neurons are present in the intermediomedial cellcolumn near lamina X. Large moderately stained neuronsalso are observed in the ventral horn in this region. Cellsare observed primarily in lamina IX, with occasionalsmaller, immunolabeled neurons observed in lamina VIII.

DISCUSSION

In situ hybridization and immunohistochemistry resultsdemonstrate a diffuse distribution of OFQ in the adultmale rat CNS. These findings are in general agreementwith early in situ hybridization studies on the distributionof the preproorphanin precursor in the rat and mouse(Houtani et al., 1996; Mollereau et al., 1996b; Nothacker atal., 1996; Pan et al., 1996; Henderson and McKnight,1997). In these studies, OFQ precursor mRNA was demon-strated in several brain areas, including the cortex, stria-tum, lateral septum, bed nucleus of the stria terminalis,thalamus, amygdala, hypothalamus, substantia nigra, cen-tral gray, raphe nuclei, pedunculopontine nucleus, supe-rior and inferior colliculi, olivary complex, spinal trigemi-nal nucleus, and spinal cord dorsal horn. There are fewareas of disagreement between the findings in the presentmapping study and initial surveys for mRNA expression.By using Northern blot analysis of the preproorphaninprecursor in the rat, Mollereau et al. (1996b) demonstratedspecific expression of preproorphanin mRNA in severalbrain areas. They found no mRNA expression in thepituitary, weak expression in the cortex and hippocampus,and strong mRNA expression in the hypothalamus andstriatum. The strong mRNA expression observed by thisgroup in the striatum is in contrast to the paucity of mRNAexpression and peptide labeling observed in the striatumof the rat in our study. In situ analysis by Houtani et al.(1996) detected orphanin mRNA in the nucleus of thelateral lemniscus and in the inferior vestibular area, twobrainstem regions in which we found no mRNA or peptideexpression. In general, however, the overall mRNA distri-bution they observed was similar to ours.

There were very few regions where mismatch occurredbetween immunostaining and mRNA expression. In layersII, III, and V of the temporal cortex; layers II and III of theoccipital cortex; the medial orbital cortex; the medialanterior olfactory nucleus; the anteroventral nucleus ofthe thalamus; the lateral habenula; the subgeniculatenucleus; the lateral interpeduncular nucleus; medial sub-nucleus B of the inferior olive; lamina V of the cervicalspinal cord; laminae IIII and VIII of the thoracic spinalcord; and the dorsal root ganglia, light-to-moderate mRNAexpression was detected, but no neuronal immunolabelingwas seen. This disparity could represent regions whereimmunolabeling was not sensitive enough to detect OFQ

peptide production within cell bodies, but it also mayrepresent regions where preproorphanin mRNA is presentbut is not being translated into the OFQ peptide. Inaddition, the precursor molecule may be undergoing post-translational processing, making it unavailable for immu-nolabeling by using our OFQ sequence. Although anantiserum directed against a different portion of thepreproorphanin sequence may generate neuronal labelingin these few regions, the general immunostaining patterngenerated with our OFQ antiserum correlates excellentlywith in situ hybridization results. In addition, immunore-activity generally was not observed in structures that weredevoid of OFQ mRNA expression, the exceptions beingscattered immunolabeled neurons observed in the striaterminalis, the stratum oriens of region CA2 of Ammon’shorn, the terete nucleus of the hypothalamus, the centralnucleus of the inferior colliculus, the rostral preolivarynucleus, the paralemniscal nucleus, and the lateral spinalnucleus. All of these regions are devoid of mRNA expres-sion but contain light neuronal immunolabeling.

Colchicine treatment

Colchicine injection into the lateral ventricle markedlyaltered OFQ immunohistochemical staining in the ratbrain, allowing us to closely analyze neuronal elementscontaining the OFQ peptide. In the untreated animals,neuronal labeling was observed in several regions (e.g.,lateral septum, hypothalamus, reticular nucleus of thethalamus, nucleus of Darkschewitsch, dorsal tegmentalnucleus, nucleus of the solitary tract, nucleus ambiguous),but intensity of labeling was significantly diminished. Incontrast, fiber and terminal labeling was more pronouncedin untreated rats than in colchicine-treated animals. Intreated animals, fiber and terminal labeling was presentin similar structures, but was diminished. Such a patternof immunolabeling is expected in animals treated with aninhibitor of microtubule-dependent axonal transport, suchas colchicine.

Although it has been used extensively in immunohisto-chemical mapping studies, recent reports have raised thepossibility that colchicine treatment induces an increasein mRNA levels for certain neuropeptides in the rat brain(Cortes et al., 1990; Ceccatelli et al., 1991; Kiyama andEmson, 1991). If such an effect does occur, then the authorsargue that some mRNA labeling seen in colchicine-treatedanimals could represent artifact rather than true mRNAexpression. In our experiences, and in the present study,we have not found that colchicine treatment produces suchan effect. An excellent correlation was observed betweenpreproorphanin mRNA distribution in untreated animalsand that of the OFQ peptide in colchicine-treated anduntreated animals. In general, every area that containedmRNAexpression also demonstrated some degree of immu-nolabeling, and more importantly, the counter also heldtrue; in no areas of the rat CNS were immunolabeledneurons detected where mRNA-expressing cells were notlocalized. Our in situ hybridization technique is generallymore sensitive in detecting cell bodies than immunohisto-chemistry, because it detects not only neurons that synthe-size OFQ but also those that have the potential to synthe-size OFQ peptide. Messenger RNA expression, therefore,is more perceptible than immunolabeling in the CNS inthose animals that were the least stressed of all (noncolchi-cine treated and nonperfused). Colchicine treatment, al-though it can provide a valuable benefit in detecting


OFQ-containing cell bodies, produced no neuronal labelingin areas devoid of mRNA expression in our hands.

