Spatial distribution of GABABR1 receptor mRNA and binding sites in the rat brain

Spatial Distribution of GABABR1Receptor mRNA and Binding Sites

in the Rat Brain

SERGE BISCHOFF,1* SABINE LEONHARD,1 NICOLE REYMANN,1

VALERIE SCHULER,1 RYUICHI SHIGEMOTO,2 KLEMENS KAUPMANN,1

AND BERNHARD BETTLER1

1Novartis Pharma AG, Nervous System, CH-4002 Basel, Switzerland2Department of Morphological Brain Science, Faculty of Medicine, Kyoto University, Japan

ABSTRACTA g-aminobutyric acid (GABA)B receptor (named GABABR1) has been recently cloned in

the rat and human brain and two variants generated by alternative RNA splicing wereidentified. In the present study, we addressed the question as to whether these variantscontribute to the diversity of GABAB receptor-mediated physiological responses and consti-tute real receptor subtypes with distinct functions. To this aim, we have mapped theGABABR1 (R1a) and GABABR1b (R1b) transcript distribution in the rat brain using in situhybridization. We have compared the mRNA distribution with the distribution of[3H]CGP54626-labeled binding GABABR1 receptor sites as assessed in adjacent cryosectionsby quantitative autoradiography. We found that GABAB receptor transcripts and binding sitesare expressed in the brain in almost all neuronal cell populations. Expression in glial cells, ifany, is marginal. We observed a good parallelism between GABABR1 mRNA transcripts andbinding sites in broad neuroanatomical entities with highest densities in hippocampus,thalamic nuclei, and cerebellum. By contrast, R1a and R1b transcripts exhibit markeddifferences in their regional and cellular distribution pattern. A typical example is thecerebellum with an almost exclusive expression of R1b in the Purkinje cells and of R1a in thegranule, stellate, and basket cells. Data pointing at a pre- versus postsynaptic localization forR1a and R1b, respectively, at some neuronal sites are presented. J. Comp. Neurol. 412:1–16,1999. r 1999 Wiley-Liss, Inc.

Indexing terms: gene expression; in situ hybridization; autoradiography; GABABR1a; GABABR1b

Inhibitory neurotransmission in the mammalian centralnervous systems (CNS) is mainly controlled by g-aminobu-tyric acid (GABA). Pharmacological and physiological stud-ies indicate that the inhibitory control of neuronal excitabil-ity by GABA is mediated by the activation of ionotropicGABAA/C and metabotropic GABAB receptors (Bowery andBrown, 1997; Kerr and Ong, 1995). GABAA/C receptors areligand-gated ion channels that are permeable to chlorideions and hyperpolarize neurons by generating the earlyphase of inhibitory postsynaptic potentials (Mody et al.,1994; Mohler, 1992). GABAB receptors couple to secondmessenger systems through G-proteins, are specificallyactivated by baclofen and insensitive to the GABAA recep-tor agonist bicuculline (Bowery et al., 1980; Hill andBowery, 1981). The predominant native GABAB receptorsare blocked by phaclofen (Kerr et al., 1986), 2-hydroxy-saclofen (Kerr et al., 1988) and derivatives of g-aminopro-pyl-phosphinic acids, such as, e.g., CGP 54626 (Bittiger etal., 1992, 1993; Froestl et al., 1992, 1996). The physiologi-

cal consequences of activating GABAB receptors have beenreviewed (Misgeld et al., 1995). For example, GABABreceptors mediate the late phase of the inhibitory postsyn-aptic potentials in hippocampal (Dutar and Nicoll, 1988;Karlsson and Olpe, 1989; Olpe et al., 1993; Thalmann,1988) and in thalamic neurons (Soltesz et al., 1988),presumably by coupling to the Kir3-type K1 channels(Luscher et al., 1997). Presynaptic GABAB receptors in-hibit neurotransmission by modulating the release ofneurotransmitters through a downregulation of the activ-ity of Ca21 channels (Dolphin and Scott, 1986; Holz et al.,1986). Three GABAB receptors, R1a and R1b (Kaupmannet al., 1997, 1998a) and GABABR2 (R2; Kaupmann et al.,

*Correspondence to: Dr. Serge Bischoff, Novartis, WKL-125.513, CH-4002 Basel, Switzerland. E-mail: [email protected]

Received 18 November 1998; Revised 29 March 1999; Accepted 5 May1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 412:1–16 (1999)

r 1999 WILEY-LISS, INC.

