Comparative Distribution of Vasopressin V1b and Oxytocin ......tocellular reticular nucleus; GP, globus pallidus; HDB, nucleus of the horizontal limb of the diagonal band; Hil, hilus

Comparative Distribution of Vasopressin V1b andOxytocin Receptor Messenger Ribonucleic Acidsin Brain*

CHRISTOPHER VACCARI, STEPHEN J. LOLAIT, AND NANCY L. OSTROWSKI

Section on Behavioral Pharmacology (C.V., N.L.O.), Biological Psychiatry Branch and Laboratory ofCellular and Molecular Regulation (S.J.L.), National Institute of Mental Health, National Institutes ofHealth, Bethesda, Maryland 20892-4090

ABSTRACTThe comparative distributions of the vasopressin V1b receptor

(V1bR) and the oxytocin receptor (OTR) messenger RNAs (mRNAs)are described in male rat brain using in situ hybridization histo-chemistry. V1bR transcripts were present in forebrain and hypothal-amus and were less abundant in mid- and hindbrain regions, similarto the gradient observed with OTR transcripts. Microscopic analysesindicated that V1bR expressing cells typically demonstrated the mor-phology of neurons and confirmed V1bR gene expression in regionsincluding the olfactory bulb, supraoptic, suprachiasmatic, and dor-somedial hypothalamic nuclei, piriform and entorhinal cortices, hip-pocampus, substantia nigra, and dorsal motor nucleus of the vagus.Most regions that expressed V1bR mRNA also expressed OTR mRNA,

although OTR gene expression was much more extensive than that ofthe V1bR. V1bR and OTR mRNA distributions were distinct fromeach other and from that of the V1a receptor mRNA in brain. A fewbrain regions express only V1bR transcripts such as the dorsomedialhypothalamic nucleus and the external plexiform layer of the olfac-tory bulb. Other brain regions, such as the fields of Ammon’s horn, thesuprachiasmatic nucleus, the substantia nigra pars compacta, andthe piriform cortex express mRNAs that encode all three receptorsubtypes (V1a, V1b, and OTR), whereas brain areas including the rednucleus and supraoptic nucleus express V1bR and OTR transcriptsonly. These data suggest functional specialization of the V1b, OTRand V1a receptors in brain. (Endocrinology 139: 5015–5033, 1998)

CENTRAL AND PERIPHERAL effects of arginine vaso-pressin (AVP) are mediated by at least three subtypes

of G protein-linked membrane-bound vasopressin receptors.These receptors fall into two classes based on second mes-senger cascades and pharmacological properties. Activationof the V2 vasopressin receptor results in the stimulation ofadenylate cyclase (1) and mediates the well established an-tidiuretic properties of AVP in kidney, where V2 receptors(V2R) (2) and V2R messenger RNA (mRNA) (3) are abun-dant. Stimulation of the V1 class of receptor subtypes resultsin the hydrolysis of phosphatidyl inositol and an increase incytosolic calcium (4). The V1a receptor (V1aR) mediates thevasoconstrictor and hepatic glycogenolytic actions of AVP.This receptor has been cloned (5), and its mRNA has beenvisualized throughout brain (3, 6), liver, and kidney (3, 7)using in situ hybridization histochemistry. The distributionof the V1aR mRNA corresponds to the distribution of ra-diolabeled binding sites for AVP and selective V1aR ligands(2, 8); this receptor is thought to be the predominant AVPreceptor in brain (9, 10).

A second subtype of the V1 class, the V1b receptor (V1bR),has been characterized in the anterior pituitary, where it

regulates AVP-mediated ACTH release by potentiating theeffects of CRH (11, 12). Recent studies have detected V1bRmRNA through RT-PCR in the thymus, heart, lung, spleen,kidney, uterus, and breast (13, 14). Furthermore, Lolait et al.(13) detected V1bR mRNA in several hypothalamic and ex-tra-hypothalamic brain regions. The cellular distribution hasnot been characterized to date.

Receptors for oxytocin are highly homologous to vaso-pressin receptors, bind both oxytocin and AVP with highaffinities, and have been localized throughout brain includ-ing regions where we have detected V1bR mRNA (8). TheOTR mRNA distribution has been characterized in rat brainusing only the human OTR gene-derived ribonucleic acidprobes (riboprobes) (15). In this paper, we describe the dis-tribution of rat V1bR-derived mRNA in male Sprague-Dawley rat brains using in situ hybridization histochemistryand compare it to the distribution of rat OTR gene expressingcells.

Materials and Methods

To determine V1bR mRNA localization, two normal adult maleSprague-Dawley rats (250 g) were euthanized by decapitation, theirbrains rapidly removed and frozen on crushed dry ice and whole brainssectioned for in situ hybridization. Distributions were confirmed inbrains of an additional four adult males that served as control animalsin another experiment (not reported here). Similarly, four adult male ratswere used to characterize the whole brain distribution of the OTRmRNA, and distributions were confirmed in brains of an additional sixmale animals used as controls in another experiment.

All work was done in accordance with National Institutes of Healthguidelines for animal use and care and following approvals of protocolsby the National Institute of Mental Health Animal Care and UseCommittee.

Received July 30, 1998.Address all correspondence and requests for reprints to: Nancy L.

Ostrowski, Ph.D., Eli Lilly and Co., Lilly Corporate Center, Building 22,Drop Code 2244, Indianapolis, Indiana 46285.

* Portions of the OTR mRNA distribution data were presented at theWenner-Gren Conference, Stockholm, Sweden, 1996. This work wasfunded by the NIMH, NIH and by grant no. MH-01050 to C. S. Carterthat provided partial support to C. Vaccari and E. Gournelos, who werealso recipients of Howard Hughes Undergraduate Fellowships from theUniversity of Maryland, College Park.