Antiserum specificity

The anti-OFQ antiserum generated for use in the pre-sent study has not been utilized in any prior studies. Afterdetailed analysis of several bleeds from both rabbits,affinity purification was performed on two bleeds. Antiserafrom bleed 6, animal 217, was chosen for this study basedon quality and reliability of staining and on specificity ofimmunolabeling. This antiserum was noted to be quitespecific in most areas studied. By preabsorbing the OFQantiserum with a 25-µM concentration of OFQ peptideprior to tissue application, immunolabeling was blockedcompletely in virtually every CNS structure analyzed.However, there were some exceptions that warrant discus-sion. In the forebrain, there is very strong fiber labeling inthe ventral lateral septum. In preabsorption control stud-ies, very small numbers of these fibers are not blockedcompletely, although they remain very lightly stained.Lateral to the paraventricular nucleus of the hypothala-mus, there is a cluster of very intensely immunolabeledneurons with scattered fibers. In preabsorption controls,fiber labeling in this region is blocked completely, butseveral of the neurons remain immunolabeled. Althoughthey are not blocked completely, the numbers of neuronslabeled are very few, and the intensity of labeling is verylight. Outside of the cerebellum (discussed below), thereare no other structures in the CNS in which immunolabel-ing is not blocked completely (Fig. 3). The two regions(discussed above) might represent cross reactivity of theantiserum with other peptides in these areas, but this isunlikely. If such cross reactivity did indeed exist, then onewould assume not only that there would be somewhatstronger immunoreactive labeling in preabsorption con-trols but also that there would be labeling in several otherCNS regions. Because this unblocked labeling was foundonly in areas of intense immunolabeling under regularconditions, the OFQ antiserum may not be blocked 100%by using a 25-µM concentration of peptide, detectingpeptide in only these regions.

In the cerebellum, immunostaining observed in thefastigial nucleus was blocked completely in preabsorptioncontrols. Specific mRNA expression also was observed inthis nucleus. In the cerebellar lobules, OFQ expression isnot so clear. Scattered mRNA-containing neurons wereobserved in the granular layer and were shown to bespecific in RNase and sense control studies, but Purkinjecells were found to have no prepro-OFQ mRNA content. Inimmunohistochemical studies, the cerebellum containednumerous, large, immunoreactive neurons within the Pur-kinje cell layer of the cerebellum. These neurons were seenin all lobules and were unblocked completely in preabsorp-tion control studies. These neurons were present in colchi-cine-treated and untreated animals, so this staining wasnot an effect of colchicine. In addition, other CNS regionswith large neuronal elements (e.g., the red nucleus, cranialnerve motor nuclei) did not demonstrate such staining, sothis was not an artifact of large neuron size. It is possiblethat, specifically within the cerebellar hemispheres wherethis unique labeling pattern is found, there may be someelements that cross react with nonorphanin-specific anti-bodies within the OFQ antiserum. In this most likelyscenario, 1) this cross reactivity is specific to the cerebellarhemisphere and is not even seen in the cerebellar deep

nuclei, 2) it is not colchicine-dependent, and 3) it is notseen in any fiber or terminal elements. The immunolabel-ing pattern in the cerebellar hemisphere, therefore, istreated as nonspecific artifact in this study. Based on theremainder of our preabsorption controls and on theirexceptional correlation with in situ expression, we areconfident that all other immunolabeling patterns in thisstudy are specific for the OFQ peptide.

Comparisons with endogenousopioid systems

The distributions of the endogenous opioid peptide pre-cursors, proopiomelanocortin, proenkephalin, and pro-dynorphin, have been studied in detail (Kachaturian et al.,1985), as has the distributions of the corresponding µ, k,and d opioid receptors (Mansour et al., 1993, 1994, 1995a,b,1996). Since its discovery, the ORL1 receptor has beenshown to differ markedly from the known opioid receptorsnot only in its structure and interactions with knownopioid agonists (Meng et al., 1996; Sim et al., 1996; Ma etal., 1997) but also in its distribution (Chen et al., 1994;Fukuda et al., 1994; Lachowicz et al., 1994; Anton et al.,1996). Likewise, the distribution of the endogenous ligandfor the ORL1 receptor, OFQ, as demonstrated in this study,is distinct from that of other opioid peptides. Close analy-sis of the distribution of these peptide-receptor systems,however, points to numerous CNS regions in which theopioid and orphanin systems may interact.

Proopiomelanocortin and the m receptor

Proopiomelanocortin, which gives rise to the endog-enous opioid, b-endorphin, has a very limited neuronaldistribution, with cell bodies encountered only in thearcuate nucleus, anterior pituitary, and nucleus of thesolitary tract. Its fiber distribution is more widespread,with most pronounced fiber labeling found in the bednucleus of the stria terminalis, central and medial nucleiof the amygdala, dorsomedial and arcuate nuclei of thehypothalamus, paraventricular nucleus of the thalamus,central gray, the raphe nuclear complex, lateral parabra-chial nucleus, mesencephalic trigeminal nucleus, andnucleus of the solitary tract (Kachaturian et al., 1985). Incontrast, the µ receptor has a much broader distributionpattern, with heavy receptor binding observed in fibersand perikarya within the neocortex, striatum, nucleusaccumbens, anterior cortical and medial amygdala nuclei,most thalamic nuclei, the presubiculum, dentate gyrus,medial mammillary nucleus, substantia nigra pars com-pacta, central gray, superior colliculus, inferior colliculus,parabrachial nucleus, dorsal and paramedian raphe, ra-phe magnus, pontine reticular formation, nucleus ambigu-ous, nucleus of the solitary tract, spinal trigeminal nucleus,and substantia gelatinosa of the dorsal horn of the spinalcord (Mansour et al., 1995a).

In the rat brain, the arcuate nucleus of the hypothala-mus and nucleus of the solitary tract are the only struc-tures in which cell bodies may express both OFQ (Figs.10A,B, 20C,D) and b-endorphin peptides, regions in whichorphanin may play a modulatory role on b-endorphin-containing neurons. The arcuate nucleus has been shownto contain abundant orphanin receptors, and OFQ hasrecently been shown to inhibit b-endorphin neurons andsecretory cells in the arcuate through activation of inwardK1 currents (Wagner et al., 1998). Within the bed nucleusof the stria terminalis, central and medial nuclei of the


amygdala, dorsomedial and arcuate nuclei of the hypothala-mus, paraventricular nucleus of the thalamus, centralgray, dorsal raphe, lateral parabrachial nucleus, andnucleus of the solitary tract, regions with abundant b-en-dorphin fibers, orphanin fiber immunolabeling is robust.Orphanin-like receptor immunoreactivity is present inthese areas as well (Anton et al., 1996), providing opportu-nity for OFQ interaction with the proopiomelanocortinsystem. Within the dentate gyrus, µ-receptor immunoreac-tivity has been shown to localize to interneurons withinthe granule cell layer (Mansour et al., 1995a), very similarto what was demonstrated for OFQ in this study (Fig.13B). Mu-Receptor immunoreactivity in cell bodies withinthe central gray, raphe nuclei, lateral parabrachial nucleus,nucleus ambiguous, solitary nucleus, and lamina II of thedorsal horn (Mansour et al., 1995a) also localizes in adistribution very similar to what was observed for orpha-nin in this study (Figs. 3E, 16A,B, 17D, 18, 20C,D, 21D). Inaddition, OFQ also is localized in neurons in the parscompacta of the substantia nigra (Fig. 8D), another area inwhich the µ receptor is localized within cell bodies andbinding is dense. Expression of OFQ within cell bodies thatcoexpress the µ-receptor protein and have dense µ bindingwould provide strong evidence for possible µ modulation ofthis peptide within these structures. Other areas in whichOFQ is strongly expressed and µ-receptor expression isrobust include the medial amygdaloid nucleus (Fig. 12A,B)and the paraventricular nucleus of the thalamus.