1998b), have been identified. Although the R1a and R1breceptor variants are blocked by available GABAB receptorantagonists (Bettler et al., 1998), the GABABR2 recep-tors are not (Kaupmann et al., 1998b). The aim of thepresent study was to get insight into the cellular andspatial distribution of the R1a and R1b mRNAs and tocompare their expression patterns with the distribution ofGABAB receptor protein, as determined in antagonist[3H]CGP54626 binding experiments. Parts of these datahave been published in abstract form (Bischoff et al.,1997b, 1998).

MATERIALS AND METHODS

Animals and tissue preparation

Adult male rats Tif (RAI [Spf]), weighing 180–200 g,were housed, handled, and killed by decapitation accord-ing to the rules stated in the authorization for animalexperiments delivered by the Veterinary Office of the Baseldistrict. The brains were removed, embedded into cryoglueTissue-Tek (Sakura Finetek, Torrance, CA) and stored at–80°C for cryosectioning. The brains were cut into 10-µm-thick coronal sections in a Cryocut 3000 cryostat (Leica,Glattbrugg, Switzerland) at 220°C to 225°C. Adjacentsections were thaw-mounted onto slides precoated with

poly-L-lysine (Electron Microscopy Sciences, Ft. Washing-ton, PA). The first three sections in a row were used for insitu hybridization (R1 pan, R1a, and R1b riboprobes), thefollowing two sections for autoradiography (total andnonspecific binding). Four to six coronal sections weremounted on a single slide. The sections used for in situhybridization were vacuum-dried overnight at room tem-perature, fixed for 5 minutes in 4% (w/v) ice-cold parafor-maldehyde and washed three times for 1 minute in 13phosphate-buffered saline (PBS). Sections were eitherused the same day for hybridization or stored in sealedboxes at 280°C.

In situ hybridization

The sequences of the DNA templates used for in vitrocRNA synthesis are described in Figure 1A. Two sense andantisense templates, derived from unique N-terminal se-quences, were prepared for each receptor variant. Twoadditional templates were derived from cDNA sequencesshared by the two receptor variants (R1 pan probes). Probesynthesis and hybridization were carried out as describedin details (Bischoff et al., 1997a). Minor modifications arethe posthybridization wash at 70–80°C and exposure ofthe slides to LM-1 liquid nuclear emulsion (LM-1, Amer-sham, England). After 10 to 30 days of exposure, the slides

Abbreviations

Amygd amygdaloid nucleiAOB accessory olfactory bulbAOD anterior olfactory nucleus, dorsalAOM anterior olfactory nucleus, medialAON anterior olfactory nucleusAsp aspartateBLA basolateral amygdaloid nucleus, anteriorCA1 CA1 field of hippocampus properCA3 CA3 field of hippocampus properCc, cc corpus callosumCF climbing fibersCG central gray nucleusCIC central nucleus, inferior colliculiCP caudate putamenDG dentate gyrusDLG dorsolateral geniculate nucleusDMD dorsomedial hypothalamic nucleus, diffuseDRN dorsal raphe nucleusEnt entorhinal cortexEpl external plexiform layerFC frontal cortexfr fasciculus retroflexusGl glomerular layerGlu glutamateGP globus pallidusGr granular layerHP hippocampus properHTM hypothalamusIgr, IGr internal granular layerIMD intermediodorsal thalamic nucleusIPN interpeduncular nucleusIsl,isl islands of CallejaLat deep lateral cerebellar nucleusLC locus coeruleusLDDM laterodorsal thalamic nucleus, dorsomedialLDVL laterodorsal thalamic nucleus, ventrolateralLH lateral habenulaLPMR lateral posterior thalamic nucleus, mediorostralLSI lateral septum nucleus, intermediateM mitral cell layerMDC mediodorsal thalamic nucleus, centralMDM mediodorsal thalamic nucleus, medialMF mossy fibersMG medial geniculate nucleusMH medial habenula