0013-7227/98/$03.00/0 Vol. 139, No. 12Endocrinology Printed in U.S.A.Copyright © 1998 by The Endocrine Society

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5016 V1b AND OXYTOCIN RECEPTOR mRNAs IN RAT BRAIN Endo • 1998Vol 139 • No 12

V1b AND OXYTOCIN RECEPTOR mRNAs IN RAT BRAIN 5017


FIG. 1. Diagram of the distribution of arginine vasopressin V1b andoxytocin receptor mRNA expressing cells in male rat brain. Distri-butions were plotted based on inspection of nuclear emulsion-dippedbrain sections (3–4 month exposures) under bright and darkfieldconditions. OTR mRNA labeled cells are presented on the left, andV1bR mRNA labeled cells on the right. Abbreviations correspond tothose in Paxinos and Watson (17): 2, 6–10, cerebellar lobules; 2n, opticnerve; 3V, third ventricle; 4V, fourth ventricle; 4&5, cerebellar lob-ules; 6, abducens nucleus; 7, facial nucleus; 7n, facial nerve; 8n,vestibulocochlear nerve; 12, hypoglossal nucleus; 12n, root of hypo-glossal; ac, anterior commissure; ACB, accumbens nucleus; aci, an-terior commissure, intrabulbar; Aco, anterior cortical amygdala nu-cleus; Acs7, accessory facial nucleus; AHC, anterior hypothalamicarea; AHi, amygdalo-hippocampal area; AHiPM, amygdala-hip-pocampal area, posteromedial part; AI, agranular insular cortex; Am,amygdala nuclei; Amb, ambiguous nucleus; AOD, anterior olfactorynucleus, dorsal part; AOM, anterior olfactory nucleus, medial part;AOP, anterior olfactory nucleus, posterior part; AOV, anterior olfac-tory nucleus, ventral part; APir, amygdalo-piriform transition area;APT, anterior pretectal nucleus; Aq, cerebral aqueduct; Arc, arcuatenucleus; AV anteroventral thalamic nucleus; BST, bed nucleus of the

stria terminalis; bsc, brachium of the superior colliculus; CA1-3, fields ofCA 1-3 of Ammon’s horn; CB, cerebellum; cc corpus callosum; CG, centralgray; Cg, cingulate cortex; CIC, central nucleus of the inferior colliculus;CL, claustrum; CnF, cuneiform nucleus; cp, cerebral peduncle; CPu,caudate putamen (striatum); Crus 1, Crus 1 of the ansiform lobule; ctg,central tegmental tract; D3V, dorsal third ventricle; DA, dorsal hypo-thalamic area; DG, dentate gyrus; DLG, dorsal lateral geniculate nu-cleus; Dll, dorsal nucleus of the lateral lemniscus; DMC, dorsomedialhypothalamic nucleus, compact part; DMSp5, dorsomedial spinal tri-geminal nucleus; DR, dorsal raphe nucleus; DSC, lamina dissecans en-torhinal cortex; ec, external capsule; ECIC, external cortex of the inferiorcolliculus; Ecu, external cuneate cortex; Ent, entorhinal cortex; EP, en-topeduncular nucleus; EPI, external plexiform layer of the olfactory bulb;f, fornix; fi, fimbria; Fr, frontal cortex; FStr, fundus striati; Gi, gigan-tocellular reticular nucleus; GP, globus pallidus; HDB, nucleus of thehorizontal limb of the diagonal band; Hil, hilus of the dentate gyrus; I,intercalated nuclei of the amygdala; ic, internal capsule; icp, inferiorcerebellar peduncle; IGr, internal granular layer of the olfactory bulb;InCo, intercollicular nucleus; IntA, interposed cerebellar nucleus; IOC,inferior olive, subnucleus C of the medial nucleus; IPI, interpeduncularnucleus, intermediate; IPC, interpeduncular nucleus, caudal; Ip1, in-ternal plexiform layer of the olfactory bulb; IRt, intermediate reticularnucleus; LC, locus coeruleus; LH, lateral hypothalmic area; LHb, lateralhabenula nucleus; LO, lateral orbital cortex; LPB, lateral parabrachialnucleus; LPGi, lateral paragigantocellular nucleus; LR4V, lateral recessof the fourth ventricle; LRt, lateral reticular nucleus; LS, lateral septalnucleus; LSO, lateral superior olive; LV, lateral ventricle; LVe, lateralvestibular nucleus; mcp, middle cerebellar peduncle; MCPO, magnocel-lular preoptic nucleus; ME, median eminence; me5, mesencephalic tri-geminal tract; mfb, medial forebrain bundle; mfba, medial forebrainbundle “a” component; MGV, medial geniculate nucleus; MHb, medialhabenula nucleus; MI, mitral cell layer of the olfactory bulb; ml, mediallemniscus; mlf, medial longitudinal fasciculus; MM, medial mammillarynucleus, medial part; MnR, median raphe nucleus; Po5, motor trigem-inal nucleus; MP, medial mammillary nucleus, posterior part; MPO,medial preoptic nucleus; MVe, medial vestibular nucleus; Oc, occipitalcortex; ON, olfactory nerve layer; Op, optic nerve layer of the superiorcolliculus; ox, optic chiasm; Par, parietal cortex; PaV, paraventricularnucleus of the hypothalamus; PCRtA, parvocellular reticular nucleus, apart; Pe, periventricular nucleus of the hypothalamus; PF, parafascicu-lar thalamic nucleus; PFI, paraflocculus; PH, posterior hypothalamicarea; Pi, pineal gland; Pir, piriform cortex; PLCo, posterolateral corticalamygdala nucleus; PMCo, posteromedial cortical amygdala nucleus;PMD, premammillary nucleus, dorsal part; Pn, pontine nucleus; PnC,pontine reticular nucleus, caudal part; PnO, pontine reticular nucleus,oral part; PoDG, polymorph layer of the dentate gyrus; PPT, posteriorpretectal nucleus; Pr5VL, principal trigeminal sensory nucleus, ventro-lateral part; Prb, nucleus of Probst’s bundle; PRh, perirhinal cortex; PrH,prepositus hypoglossal nucleus; PrS, presubiculum; PVA, paraventricu-lar nucleus of the thalamus, anterior part; py, pyrimidal tract; RCh,retrochiasmatic area; RMC, red nucleus, magnocellular part; RMg, ra-phe magnus nucleus; ROb, raphe obscurus nucleus; RPC, red nucleus,parvocellular part; RPO, rostral periolivary region; RR, retrorubral nu-cleus; RRF, retrorubral field; RSA, retrosplenial agranular cortex; Rt,reticular thalamic nucleus; RtTg, reticulo-tegmental nucleus of the pons;S, subiculum; s5, sensory root of the trigeminal nerve; scc, splenium ofthe corpus callosum; SCh, suprachiasmatic nucleus; scp, superior cer-ebellar peduncle, brachium conjunctivum; SFO, subfornical organ; SI,substantia inominata; Sim, simple lobule; sm, stria medullaris of thethalamus; SNR, substantia nigra, reticular part; SNC, substantia nigra,compact part; SO, supraoptic nucleus; Sol, nucleus of the solitary tract;sol, solitary tract; SOR, supraoptic nucleus, retrochiasmatic; SPTg, sub-peduncular tegmental nucleus; SPO, superior periolivary nucleus; sp5,spinal trigeminal nucleus; Sp51, spinal trigeminal nucleus, caudal part;Sp5O, spinal trigeminal nucleus, oral part; st, stria terminalis; Stg,stigmoid hypothalamic nucleus; STh, subthalamic nucleus; SuM, su-pramammillary nucleus; Te, temporal cortex; tfp, transverse fibers of thepons; TM, tuberomammillary nucleus; ts, tectospinal tract; Tu, olfactorytubercle; tz, trapezoid body; VCA, ventral cochlear nucleus, anteriorpart; VEn, ventral endopiriform nucleus; VL, ventrolateral thalamicnucleus; VLO, ventrolateral orbital cortex; VMH, ventromedial nucleusof the hypothalamus; VP, ventral pallidum; VPM, ventral posteromedialthalamic nucleus; VTA, ventral tegmental nucleus (Tsai); VTg, ventraltegmental nucleus (Gudden); ZI, zona incerta.