Prodynorphin and the k receptor

Prodynorphin-containing cell bodies are expressed pri-marily in the striatum, dentate gyrus, central nucleus ofthe amygdala, paraventricular, supraoptic, ventromedialand arcuate nuclei of the hypothalamus, lateral hypotha-lamic area, anterior pituitary, substantia nigra pars reticu-lata central gray, parabrachial nucleus, lateral reticularnucleus, nucleus of the solitary tract, spinal trigeminalnucleus, and dorsal horn of the spinal cord. Fibers contain-ing prodynorphin are very heavy in the forebrain innucleus accumbens, ventral pallidum and globus pallidus,CA3 area of Ammon’s horn, paraventricular nucleus of thehypothalamus, and pars reticulata of the substantia, butthey are less dispersed in the brainstem, with moderatefiber plexuses found in the central gray, the raphe nuclearcomplex, parabrachial nucleus, solitary nucleus, spinaltrigeminal nucleus, and dorsal horn (Kachaturian et al.,1985). The densest concentrations of k-receptor bindingand cell immunolabeling are found in the nucleus accum-bens, olfactory tubercle, medial preoptic area, bed nucleusof the stria terminalis, medial nucleus of the amygdala,paraventricular nucleus of the thalamus, several hypotha-lamic nuclei, including the supraoptic, paraventricular,ventromedial, and medial mammillary nuclei, superiorcolliculus, ventral tegmental area, substantia nigra, cen-tral gray, dorsal raphe, parabrachial nucleus, dorsal teg-mental nucleus, caudal nucleus of the solitary tract, andspinal trigeminal nucleus (Mansour et al., 1995b, 1996).

There are several regions in which orphanin-containingcell bodies could coexist or colocalize with dynorphin-containing neurons, including the dentate gyrus (Fig.13B), central nucleus of the amygdala (Fig. 12C,D), para-ventricular and arcuate nuclei of the hypothalamus (Fig.10), lateral hypothalamic area, pars reticulata of thesubstantia nigra (Fig. 3C), central gray (Fig. 16A,B),interpeduncular nucleus (Fig. 15A,B), paramedian raphe

and raphe magnus (Fig. 18), lateral reticular nucleus,solitary nucleus (Fig. 20C,D), spinal trigeminal nucleus,(Fig. 21A,B) and dorsal horn of the spinal cord (Fig. 21C,D). Within the CA3 region of Ammon’s horn, the centralgray, parabrachial nucleus, caudal spinal trigeminalnucleus, nucleus of the solitary tract, and dorsal horn ofthe spinal cord, orphanin is strongly expressed in cells andfibers, and the ORL1 receptor also is intensely immunola-beled (Anton et al., 1996). These regions also containrobust dynorphin fiber labeling and moderate k-receptorlocalization and binding, providing possible sites of dynor-phin and orphanin system interactions. The core of thenucleus accumbens (Fig. 6C,D), medial preoptic area (Fig.9F), bed nucleus of the stria terminalis (Fig. 9C,D), para-ventricular and ventromedial nuclei of the hypothalamus,median eminence (Fig. 1B), and medial nucleus of theamygdala (Fig. 12A,B) contain abundant OFQ fiber andcell staining and robust k-receptor localization and bind-ing. These distribution patterns also provide evidence forpossible k modulation of OFQ within these structures. It isof interest to note that the substantia (nigra) pars reticu-lata contains a very dense prodynorphin cell and fiberplexus. Although this structure contains only light kbinding and receptor content, it possesses a moderateamount of OFQ peptide and abundant ORL1-containingfibers, providing a likely milieu for orphanin-opiate inter-action in this basal ganglia structure.

Proenkephalin and the d receptor

Proenkephalin is the most widely distributed of theendogenous opioid peptides (Kachaturian et al., 1985),with the densest concentration of enkephalin-containingcell bodies in the cingulate and neocortex, anterior olfac-tory nucleus, olfactory tubercle, piriform cortex, striatum,nucleus of the diagonal band, bed nucleus of the striaterminalis, central nucleus of the amygdala, hippocampalformation, paraventricular, arcuate, and ventromedial nu-clei of the hypothalamus, central gray, interpeduncularnucleus, inferior colliculus, raphe nuclei, parabrachialnucleus, dorsal tegmental nucleus, medial vestibularnucleus, lateral reticular nucleus, nucleus of the solitarytract, spinal trigeminal nucleus, and dorsal horn of thespinal cord. Enkephalin-containing fibers are most pro-nounced in the nucleus accumbens, globus pallidus, hippo-campal formation, ventromedial nucleus of the hypothala-mus, central gray, solitary nucleus, spinal trigeminalnucleus, and dorsal horn. Delta-Receptor localization andbinding are most prominent the neocortex, anterior olfac-tory nucleus, nucleus accumbens, striatum, olfactory tu-bercle, medial and basolateral nuclei of the amygdala,hippocampal formation, interpeduncular nucleus, pontinenuclei, nucleus of the trapezoid body, lateral reticularnucleus, spinal trigeminal nucleus, and dorsal horn of thespinal cord (Mansour et al., 1995b).

Enkephalin-containing neurons have a high likelihoodof colocalizing or coexpressing with OFQ in several struc-tures in which orphanin-containing neurons are present,including the cingulate and piriform cortices (Fig. 6A),diagonal band, bed nucleus of the stria terminalis (Fig.9C,D), central nucleus of the amygdala (Fig. 12C,D), hippo-campal formation (Fig. 13), paraventricular and arcuatenuclei of the hypothalamus (Fig. 10), central gray (Fig.16A,B), interpeduncular nucleus (Fig. 15A,B), paramedianraphe and raphe magnus (Fig. 18), lateral reticular nucleus(Fig 20E), solitary nucleus (Fig. 20C,D), spinal trigeminal


nucleus (Fig. 21A,B), and dorsal horn of the spinal cord(Fig. 21C,D). Enkephalin-containing fibers in the nucleusaccumbens, hippocampus ventromedial nucleus of thehypothalamus, central gray, interpeduncular nucleus, soli-tary nucleus, and dorsal horn are within regions whereorphanin-containing fibers also are present. These regionsalso contain heavy ORL1 immunoreactivity (Anton et al.,1996) and may be areas of orphanin modulation of enkepha-lin activity. Although not yet demonstrated in the brain,OFQ has been shown to modulate enkephalin release inmyenteric plexus preparations (Gintzler et al., 1997). Theneocortex, cingulate cortex, nucleus accumbens, ventrome-dial nucleus of the hypothalamus, medial nucleus of theamygdala, interpeduncular nucleus, spinal trigeminal nucleus,and substantia gelatinosa of the dorsal horn contain strongOFQ neuronal and/or fiber labeling as well as numerousneurons with d-receptor protein and robust binding. Thestrong overlapping distribution of the d receptor withorphanin fibers in these nuclei provides sites for enkepha-lin or other opioid modulation of the orphanin peptide.

Functional considerations

Comparison with ORL1 receptor distribution. Intheir detailed analysis of the distribution of the ORL1receptor, Anton et al. (1996) did not include an analysis oforphanin receptor mRNA distribution but, rather, focusedon the distribution of the receptor protein. Similar to ourfindings with OFQ, those authors found that the orphaninreceptor was distributed diffusely the rat CNS. In theirstudy, Anton et al. reported very few neurons containingORL1 receptor immunoreactivity, with virtually all immu-nostaining confined to fibers and puncta. Orphanin recep-tor immunoreactivity was observed within somata in thedentate gyrus of the hippocampus, the red nucleus, thepontine reticular nucleus, gigantocellular and paragiganto-cellular reticular nuclei, lamina IX of the spinal cord, andthe central cervical nucleus. This is in sharp contrast tothe diffuse distribution of OFQ-containing perikarya re-ported in our analysis. Densest ORL1 receptor fiber andterminal staining was observed in the neocortex, cingulatecortex, piriform cortex, entorhinal cortex, dorsal and ven-tral pallidum, triangular and medial septum, medial pre-optic area, central and medial nuclei of the amygdala,cortical amygdala, Ammon’s horn, subiculum, anteriorolfactory nucleus, claustrum, endopiriform nucleus, mam-millary bodies, parafascicular and posterior thalamic nu-clei, substantia nigra, ventral tegmental area, interpedun-cular nucleus, central gray, dorsal raphe, trigeminal motornucleus, suprafacial nucleus, nucleus ambiguous, nucleusof the solitary tract, dorsal nucleus of the vagal nerve,hypoglossal nucleus, parvocellular reticular nucleus of themedulla, and lamina II of the spinal cord. Lighter fiber andterminal labeling was observed in numerous other struc-tures diffusely throughout the CNS. Mismatch betweenthe receptor and neurotransmitter distributions like whatwe observed with the orphanin system is not unique andhas been demonstrated extensively in the opioid peptideand receptor systems (Kachaturian et al., 1985; Mansouret al., 1995b). When comparing ORL1 receptor immunohis-tochemical distribution with OFQ peptide distribution, itis clear that they differ markedly in their anatomical andstructural localization. However, on close analysis, mul-tiple anatomical circuits become evident in which peptide-receptor interactions may provide insights into functionalroles for this new orphanin system.

Limbic-hypothalamic-pituitary adrenal axis. Thefunction of the limbic-hypothalamic-pituitary adrenal (L-HPA) axis has been studied extensively, and much isknown concerning its role at the behavioral, anatomic, andcellular levels (Herman et al., 1996; Herman and Cullinan,1997). It is generally accepted that two types of stressorsare involved in the initiation of the stress response: thoseinvolving an immediate physiologic threat (systemic stress-ors) and those requiring interpretation by higher brainstructures (processive stressors). Whereas processivestressors are likely channeled through limbic forebraincircuits involved in the L-HPA axis and then to theparaventricular nucleus of the hypothalamus, systemicstressors probably are relayed directly to the paraventricu-lar nucleus through brainstem cholinergic neurons. TheL-HPA axis is a steroid-sensitive system (Herman andCullinan, 1997) because many of these stress-relatednuclei are sensitive to circulating glucocorticoid levels.

Several forebrain nuclei are known to be involved instress-excitatory circuits. Of these, the medial, central,and posterior medial cortical nuclei of the amygdala andthe lateral bed nucleus of the stria terminalis possess bothmineralocorticoid receptors and OFQ-containing neuronaland fibers elements. Glucocorticoid receptors also arefound in the central nucleus. Stress-inhibitory circuitsinclude the medial bed nucleus of the stria terminalis, themedial preoptic area, and the dorsomedial, ventromedial,arcuate and suprachiasmatic nuclei of the hypothalamus.Mineralocorticoid receptors are found in the medial preop-tic area, and glucocorticoid receptors are found in themedial preoptic area, arcuate nucleus, bed nucleus of thestria terminalis, and ventromedial nucleus. With theexception of the suprachiasmatic nucleus, OFQ immunola-beling and mRNA expression are seen in all of these nuclei.Other forebrain regions with direct input into this cir-cuitry are the ventral subiculum, prefrontal cortex, andhippocampal formation, all of which have orphanin-containing neurons and glucocorticoid receptors.

There is considerable evidence supporting a role forbrainstem catecholaminergic neurons in the regulation ofthe stress axis (Plotsky et al., 1989). Their action is onL-HPA activation and appears to be stimulatory by way ofa-1-adrenergic receptors. Direct noradrenergic input ontoneurons in the parvocellular paraventricular nucleus isthrough the ventral noradrenergic bundle, which is sup-plied primarily by the A2, C1, C2, and C3 cell groups in thebrainstem. Orphanin peptide is not expressed in theseregions, making it unlikely that OFQ plays a modulatoryrole in these direct inputs. The locus coeruleus, one of themost stress-responsive nuclei in the brain (Herman et al.,1996), is thought to modulate L-HPA activation indirectlythrough multisynaptic connections with forebrain regions,such as the prefrontal cortex, hippocampus, and amyg-dala. Orphanin mRNA and peptide is localized in all ofthese regions, providing possible regions in which OFQmay modulate noradrenergic input into the stress axis. Arole for serotonin in L-HPA activation is supported by thefinding that destruction of serotonin neurons in the dorsalraphe reduces corticosterone and adrenocorticotropin re-sponses to stressful stimuli (Herman et al., 1996), andneurons within the raphe complex provide direct projec-tions to the parvocellular paraventricular nucleus. Orpha-nin peptide and mRNA are localized within many neuronaland/or fiber elements within the raphe system, allowing


involvement of OFQ in the modulation of brainstem sero-tonergic activation of the L-HPA axis.