Probes

The region of the AVP and OT receptor genes demonstrating the leasthomology, (i.e. a sequence corresponding to the third intracellular loopof the receptors) was included in each of the riboprobes used. The V1bRprobe was a 618-bp Asp718/blunt end sequence (extending from thesecond intracellular loop to the seventh transmembrane region of the ratcDNA) subcloned into pGEM3Z (13). The OTR cDNA was a 633-bp PCRfragment (spanning the second to sixth transmembrane regions) sub-cloned into pGEM4Z (16). Sense and antisense probes for both V1bR andOTR constructs were synthesized using 35S radiolabeled-UTP. One ad-ditional V1bR probe and two additional OTR probes were prescreenedin liver, brain, kidney, uterus, and spleen. All probes yielded similardistributions of their respective mRNAs. The probes used in these ex-periments demonstrated the greatest signal to noise ratios indexed bythe density of selective tissue labeling relative to background [(Anti-sense-background)–(sense-background)]. In addition to “sense” controlexperiments, V1b, V1a, V2, and OTR transcript labeling was measuredin liver, kidney and pituitary.

In situ hybridization histochemistry

Whole brains were cryosectioned (24 mm-thick), thaw-mounted(three per slide) onto glass microscope slides subbed with gelatin-chrome-alum, and frozen at 270 C until used. Slide-mounted sectionswere rapidly thawed to room temperature, immersed in 4% formalde-hyde solution, treated with acetic anhydride, rinsed, dehydrated, anddelipidated in a series of graded ethanol solutions followed by immer-sion in chloroform. Slide-mounted tissue was covered with 35S-labeledprobe (1.25 million cpm per slide), coverslipped, and incubated for 22 hat 55 C. Posthybridization washes consisted of a series of 43, 23, 13,and 0.53 saline-sodium citrate buffer (SSC) washes, incubation in a 20mg/ml RNase solution at 37 C, followed by a series of 0.13 SSC washescontaining dithiothreitol at 65 and 67.5 C. Tissue was dehydrated in aseries of graded alcohol solutions containing 300 mm ammonium ace-tate, then dried, apposed to autoradiographic film (Kodak B-Max; East-man Kodak, Rochester, NY), and exposed for 14 days.

To visualize the hybridization signal at the cellular level, slides weredipped in nuclear emulsion (Kodak NTB-2; Eastman Kodak) and ex-posed at 4 C for 3 (V1bR) or 4 (OTR) months. Slides were developed(Kodak D19; Eastman Kodak) and stained with thionin, and in somecases, counterstained with eosin and coverslipped. Slides were exam-ined with a light microscope, under bright and dark field illumination.Distributions of cells expressing the respective mRNAs were plottedonto templates modified from the rat brain atlas of Paxinos and Watson(17) using Adobe Photoshop and templates similar to those used forV1aR mRNA (6).

Data analysis

Hybridization signal was quantified using the NIH Image program.Optical densities (corrected for background signal) were obtained for theleft and right hemispheres for each region analyzed. To assess theamount of hybridization signal in emulsion-dipped slides, individualgrains were counted. Cells covered by at least two times as many grainsas background were designated as expressing the given receptor mRNA.

Hybridization experiments for OTR and V1bR mRNAs wereconducted separately, permitting qualitative, but not quantitativecomparisons.