At present, little has been shown behaviorally to supportan role for orphanin in the stress response. Recently, Jencket al. (1997) demonstrated that OFQ can modulate anxietystates generated by acute stress. Those authors concludedthat OFQ may act as an anxiolytic in certain stressfulcircumstances. Orphanin indeed may play a modulatoryrole on the stress response through inhibitory effects onL-HPA activation. If so, then its role as an anxiolytic agentcould be explained.

Learning and memory. The hippocampal formationis not only intimately involved in the function of the L-HPAaxis, it is also a critical structure in learning and memory.OFQ is found throughout the hippocampal formation, andOFQ-containing neurons are also observed in the entorhi-nal cortex, which has direct projections to the entirehippocampal formation. In addition, ORL1 receptor immu-noreactivity is strong in all hippocampal regions (Anton etal., 1996). Orphanin has been reported to reversibly in-hibit voltage-gated calcium channels in pyramidal neu-rons from areas CA1 and CA3 (Knoflach et al., 1996) and toinhibit synaptic transmission and long-term potentiationin area CA1 of Ammon’s horn and the dentate gyrus (Yu etal., 1997). The latter effect is thought to be postsynaptic innature (Yu and Xie, 1998). Microinjections of OFQ into theCA3 region of adult rats have been shown to impair spatiallearning (Sandin et al., 1997). These findings remaincontroversial, because the effects of orphanin on motorbehavior were not taken into account (see below). How-ever, in support of the findings by Sandin et al., recentstudies on knockout mice lacking the orphanin receptordemonstrated enhancement of spatial attention in thewater-finding test (Mamiya et al., 1998) and facilitation oflong-term potentiation and memory (Manabe et al., 1998).Initial studies support a role for this peptide in learningand memory, and its distribution throughout the hippocam-pal formation and entorhinal cortex supports this conceptas well.

Motor systems. Orphanin-containing neurons arelocated in the globus pallidus, entopeduncular nucleus,and substantia nigra, and significant OFQ-containingfibers are found in these structures and in the striatum. Inaddition, the lateral bed nucleus of the stria terminalis, acomponent of the extended central amygdaloid complex(Alheid et al., 1995), has abundant OFQ peptide contentand has direct projections to the retrorubral field, ventraltegmental area, and substantia nigra, pars compacta.These structures possess abundant ORL1 receptor density(Anton et al., 1996) and give rise to major dopaminergicprojections. Early behavioral effects observed with centraladministration of this peptide may reflect a conceivablerole of OFQ in the modulation of gross locomotive behavior.

OFQ also is localized within brainstem structures thatdirect several aspects of fine motor control and balance,including vestibular nuclei, deep cerebellar nuclei, and theinferior olive. Proprioceptive input from the dorsal col-umns of the spinal cord is integrated in the dorsal olivarynucleus, which relays information to the cerebellar vermis,which, in turn, has very strong inputs into the medial(fastigial) cerebellar nucleus. The fastigial nucleus projectsheavily to nuclei involved with visual integration and tothe spinal vestibular nucleus. Orphanin-containing cellbodies are expressed consistently throughout this cir-cuitry, and, although ORL1 immunoreactivity is light in

these areas (Anton et al., 1996), orphanin binding isprominent in many of these structures (personal observa-tions). Input into the principal olivary nucleus originatesprimarily in visual integration areas and association corti-ces. This nucleus projects to the lateral cerebellar hemi-spheres, which have strong input to all vestibular nucleiexcept the lateral nucleus. Orphanin peptide content isvery strong in the visual nuclei, which project to theprincipal olivary nucleus (nucleus of Darkschewitsch, inter-stitial nucleus of the medial longitudinal fasciculus, andmedial accessory oculomotor nucleus), as well as withinthe principal olivary nucleus itself. The vestibular nucleiprovide integration of proprioceptive input and provideefferents from the lateral vestibular nucleus to the inferiorolive and from the spinal vestibular nucleus to the cerebel-lum and reticular formation. The medial and superiorvestibular nuclei have projections to the cerebellum, spi-nal cord, thalamus, and lateral paragigantocellular reticu-lar nucleus.

The effects of orphanin on gross motor function aresomewhat controversial and are largely unknown at thispoint. Orphanin has been implicated in producing hypolo-comotion in rats (Devine et al., 1996a) and in stimulatinglocomotion and exploratory behavior in mice (Florin et al.,1996). Many early behavioral tests using intraventricularorphanin may have been confounded by the fact thathigher doses of this peptide have been shown to seriouslydisrupt motor behavior in adult rats (D.P. Devine, personalcommunication). Indeed, OFQ is localized within manystructures in the CNS that are intimately involved inlocomotion, balance, and proprioception. It remains to bedetermined exactly what role orphanin plays in motorfunction, but the presence of OFQ in the basal ganglia andin the vestibular and visual motor systems supports amodulatory role for this peptide in these related behaviors.The effects of this peptide on motor function should beconsidered when analyzing other behavioral effects.

Reinforcement and reward. The nucleus accumbenshas been implicated in a number of functions, includingdrug reinforcement and locomotor behavior (Koob et al.,1991). Little is known concerning possible motivational orreward effects of OFQ in relation to this region. Rats havebeen shown to develop tolerance to locomotor effectsinduced by orphanin (Devine et al., 1996a), but they failedto demonstrate conditioned place preference when givenintraventricular injections of this peptide (Devine et al.,1996b). In addition, it has been reported that injections ofOFQ into the lateral ventricle of the rat suppressesdopamine release in the nucleus accumbens (Murphy et al,1996). Orphanin is strongly expressed in several struc-tures involved in this circuitry, including the substantianigra and the ventral tegmental area. These regionscontain direct projections to the nucleus accumbens andventral pallidum, both of which have abundant ORL1receptor (Anton et al., 1996). The inability to generateconditioned place preference or aversion in earlier studiesin the rat may have been limited by locomotor effects ofthis peptide (D.P. Devine, personal communication), andthe abundance of OFQ within this mesoaccumbens cir-cuitry still may indicate a modulatory role for this peptidein reinforcement behaviors.