ResultsSpecificity of the probes

Control experiments showed that liver expressed onlyV1aR transcripts, whereas pituitary expressed V1aR, V1bR,and OTR mRNAs in different cell populations. Renal tissuewas the most informative, in that all four transcripts weredifferentially distributed (data not shown): V1aR mRNA waslocalized primarily to vascular elements including vasa recta;V2R mRNA was abundant in the collecting ducts and thickascending limbs of the Loops of Henle; OTR transcripts were

found in macula densa cells in renal cortex, and, in animalspretreated with estrogen, in S2 and S3 segments of proximaltubules (see Ref. 16). V1bR mRNA was not detected in pa-renchymal tissue but was localized in some large renal bloodvessels and in the transitional epithelium of the pelvic wall.Estrogen treatment neither induced nor changed V1bR la-beling in kidney.

V1bR sense labeling was undetectable except in the den-tate gyrus of the hippocampus where low levels of signalwere detected similar to those observed using the antisenseprobe. Microscopic evaluation failed to localize dentate an-tisense or sense labeling to cell bodies; antisense levels didnot reach criteria for specific labeling. OTR sense labelingwas typically near film background levels. Film autoradio-graphs showed above-background levels of sense labeling inthe dentate gyrus and the cerebellum. In the dentate, anti-sense accumulation over cell soma indicated specificlabeling.

Table 1 presents brain regions that express V1bR mRNA.Regions are rank-ordered according to film optical densitymeasurements for the V1bR hybridization signal. The coex-pression of OTR mRNA was determined by microscopicanalyses. The dorsomedial hypothalamic nucleus and theexternal plexiform layer of the olfactory bulb express onlyV1bR mRNA. However, most other regions that expressV1bR transcripts also express the OTR gene.

Figure 1 presents a schematic diagram of the localizationsof the V1bR and OTR transcripts in emulsion-dipped tissues.Qualitative differences were observed in the hybridizationsignals of V1bR and OTR mRNAs. Specifically, in regionswhere V1bR mRNA was detected, signal intensity was usu-ally homogeneous from cell to cell, with a preponderance ofgrains localized to the nucleus and soma of the cells. Signalintensity varied slightly from cell to cell, and film densitiestypically reflect the number of cells in a given region ex-pressing the gene. In contrast, the OTR hybridization signalintensity varied from region to region and from cell to cellwithin specific regions. The signal was not only localized tonuclei and soma but also to the cytoplasm of the cell.

Microscopic analyses confirmed localization of both V1bRand OTR transcripts to cell populations in the olfactory bulb,forebrain, hypothalmus, hippocampus, midbrain, and hind-brain. Both receptor mRNAs were more abundant in anteriorthan in caudal brain. In addition, scattered cells were de-tected on emulsion-dipped slides that were undetectable onfilm autoradiographs. V1bR-labeled cells tended to be large,with large, round nuclei, and demonstrated light stainingNissl substance with a granular appearance. OTR-labeledcells demonstrated no consistently unique morphology.

Olfactory bulb

V1bR mRNA was found in periodically spaced cells in themitral cell layer (Fig. 2, A and C) of the olfactory bulb withdensest label in the ventrolateral bulb. The external plexiformlayer weakly labeled. In contrast, OTR mRNA was distrib-uted throughout the granule cell layer, with a denser ex-pression in the mitral cell layer. The OTR gene was expressedin some periglomerular cells (See Fig. 2, B and D).


Forebrain

V1bR transcripts were found in the tenia tecta and piri-form cortex (Pir). In the tenia tecta, label was greatest inthe most anterior sections, and decreased caudally. Theentire piriform cortex labeled, with strongest signal de-tected in the anterior forebrain and decreasing in intensitycaudally. Several cortical areas expressed light to inter-mediate hybridization signal, including the outer layers ofagranular insular cortex, ventrolateral orbital cortex, lat-eral orbital cortex, and occasional scattered cells in theparietal and frontal cortices. Cells in the outer layers of theventrolateral and lateral aspects of the orbital cortexlightly labeled, primarily around 3.7 mm anterior toBregma. At this level, a string of labeled cells coursed

around the superficial layers of cortex, with label densestin the agranular insular cortex and more scattered in cellsof the parietal and frontal cortices.

The anterior olfactory nucleus (Fig. 3), tenia tecta, andpiriform cortex expressed high levels of OTR mRNA. V1bRtranscripts were not visualized in the anterior olfactory nu-cleus. The distribution of OTR mRNA was similar to that ofV1bR mRNA in the piriform cortex (see Fig. 4) but, whereasOTR transcripts were abundant in the olfactory tubercle,V1bR mRNA was not detected.

OTR expression in frontal cortex exhibited a gradient withscattered cells in layers 2 and 3 labeling throughout; deepercells exhibited less intense label and were more sparselydistributed. Cortical expression was less intense at morecaudal levels. Cingulate cortex expressed moderate levels oftranscripts.

The lateral septal nucleus, ventral pallidum, globus pal-lidus, and accumbens nucleus expressed very low densitiesof the OTR gene. In the caudate putamen, only the dorso-medial portion expressed low levels of the OTR gene.

Hypothalamus and diencephalon

V1bR mRNA was in numerous cells of the suprachias-matic and supraoptic nuclei (SON) of the hypothalamus (Fig.5, A and C). There was light labeling of cells in the periven-tricular nucleus. Grain counts were slightly elevated abovebackground but did not reach criterion for labeling in theparaventricular nucleus of the hypothalamus (Fig. 6, A andC). V1bR transcripts were evident in cells of the medialpreoptic area, lateral hypothalamus, anterior amgydala, andthe region lateral to the SON.

More caudally, label was detected in the hypothalamus atapproximately 3.3 mm posterior to Bregma, where V1bRmRNA transcripts were well labeled in the compact portionof the dorsomedial hypothalamic nucleus. The ventromedialnucleus of the hypothalamus did not express V1bR mRNA.Further caudally, signal was seen in the tuberomammillarynucleus, with light labeling in the dorsal premammillarynucleus.

At approximately 4.3 mm caudal to Bregma, labeled cellsof the piriform cortex and cells in the perirhinal cortex ap-peared contiguous. The posteromedial cortical amygdala nu-cleus (PMCo) expressed moderate amounts of V1bR mRNA,and the posterolateral cortical amygdala nucleus (PLCo) ex-pressed low levels.

In thalamus, only a few cells in the anterior paraventricularnucleus expressed hybridization signal.

Similar to the distribution of V1bR mRNA, OTR tran-scripts were also abundant in the suprachiasmatic and su-praoptic nuclei (See Fig. 5, B and D). OTR, but not V1bRmRNA, was expressed in the paraventricular nucleus (Fig. 6).

OTR transcripts were densest in the ventromedial nucleus(VMN) of the hypothalamus (Fig. 7), with the ventrolateraldivision expressing higher levels of transcript than the dor-somedial division. Notably, the entire VMN, ranging fromthe most anterior to the caudal poles, expressed OTR signal.

OTR gene expression could be visualized in scatteredcells throughout the ventral hypothalamus with high ex-pression in the retrochiasmatic area, the premammillary

TABLE 1. Distribution of arginine vasopressin V1b receptor(V1bR) mRNA in rat brain

Greatest V1bR mRNA labeling

Olfactory bulb: mitral cell layerSuprachiasmatic nucleusPiriform cortexCA2Dorsomedial hypothalamic nucleus (compact region)a

Tenia tectaDorsal rapheSupraoptic nucleusCA3Pontine nucleusTuberomammillary nucleus

Intermediate V1bR labeling

Lateral reticular nucleusSubstantia nigra, pars compactaCA1Dorsal motor nucleus of vagusEntorhinal cortexAgranular insular cortexRed nucleus, magnocellularTrigeminal nucleus, oralVentral tegmental areaPosteromedial cortical amgydala nucleusAmgydala-piriform transitionFacial nucleus

Lowest V1bR labeling

Inferior olivary nucleusLateral hypothalamusVentrolateral orbital cortexPeriventricular hypothalamic areaLateral orbital cortexLocus coeruleusTrigeminal nucleus, interpolarOlfactory bulb: external plexiform layera

Dorsal premammillary nucleusPosterolateral cortical amgydala nucleusGigantocellular reticular nucleus

Film autoradiographs (Kodak, B-MAX; 14 day exposures) wereanalyzed and brain regions rank ordered according to optical densitymeasurements (NIH Image Program). Microscopic analysis of nuclearemulsion dipped tissues was conducted to confirm the cellular local-ization of silver grains. Experiments conducted with the control(sense) mRNA probes showed background levels of signal on films andno regional or cellular accumulation of silver grains. Emulsion-dippedtissue sections were microscopically analyzed to determine whetherOTR mRNA was present or absent in regions that express the V1bRgene. All regions listed above except those marked with an a alsoexpressed OTR mRNA.


nucleus and mammillary complex, the arcuate nucleus,and periarcuate area. A restricted portion (probably in-termediate) of the bed nucleus of the stria terminals ex-pressed some of the densest OTR mRNA labeling in brain.The bed nucleus of the stria terminalis and SON expressedgreater levels of OTR transcripts than the median preopticnucleus, the horizontal nucleus of the diagonal band, themedial preoptic nucleus, and the olfactory tubercle. Themedial habenula exhibited moderate gene expression.Other regions that expressed lower levels of OTR hybrid-ization signal included the central nucleus of the amyg-dala, ventral subiculum, cortical and medial amygdala,subfornical organ, and entorhinal cortex.

Thalamic OTR transcript labeling was sparse with detect-able signal primarily in the anterior paraventricular nucleusof the thalamus (see Fig. 1).

Hippocampus

V1bR mRNA was most abundant in CA2. It was uniformlyexpressed in CA1 (Fig. 8, A, C, and E) whereas signal inten-sity in CA3 became progressively greater more caudally. Thedentate gyrus showed low levels of V1bR gene product thatcould not be localized to cell bodies and did not reach criteriafor specific labeling.

Notably, OTR transcripts were evident in the cell-denseareas of the hippocampus with the dentate gyrus, CA2 andCA3 expressing greater levels than CA1 (Fig. 8, B, D, and F).

Midbrain

Low levels of V1bR mRNA were in cells in the ventraltegmental area (VTA) and the substantia nigra, pars compacta(SNC), whereas only occasional cells expressed transcript in

FIG. 2. Olfactory bulb. V1bR (left) and OTR (right) mRNA distributions differ in the olfactory bulb. Darkfield (A; 4003) and correspondingbrightfield (C; 4003) photomicrographs show V1bR transcripts are in large, lightly thionin-stained cells at the periphery of the mitral cell layer.Granule cells (bottom of photograph) exhibit lower levels of signal. In contrast, OTR mRNA is dispersed throughout the olfactory bulb (B; 253)with hybridization signal concentrated throughout the mitral cell layer (arrow), moderate in periglomerular cells (arrowhead), and least densein the granule cells (bottom of photograph in D; 4003). Silver grains appear white in darkfield and black in brightfield images.


the pars reticulata (Fig. 9). The magnocellular aspect of thered nucleus (Fig. 10) and outer layers of the entorhinal cortexmoderately labeled. Labeling in the entorhinal cortex becamestronger progressing caudally and extended into the deeperlayers of cortex. Strong labeling of V1bR mRNA was evidentin the dorsal raphe nucleus, with lighter labeling in theamgydala-piriform transition area.

OTR mRNA was detected in the red nucleus, dorsal raphenucleus, SNC (Fig. 9), VTA and the central gray.