One particular reinforcing behavior that appears to beinfluenced by the accumbens nucleus is that of feedingbehavior. Feeding as a behavioral act is complicated,requiring several somatomotor and autonomic events re-


lated to searching for and handling of food, eating, anddigesting. This process involves multiple CNS circuits thatare involved in the integration of olfactory, visual, andauditory information and many brainstem centers that areinvolved in the acts of mastication and digestion. Thelateral septum, a region with high OFQ content, has directprojections to the medial hypothalamus and the lateralhypothalamic area. It has been postulated that thesestructures are central in the initiation and control of foodintake (King and Nance, 1986). In addition, some mesoac-cumbens-lateral hypothalamic interactions may be morecentral in motivating effects of food intake. A recent reportby Cabeza de Vaca and Carr (1998) demonstrated adefinite enhancement in the central rewarding effect ofabused drugs in animals undergoing food restriction.Although the nucleus accumbens is thought to be centralin the mechanism, those authors also argue for a majorrole of the lateral hypothalamus, a region with abundantorphanin content.

Pomonis et al. (1996) initially demonstrated that injec-tions of OFQ into the lateral ventricle of the rat stimulatedfeeding to a degree similar to that seen with injection ofother opiates. This response however, was blocked bynaloxone, making it more likely that it may have been anopiate-driven event. More recently, Stratford et al. (1997)demonstrated that injections of orphanin into the ventro-medial hypothalamic nucleus or the nucleus accumbensincreased food intake in rats. By using small doses oforphanin (2.5–25 nM), they demonstrated orphanin-specific stimulation of this motivational behavior withinjections into both regions. The role of OFQ in stimulationof feeding behavior gains further support in a recent studyby Leventhal et al. (1998), which demonstrates a reductionof OFQ-induced hyperphagia by central administration ofan ORL1 antisense probe. Much remains to be answeredconcerning the possible role of OFQ in mechanisms ofmotivation and reinforcement. However, early behavioralfindings, coupled with the presence of OFQ and its ORL1receptor to varying degrees within many structures in-volved in this circuitry, support involvement of the orpha-nin system.

Sexual behavior. The anatomic distribution of OFQalso argues for a possible functional role in sexual behaviorand sexually dimorphic pathways. Evidence for sexualdimorphism was first reported in the medial preoptic areaby Gorski et al. (1980). Sexual dimorphism emerges earlyin development, is related to differences in neuronalproduction, and appears to be regulated by circulatinggonadal steroids (Jacobson and Gorski, 1981; Davis et al.,1996). A steroid-sensitive, enkephalinergic sexual dimor-phism has also been described in the medial preopticregion (Watson et al., 1986; Simerly et al., 1988). Otherforebrain nuclei that have major connections with themedial preoptic area also demonstrate sexual dimorphism,including the medial amygdala (Nishizuka and Arai, 1983),medial and lateral parts of the bed nucleus of the striaterminalis (Guillamon et al., 1988), and several hypotha-lamic nuclei, including the arcuate nucleus, ventromedialnucleus, and tuberomammillary nucleus (Urban et al.,1993; Borisova et al., 1996; Gonzales et al., 1996). Thecircuitry that is involved primarily in the medial preopticarea, bed nucleus of the stria terminalis, and medialnucleus of the amygdala is not only sexually dimorphic,but it also has been shown to be critical for normal sexualbehavior in the male hamster (Neal and Newman, 1989).

The medial preoptic area also shares inputs with thearcuate and ventromedial nuclei of the hypothalamus.These limbic regions contain some of the highest amountsof orphanin in the forebrain. Furthermore, the lateralseptum, which contains abundant orphanin peptide andmRNA expression, also is known to possess androgen andestrogen receptors and vasopressin immunoreactivity. Thisnucleus is also sexually dimorphic and sex-hormone sensi-tive (Jakab and Leranth, 1995). The lateral septum hasprofound hypothalamic projections and receives concen-trated input from the hippocampal formation, a regionknown to be sexually dimorphic (Madeira et al., 1991).What role orphanin plays in sexual dimorphism or insexual behavior is not known. However, its heavy contentthroughout circuitry that is not only sexually dimorphicbut also intimately involved in sexual behavior makes itlikely that orphanin plays a major role in the developmentor maintenance of this behavior. Supporting this notion, ithas been reported recently that OFQ microinjections intothe ventromedial nucleus of the hypothalamus facilitatelordosis in female rats (Sinchak et al., 1997).

Pain perception. Several studies have demonstrateda modulatory role for orphanin in pain perception, al-though its effects on the pain response remain controver-sial (Henderson and McKnight, 1997). Studies to datehave shown that orphanin plays an important modulatoryrole in pain perception, producing analgesia in somemodels (King et al., 1997; Rossi et al., 1997; Tian et al.,1997a,b; Yamamoto et al., 1997) and antagonizing analge-sia in others, tempting some to depict its actions asantiopioid (Grisel et al., 1996; Mogil et al., 1996a,b; Xu etal., 1996; Dawson-Basoa and Gintzler, 1997; Heinricher etal., 1997; Morgan et al., 1997; Jhamandas et al., 1998).OFQ also has been implicated as an antagonist of mor-phine analgesia (Hao et al., 1997; Tian et al., 1997b; Zhu etal., 1997), although morphine analgesia has been demon-strated in mice lacking the ORL1 receptor gene. It isinteresting to note that these ORL1-deficient animalswere unable to develop tolerance to the analgesic affects ofmorphine after repeated doses (Ueda et al., 1997). Recentstudies have also implicated orphanin in pain inductionfrom nonnoxious stimuli (allodynia; Okuda-Ashitaka etal., 1996; Hara et al., 1997; Minami et al., 1997).

Much is known concerning the ascending and descend-ing pain pathways, particularly those involving endog-enous opioid systems (Mansour et al., 1995b). In thiscontext, somatosensory inputs from laminae I and II of thedorsal horn of the spinal cord and from the spinal trigemi-nal nucleus ascend directly to the posterior and centrolat-eral nuclei of the thalamus and then are relayed to thesomatosensory cortex. This ascending information is modu-lated at many levels, including, initially, within thesubstantia gelatinosa, and by passing inputs from thereticular formation. Descending information from the so-matosensory cortex ultimately terminates in the dorsalhorn of the spinal cord, relaying feed-back information fornociception modulation. These inputs are not direct andarrive at the spinal cord by way of the central gray, raphemagnus, and alpha division of the gigantocellular reticularformation (Mansour et al., 1995b). These projections aremodulated further in passing by inputs from the dorsalraphe, median raphe, lateral parabrachial nucleus, andreticular formation. It has long been known that endog-enous opioid peptides and receptors strongly influencethese nociceptive pathways. It is also important to note


here that OFQ is physically present along all parts of thispain circuitry as well, with OFQ-containing neurons andfibers found in the central gray, the raphe nuclei, thelateral parabrachial nucleus, the reticular formation nu-clei, the spinal trigeminal nucleus, and the superficiallaminae of the spinal cord.

Although colocalization has not been demonstrated,OFQ has been shown to have an overlapping distributionwith opioid peptides in several pain modulatory regions,including the dorsal horn of the spinal cord, spinal trigemi-nal nucleus, raphe nuclei, locus coeruleus, and centralgray (Schulz et al., 1996). In addition, there is an accumu-lating body of evidence supporting modulatory influencesof OFQ on painful inputs into the CNS as both an agonistand an antagonist to analgesia. It is presently hypoth-esized that the outer two-thirds of the substantia gelati-nosa play a major role in the modulation of nociception,and this region contains a myriad of various neurotransmit-ters, including opioid peptides (Ribeiro-da-Silva, 1995).Orphanin immunoreactivity has been demonstrated quan-titatively in lamina II, and OFQ-containing fibers parallel-ing endogenous opioids have been identified (Riedl et al.,1996; Schuligoi et al., 1997). Cell recordings from substan-tia gelatinosa neurons have shown that exogenous OFQdepresses excitatory postsynaptic potentials evoked bystimulation of dorsal root ganglia (Lai et al., 1997; Liebelet al., 1997). Intrathecal orphanin also has been shown toinhibit C-fiber-evoked discharge of dorsal horn neurons(Stanfa et al., 1996). These studies clearly support a rolefor orphanin inhibition of lamina II neurons within thespinal pathway of the rat spinal cord.

Opioid- and orphanin-containing cell bodies also arenumerous in the rostral ventromedial medulla, a region inwhich the ORL1 receptor also has been identified (Anton etal., 1996). This region includes reticular formation andraphe neurons that are known to have an antinociceptiveinfluence to noxious stimuli. Electrophysiologic and phar-macologic studies in this region have demonstrated aninhibitory role of OFQ on the firing of these neurons in thepresence of morphine activation, leading the authors tohypothesize that OFQ exerts an ‘‘antiopioid’’ effect bysuppressing firing of these cells (Heinricher at al., 1997).Finally, microinjections of morphine and OFQ into thecentral gray have demonstrated that OFQ attenuatesmorphine-induced analgesia in awake rats (Morgan et al.,1997). Separate studies have shown that microinjections ofOFQ alone demonstrate inhibitory actions on central grayneurons (Vaughan et al., 1997).

In conclusion, the anatomic localization of OFQ withinimportant structures along the ascending and descendingpain pathways, its proximity to the endogenous opioidpeptide systems, and mounting physiologic and behavioralevidence appear to support a major role for orphanin inpain modulation. It is important to note that it has beendemonstrated recently that the ORL1 and µ antisera labeldifferent fibers in pain-processing regions of the rat brain(Monteillet-Agius et al., 1998). Therefore, it is most likelythat ORL1 and µ influences on pain perception occurthrough predominantly different fiber systems.

Autonomic and physiologic functions. There havebeen several studies supporting a role for orphanin incontrol of basic autonomic and physiologic mechanisms,including cardiovascular control (Gumusel et al., 1997;Champion and Kadowitz, 1997a, b; Champion et al., 1997and water and electrolyte balance (Kapusta et al., 1997).

Application of OFQ onto arterial rings from the cat demon-strates vasorelaxant properties (Gumusel et al., 1997), andthis peptide has been shown to induce hypotension (Cham-pion and Kadowitz, 1997a,b) and to decrease cardiacoutput (Champion et al., 1997) in rats. Although the role ofOFQ in autonomic circuitry is not clear, L-glutamatestimulation of the zona incerta has been shown to decreaseheart rate and blood pressure (Spencer et al., 1988) in therat. Intraventricular administration of OFQ also has beenshown to induce diuresis and antinatriuresis in the rat,implicating this peptide in the central control of waterbalance and possibly in the regulation of blood pressure(Kapusta et al., 1997). Peripheral intravenous infusion oforphanin also produces diuresis and inhibits salt wasting,implying that this effect also may have a peripheralcomponent. It should be noted, however, that the OFQdoses used by Kapusta et al. were magnitudes higher thanthose used in studies that elicited analgesic, nociceptive,and motor effects. In support of a role for OFQ in waterbalance, the ORL1 receptor is highly expressed in thesupraoptic and paraventricular nuclei of the hypothala-mus (Anton et al., 1996). The diuretic and antinatriureticeffects of OFQ may be vasopressin mediated, because OFQhas been shown to inhibit firing of vasopressin-containingneurons in the supraoptic nucleus of the hypothalamus(Doi et al., 1998), or it may involve peripheral modulationof renal sympathetic nerves.

Orphanin involvement in noradrenergic and serotoner-gic systems also may play a role in cardiovascular controland fluid balance. Treatments that increase central extra-cellular serotonin increase sodium excretion, and renaldenervation attenuates natriuresis produced by intraven-tricular administration of serotonin (Montes and Johnson,1990). In addition, activation of serotonergic neurons inthe ventromedial medulla produces a drop in blood pres-sure and heart rate by inhibiting central sympatheticoutflow (Kalkman and Fozard, 1991). This effect is limitedto the medial serotonergic cell group in this region, withstimulation of laterally located neurons inducing hyperten-sion in these same animals. Numerous orphanin-contain-ing neurons are located in the region of the noradrenergicA5 cell group in the caudal mesencephalon and meten-cephalon. This cell group is known to project to autonomicnuclei of the brainstem and spinal cord (Byrum andGuyenet, 1987), with direct spinal projections to theintermediate gray and intermediolateral (sympathetic pre-ganglionic) cell column in the spinal cord and to thenucleus of the solitary tract. Chemical stimulation ofneurons in the A5 region has been reported to induce a fallin blood pressure (Neil and Loewy, 1982). The localizationof orphanin within neurons in the dorsal raphe andventromedial medulla and within the region of the A5 cellgroup raises the possibility of colocalization with noradren-ergic or serotonergic projection neurons controlling bloodpressure, cardiac output, and sodium balance.