Hindbrain

Both V1bR and OTR transcripts were less abundant inhindbrain than more anterior brain regions. Pontine nuclei

exhibited moderate V1bR expression, whereas light to in-termediate levels of V1bR signal were detected in the locuscoeruleus, facial nucleus, dorsal motor nucleus of the vagus(DMV), gigantocellular reticular nucleus, lateral reticular nu-cleus (LRt), and trigeminal nucleus (oral and interpolar por-tions). Labeled cells in the regions of the DMV appearedcontiguous with a few labeled cells in the nucleus of thesolitary tract (NTS) and hypoglossal nucleus in emulsion-dipped slides.

Hindbrain OTR transcripts were highest in the dorsal mo-tor nucleus of the vagus (DMV) and Nucleus O. The locusceruleus, and trigeminal and lateral reticular nuclei ex-pressed intermediate levels of transcript, whereas the pon-

FIG. 3. Anterior olfactory nucleus.Brightfield (A; 203) and darkfield (B;203) photomicrographs show OTRtranscripts are in all regions of the an-terior olfactory nucleus (AON) withdenser signal in the ventral quadrants.Arrow points to rostral piriform cortex.Note low levels of OTR mRNA in over-lying cortex.


tine, gigantocellular reticular, medial vestibular, hypoglossalnuclei, and the lateral aspect of the inferior olive nucleusexpressed low hybridization signal.

OTR mRNA was also detected at low levels in blood ves-sels on the ventral surface of the brain, in the pineal, inintermittent large cells (may be Purkinje cells) at the borderof the granule cell layer and in the molecular cell layer of thecerebellum, and cells in the lining of the ventricular walls.

Discussion

Until recently, pharmacologic discrimination of the V1a,V1b, V2, and OTR receptors relied heavily on radioligand

binding assays using AVP, OT, and relatively selective ago-nists and antagonists (8, 10, 11, 18–20). With the cloning ofall four of the rat complementary DNAs for AVP and OTreceptors (V1a: 5; V1b: 13; V2: 21; OTR: 22) in situ hybrid-ization can provide a method of localizing and distinguish-ing among cells that express the genes encoding these re-ceptors. Further, in situ hybridization histochemistryprovides a sufficiently high level of resolution to begin toaddress questions regarding the phenotypes of cells in thebrain that express the AVP and OT receptor subtypes;whether there is regional overlap in their distributions; and,eventually, whether individual cells coexpress these receptor

FIG. 4. Piriform cortex. Photomicro-graphs depicting OTR mRNA labelingin the piriform cortex (PIR) and lateralolfactory tubercle (t, open arrow). A(503), brightfield photomicrographshowing the lateral olfactory tract(LOT), piriform cortex (PIR), and lat-eral aspect of the olfactory tubercle (t).B (503), corresponding darkfield photo.V1bR transcripts were found through-out the piriform cortex but were not ex-pressed in the olfactory tubercle (notshown).


FIG. 5. Supraoptic and suprachiasmatic nuclei of the hypothalamus. V1bR (left) and OTR (right) transcripts have similar distributions in theSON and SCN depicted in the top darkfield panels (A and B, 253). Silver grains appears white. Greater magnification (6303) of cells in theSON (C, D) and SCN (E and F) show differences in V1bR and OTR labeling patterns. Note the clustering of V1bR label near thionin-stainednuclei in C and E. In contrast, OTR mRNA, while densest over stained nuclei, tends to be more diffusely localized (D and F).


FIG. 6. PVN. The PVN expresses OTR mRNA (right) but not V1bR mRNA (left). A and B are brightfield photomicrographs (503) and C andD are corresponding darkfield photomicrographs of the PVN (503). Both the magnocellular (m) and parvocellular (p) regions express OTRhybridization signal. E and F, high magnification (6303) photos of cells at the border between the magnocells and parvocells. Note backgroundlevels of V1b signal over cells (arrows) and diffuse OTR label over individual nuclei (arrows).


FIG. 7. Ventromedial nucleus. The top left darkfield image (A, 503) shows that V1bR transcripts are undetectable in the ventromedial nucleusof the hypthalamus (VMN). High magnification of VMN cells from the ventrolateral subdivision of this nucleus confirms sparse label. Note lowlevels of grains over cells in D (arrow and arrowhead) and G (arrow and arrowhead). In contrast, the ventrolateral (vl) VMN, but not thedorsomedial (dm) VMN from an adult male shows abundant OTR gene expression (B and C, 503). High magnification brightfield (E) anddarkfield (H) images show label surrounding cells. The entire VMN expresses OTR transcripts; both the anterior (not shown) and posterior (p)portions (C) of the VMN express dense accumulations of transcripts over individual cells (F, arrow) and in the surround (I). High magnificationsare 6303.


FIG. 8. Hippocampus. OTR mRNA (right) is more abundant in the hippocampus than V1b mRNA (left). OTR transcripts are moderately densein the dentate gyrus (DG) (D, 253). As can be discerned in panel D, labeling is slightly more abundant in CA2 than CA1 and CA3. Similarly,V1bR transcripts are slightly greater in CA2, followed by CA3, CA1, and the DG (C). E and F, High magnification (4003) of cells sampled atarrows in A and B. Note the restricted localization of V1bR signal and the diffuse distribution of the OTR hybridization signal in CA2 depictedin E and F, respectively.


FIG. 9. Substantia nigra and ventral tegmental area. Both V1bR and OTR transcripts are expressed in the SNC and ventral tegmental area(VTA). Note sparse V1bR mRNA in the medial SNC and VTA in darkfield photomicrograph in A and C (magnification, 253). OTR-labeled cellscan be discerned at 503 magnification (B and D) in the region corresponding to the pars compacta (arrows, C, D). Higher magnification (6303,E and F) shows sparse V1bR (E) and diffuse OTR gene expression (F) in individual cells in the medial SNC (arrows).


mRNAs. In this paper we described the localization of theV1bR and OTR mRNAs in rat brain. We have previouslyreported the localization of the V1aR mRNA (6).