The nucleus of the solitary tract contains numerousOFQ mRNA-expressing cells and immunoreactive neuronsand abundant ORL1 receptor (Anton et al., 1996). Auto-nomic efferents from this nucleus project to the intermedio-lateral cell column of the spinal cord, nucleus ambiguous,dorsal motor nucleus of the vagal nerve, ventrolateralmedulla, A5 cell group, parabrachial nucleus, and numer-ous forebrain regions (Saper, 1995). Solitary nucleus projec-tions to the preganglionic parasympathetic neurons in themedulla and sympathetic neurons in the spinal cord


influence numerous autonomic responses. In addition,efferents to the medullary reticular formation influencegastrointestinal, cardiovascular, and respiratory reflexes.The brainstem orphanin system is localized diffusely withinmajor autonomic control centers. Although initial studieshave demonstrated an OFQ influence on primarily cardio-vascular function and water balance, this peptide may beinvolved in several autonomic and physiologic functions,including general arousal, cardiovascular tone, respira-tory drive, gastrointestinal function, and general visceralsensory feedback.

Special sensory systems. Brainstem special sensorysystems provide extensive input to higher centers throughstrong olfactory, gustatory, and visceral sensory informa-tion that regulates the medial preoptic area, anteriorhypothalamus, dorsomedial hypothalamus, and ventrome-dial hypothalamus and through visual and auditory infor-mation to the mammillary bodies. OFQ is present inseveral nuclei within these circuits and probably playssome role in the integration and/or processing of thisinformation.

OFQ is highly expressed within nuclei that integratevisual information, particularly the nucleus of Darks-chewitsch, pretectum, and superior colliculus, providing amedium for OFQ involvement in visual sensory integra-tion, as well modulation of output to the spinal cord, themidline pontine and medullary nuclei, the hypothalamus,and the intralaminar thalamic nuclei. In the forebrain, thelateral mammillary nucleus contains a very dense plexusof OFQ-containing neurons and fibers. This nucleus isinfluenced strongly by both visual and auditory stimuliand is thought to be involved the processing of visual andauditory information, and it has been shown to have directcortical connections (Simerly, 1995). Although the majorityof brainstem auditory regions have little OFQ and ORL1content, structures primarily involved with the integra-tion of high frequency sound in the rat (Aitkin et al., 1984)contain abundant OFQ expression. Such information isintegrated directly in the trapezoid body, lateral superiorolive, and inferior colliculus. Orphanin expression is strongin the nucleus of the trapezoid body, which projects directlyto the lateral superior olive, which, in turn, projects to theinferior colliculus. These structures contain moderateamounts of ORL1 immunoreactivity (Anton et al., 1996) aswell as OFQ-containing cell bodies, supporting a role forOFQ in the integration of high-frequency sounds. It isimportant to note that, given the presence of OFQ and theORL1 receptor within the auditory nuclei described above,as well as in the inferior olive and the vestibular system,the OFQ system may be involved in the integration ofauditory and vestibular information and possibly plays amajor role in an animal’s ability to adapt to visual,proprioceptive, and auditory cues in its environment.Little is known concerning a role for OFQ in visual orauditory processing, but a recent study in ORL1-deficientmice has shown that these animals demonstrate an insuf-ficient recovery of hearing ability after adaptation to soundexposure (Nishi et al., 1997).

A brief mention should be made of the possible role oforphanin in the integration of olfactory and gustatoryinformation. Cranial nerves that carry gustatory andsomatosensory axons from the oral cavity are distributedthroughout the nucleus of the solitary tract in a topographi-cal fashion (Altschuler et al., 1989). Gustatory informationfrom the solitary nucleus is relayed to the parabrachial

nucleus. Gustatory neurons within the parabrachialnucleus are located primarily in the medial part, whereOFQ is not present, but abundant ORL1 receptor immuno-reactivity (Anton et al., 1996) is observed. The parabra-chial nucleus relays gustatory information to the ventralposteromedial thalamus, then to the dorsal insular cortex,and also directly to the central nucleus of the amygdala.Olfactory stimulus perception and integration also may bemodulated by OFQ. The entorhinal cortex, which containsneurons with OFQ-like immunoreactivity and OFQ mRNAexpression, receives substantial olfactory input from theolfactory bulbs and the piriform cortex. This structure, inturn, projects by way of the perforant pathway to the hippocam-pal formation and to other higher centers (Schwerdtfeger etal., 1990). There are no data to date supporting a role fororphanin in the processing of olfactory or gustatory informa-tion. However, as discussed above, injections of OFQ into thelateral ventricle of the rat appear to increase feedingbehavior (Pomonis et al., 1996). It is highly likely thatsignals from gustatory and olfactory centers are importantin the complex series of behaviors involved with feeding.Orphanin FQ, by its presence within major nuclei in thesesystems, may be involved in the stimulatory effects ofintraventricular orphanin on feeding behavior.

CONCLUSIONS

We have demonstrated that OFQ, the novel endogenousagonist of the ORL1 receptor, is distributed widely through-out the CNS of the adult male rat. We have also establisheda very close correlation between preproorphanin mRNA andOFQ peptide expression. In addition, analysis of the immuno-histochemical distribution of the ORL1 receptor demonstratescorrespondence of the orphanin peptide with its endogenousreceptor, providing the groundwork for study of specificanatomic systems in deciphering the multiple behavioraland physiologic roles of this neuropeptide. Orphanin FQand the ORL1 receptor are expressed in critical compo-nents of numerous neural circuits, supporting their pos-sible involvement in various CNS systems, including noci-ception, modulation of the L-HPA stress axis, motivationand reward, learning and memory, gross motor control,balance and proprioception, sexual, aggressive, and inves-tigatory behaviors, control of autonomic and physiologicfunctions, and integration of special sensory input.

Although much claim has been made on naming thismolecule ‘‘nociceptin’’ due to its possible antagonism ofanalgesic modalities, the distribution of the orphaninsystem in the CNS is very diffuse and includes numerousforebrain and brainstem circuits and systems. OrphaninFQ and its receptor most likely play a major role in manybehaviors and physiologic functions, making nociceptin toolimiting a nomenclature for such an important neuropeptide.We support the continued use of ‘‘orphanin FQ’’when referringto this neuropeptide as we continue to unravel its role in theCNS.

ACKNOWLEDGMENTS

The authors thank Sharon Burke, Robert Pavlic, andJames Stewart for their superb technical assistance. Thiswork was sponsored by a National Institute of MentalHealth Training grant to C.R.N. (NIMH 5 T32 MH15794)and by a National Institute of Drug Abuse grant to S.J.W.(NIDA RO1 DA08920).


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Localization of Orphanin FQ (Nociceptin) Peptide and