Localization of V1bR mRNA

Cells that express V1bR transcripts are present in anatom-ically discrete brain regions (e.g. suprachiasmatic nucleus,the red nucleus, the substantia nigra pars compacta, and thesupraoptic nucleus) and have the morphology of neurons.V1bR transcripts are less abundant and more restricted intheir distribution than the OTR and the V1aR transcripts.Although the OTR gene is expressed by numerous cells andin regions that express V1bR mRNA, the overall distributionpattern supports distinct receptor gene products. Specifi-cally, the anterior olfactory nucleus and the ventromedialnucleus of the hypothalamus, regions that expressed thehighest levels of OTR transcripts did not express V1bR

mRNA, excluding the possibility that the V1bR probe cross-hybridized with OTR mRNA. Furthermore, the brain distri-bution of V1bR transcripts also differed from that of V1aRmRNA making it improbable that the V1bR riboprobe cross-hybridized with V1aR mRNA. Again, V1bR transcripts wereundetectable in the lateral septum and the anterior olfactorynucleus, two regions that express high levels of V1aR tran-scripts (6). In contrast to a previous study, (23), we have notdetected specific labeling of V2R mRNA in adult rat brain (6,Ostrowski, unpublished).

Following the cloning of the V1bR from pituitary cDNAlibraries, the receptor mRNA was detected by RT-PCR in therat thymus, heart, lung, spleen, kidney, uterus, and breast(13, 14). Lolait et al. (13) have published RT-PCR data pro-viding evidence for extrapituitary expression of V1bRmRNA in the olfactory bulb, caudate putamen, septum, cor-tex, hypothalamus, hippocampus, and cerebellum in the rat.

FIG. 10. Red nucleus. V1bR-mRNA expressing cells in the red nucleus are typical of many cells that express V1bR mRNA. As can be seen inA (103 magnification) the cells tend to lightly stain with thionin, have large cell bodies (B, 4003) and have nuclei with a lightly granularappearance. V1bR-labeled cells in the red nucleus appear to correspond to the magnocells; cells with other morphologies did not label. Large,scattered, labeled cells, similar to those depicted here (arrows), were occasionally detected throughout the midbrain and cortex.


Here, we visualized V1bR transcripts in cells in regionsshown by RT-PCR to express V1bR mRNA with the excep-tion of the caudate putamen, cerebellum and septum wherewe failed to detect transcript. While the reason for this dis-crepancy remains unclear, it is possible that the number ofcopies of transcripts were too low to be visually detectedusing emulsion autoradiography or that the RT-PCR samplesincluded adjacent V1bR-expressing tissues. Confirmation ofexpression of the V1bR protein throughout brain will requirethe development of selective or specific V1bR radioligandsand/or V1bR-specific antibodies.

Permutations in the patterns of coexpression of V1aR,V1bR, and OTR genes in specific brain regions suggest thatthese three gene products may be independently regulated.Moreover, their expression in structures including the hip-pocampus, arcuate and suprachiasmatic nuclei, may suggestalternative interpretations of some radioreceptor bindingdata and pharmacological effects observed after local appli-cation of AVP and other selective agonists and antagonistsdirectly to brain (24, 25).

An interesting observation (See Table 1) was that mostbrain regions that expressed V1bR signal also expressed OTRtranscripts. This is intriguing considering that V1bR mRNAwas extremely restricted in its distribution, in that the ma-jority of discrete brain regions do not express the V1bR gene.The coexpression of OTR in virtually all V1bR-labeled re-gions suggests that there may be a physiological basis fortheir coexistence.

V1bR mRNA and AVP-immunoreactive pathways

AVP immunoreactive cells or processes have been re-ported in most regions where we detect V1bR mRNA. AVPand OT secreting cells originate primarily in the SON andPVN and project to the neural lobe of the pituitary where thehormones are released directly into the bloodstream. CentralAVP projections originate from AVP-synthesizing parvocel-lular neurons in the hypothalamic PVN, a few magnocellularneurons in the SON, the bed nucleus of the stria terminalis(BNST), the dorsomedial hypothalamus (DMH), medial am-gydala, suprachiasmatic nucleus (SCN), and locus coeruleus(26).

V1bR mRNA was not visualized in magnocellular or par-vocellular neurons in the PVN. The V1bR, therefore, is anunlikely candidate for directly regulating AVP or OT releasefrom PVN neurons. In contrast, both OTR and V1aR (6)mRNAs were found in the PVN; the OTR mRNA was de-tected throughout the PVN, whereas V1aR transcripts werelocalized primarily in the parvocellular region. This distri-bution is consistent with a role for OTR in the regulation ofAVP release from the PVN.

OT-containing cells in the SON project to the posteriorpituitary and are well established to play a role in milkejection during lactation (27, 28). The SON expressed bothV1bR and OTR transcripts (but not V1aR, 6). Afferents in-clude noradrenergic projections from the ventrolateral me-dulla (29) and smaller projections from the median preopticnucleus, BNST and dorsomedial hypothalamic nucleus (30),the latter two of which contain AVP-synthesizing neurons.The expression of V1bR and OTR transcripts in the SON

raises the possibility that the V1bR and/or the OTR may beinvolved in regulating OT or AVP release.

The differential localization of OTR mRNA with V1bRtranscripts in the SON and with V1aR gene products in thePVN raises interesting possibilities regarding regulation andcross-talk in AVP- and OT-containing brain systems. Exper-imental data narrowing the possible interactions (e.g. V1bautoreceptor function; mediation of selective effects on AVPor OT transcription or release; regulation of OTR gene ex-pression, etc.) await more precise colocalization data andphysiological experiments using highly selective receptorsubtype agonists and antagonists. Clearly, the selective lo-calization of V1aR and V1bR transcripts in the PVN andSON, respectively, could function to provide a high degreeof specificity in the cellular responses to brain AVP in thesemagnocellular nuclei.

V1bR, OTR (shown here), and V1aR (6) transcripts wereexpressed by cells in the SCN, where AVP-producing neu-rons have also been described (31, 32). The SCN plays a rolein circadian rhythmicity in hormone secretions (33), bodytemperature (34); and sleep and waking cycles (35). AVPexhibits a rhythmic pattern of secretion, with highest levelsduring light periods and lowest levels during dark periods(36). The V1aR mRNA in the SCN also exhibits a circadianrhythm but it is 12 h out of phase with AVP secretion and isindependent of AVP levels (37).

AVP is synthesized in the locus coeruleus and the DMH(26), two regions that also express V1bR transcripts. TheBNST, while a source of AVP, does not express V1bR mRNA.Other areas where AVP immunoreactive processes and V1bRmRNA have been found include the olfactory bulb, arcuatenucleus of the hypothalamus, ventral tegmental area, hip-pocampus, and substantia nigra pars compacta.

Overlap of V1bR with other AVP/OT receptors

The V1a receptor has been thought to mediate most of theeffects of AVP in brain (8, 9, 38–41). The finding that anumber of brain regions express V1aR, V1bR and OTR mR-NAs suggests that some of AVP’s effects on memory andlearning (42), antipyresis (43), selective aggression and part-ner preference (44, 45), cardiovascular responsivity (46),blood flow to the choroid plexus and cerebrospinal fluidproduction (47), smooth muscle tone in superficial brainvasculature (48), and analgesia (49) may involve multiplereceptor subtypes. All three subtypes are expressed in thehippocampus (Fields of Ammon’s horn), arcuate nucleus,locus coeruleus, substantia nigra pars compacta, ventral teg-mental area, suprachiasmatic nucleus, dorsal motor nucleusof the vagus, and piriform cortex. Other regions, such as theolfactory bulb and the hypothalamus express all three tran-scripts but in markedly different patterns suggesting discretefunctions for each receptor subtype. The possibility that sin-gle cells coexpress all three receptor genes remains to bedetermined. Some brain regions such as the suprachiasmaticnucleus, piriform cortex (V1a, V1b, OTR), and the supraopticnucleus (V1b, OTR) appear to be likely candidates for coex-pression based on the large numbers of cells that label witheach respective hybridization probe.


Role for V1bR in brain

To date, the only function attributed to the V1bR has beenthe potentiation of CRH induction of ACTH release in thepituitary (11, 12), and it is possible that V1bR may interactwith CRH receptors at other sites in brain. Several regionsthat are involved in processing olfactory signals express bothCRH receptors (50) and V1bR mRNA such as the olfactorybulb (external plexiform layer), amygdala (PLCo, PMCo,APir, AA), medial preoptic area, cortex (somatosensory, stri-ate, entorhinal, piriform), and pons. A role for V1bR in au-tonomic regulation is suggested by its presence in hindbrainregions, some of which are involved in cardiovascular func-tion. V1bR transcripts were detected in the facial nucleus,lateral reticular nucleus, the dorsal motor nucleus of thevagus (DMV), the nucleus of the solitary tract (NTS) and thetrigeminal nucleus, areas important in AVP’s and CRH’sautonomic effects, including those on heart rate, mean arte-rial blood pressure, and plasma concentrations of epineph-rine, norepinephrine, glucose, and glucagon (46, 51). V1bRtranscripts were in cells of the locus coeruleus (LC) whereAVP and norepinephrine may be colocalized (52) and wherevasopressinergic neurons may be involved in adaptation torepeated stress (53). V1bR mRNA was also detected at lowlevels in the dopamine-synthesizing regions of the substantianigra par compacta and ventral tegmental area. The coex-pression of V1bR and OTR in many brain regions suggeststhat the V1bR may play an interactive role with OT receptivebrain systems, possibly up- or down-regulating them in con-junction with AVP release in specific pathways.

Distribution of OTR mRNA in CNS

We detected OTR transcripts in virtually all brain regionswhere Yoshimura et al. (15) reported hybridization signalusing the human OTR-derived riboprobe (54). In contrast totheir study, we also visualized OTR transcripts in the su-prachiasmatic nucleus and portions of the inferior olivarynucleus. Whereas they described OTR transcripts dorsal tothe substantia nigra pars compacta, we visualized signalthroughout the medial pars compacta and ventral tegmentalarea. Finally, we found OTR transcripts throughout the PVNwhereas Yoshimura and collaborators detected signal onlyover magnocells in the dorso-medial portion. The subtle na-ture of the differences between our results and those ofYoshimura et al. (15) suggest that they may be attributable tothe plane of section of tissue, gender or strain differences.Overall, the degree of correspondence, rather than the di-vergence, is noteworthy.

In summary, in situ hybridization data indicate that theV1bR mRNA is expressed in a restricted number of mor-phologically discrete brain regions. Regions expressing thehighest levels of V1bR transcripts also expressed OTR mRNAalthough the pattern of OTR gene expression was far moreextensive than that of V1bR. Both transcripts, however, weremore abundant in forebrain and hypothalamus than mid-and hindbrain. The differential distributions of the genesencoding the vasopressin V1a, V1b, and OT receptors con-firm that these receptor subtypes are distinct gene productsand suggest that permutations in their regional coexpressionis may influence brain region-specific regulation and func-

tions. It remains to be determined whether V1aR, V1bR, andOTR transcripts colocalize in individual cells. The respectiveroles for these receptor subtypes in influencing cellular ac-tivity and their potential for receptor subtype interactions atthe physiological level require further investigation.1

Acknowledgments

Thanks are extended to Elena Gournelos for her technical assistance,Ricardo Dreifuss for his expert photographic work, and to Drs. Agu Pertand Robert Post for their sponsorship of this project. Appreciation isextended to C. S. Carter for her support and encouragement.

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Comparative Distribution of Vasopressin V1b and Oxytocin ......tocellular reticular nucleus; GP, globus pallidus; HDB, nucleus of the horizontal limb of the diagonal band; Hil, hilus