MOB main olfactory bulbMol molecular layerMolDG molecular layer of the dentate gyrusMM mammillary bodyNAc nucleus accumbensOc occipital cortexP Purkinje cellsPAG periacqueductal grayPar parietal cortexPaV paraventricular hyptohalamic nucleus, ventral partPF parallel fibersPir piriform cortexPl Purkinje cell layerPN pontine nucleusPnC pontine reticular nucleusPVP paraventricular thalamic nucleus, posteriorRe reuniens thalamic nucleusRN raphe nucleusRPN raphe pontine nucleusRt reticular thalamic nucleusSC superior colliculiSept septumSNc substantia nigra, pars compactaSNr substantia nigra, pars reticulataSO superior olive nucleiSP50 spinal trigeminal nucleusSth subthalamic nucleusSub subiculumSuG superficial gray layer, superior colliculiThal thalamic nucleiTu olfactory tubercleTT tenia tectaVCO vestibulocochlear nucleusVLG ventrolateral geniculate nucleusVM ventromedial thalamic nucleusVMHDM ventromedial hypothalamic nucleus, dorsomedialVMHVL ventromedial hypothalamic nucleus, ventrolateralVP ventral pallidumVPL ventral posterolateral thalamic nucleusVPM ventral, posteromedial thalamic nucleusVTA ventral tegmental areaZi zona incertaI, II, III, IV,

V, VI cortical layers

2 S. BISCHOFF ET AL.

were developed and the sections were counterstained withcresyl violet for histomorphological discrimination be-tween neurons and glial cells (Gerfen, 1989). Glial cellswere excluded from the quantitative measurements ofneuronal gene expression. Sections stained with cell mark-ers, such as calbindin, calretinin, and tyrosine hydroxylasewere used for the identification of particular cell popula-tions. Brain regions and nuclei were identified according toPaxinos and Watson (1986) and Swanson (1992).

Determination of mRNA expression levels

Quantitative measurement of transcript expression lev-els (represented by the density of silver grains) within agiven cell (visualized by the counterstaining) was per-formed on emulsion-dipped slides. This allowed the distinc-tion between high or low levels of expression from high orlow densities of cells. The analysis was performed ondigitized images of the sections under light microscope(Axioplan, Zeiss AG., Zurich, Switzerland) at a 200-foldmagnification, using a DECpc computer and KS400 image

analysis software (Kontron Elektronik, Eching, Germany).Identical images taken under bright- and darkfield illumi-nation were captured by means of a CCD camera (C 5810color chilled, Hamamatsu Photonics, Herrsching, Ger-many). Subsequently the expression levels were measuredand the data processed for distribution analysis. Thisoperation was performed with an interactive macro. Theprinciple of this processing is to create a relationshipbetween the level of expression and the density of silvergrains spread over a given cell. Simply counting thenumber of grains over a given cell is biased when thegrains form clumps. We therefore measured the surface ofthe cells covered by the grains. For this purpose, theindividual counterstained cells were circled on the bright-field image. A mask was created on which the cells appearas holes in a black matrix. This matrix was then superim-posed over the darkfield image revealing the silver grains.Only the grains that cover the cells were included in theanalysis. For each cell, the surface occupied by the grainswas measured and expressed as the percentage of the total

Fig. 1. Technical specifications. A: Hybridization probes used formRNA expression analysis. Riboprobes were synthesized from ratg-aminobutyric acid (GABA)B receptor (GABABR1a) and GABABR1bcDNA sequences, accessions Y10369 and Y10370, respectively. Barsdenote coding sequences, lines correspond to 5’ and 3’ untranslatedsequences. Probes were synthesized from restriction fragments sub-cloned into Bluescript (Stratagene, La Jolla, CA). The restrictionendonucleases used and the size of the fragments are indicated. Splicevariant selective probes were synthesized corresponding to theN-terminal regions which are unique to GABABR1a and GABABR1b

(open bars). The pan probes detecting both splice variants werederived from sequences common to both receptor splice variants(closed bars). Specificity of probes is illustrated using sense andantisense probes on coronal sections under darkfield microscopyillumination. B: Quantitative autoradiography: the quantitation ofthe density of [3H]CGP54626-labeled GABAB receptors on the autora-diograms is performed by means of the calibration scale with pseudo-colors corresponding to a density of binding sites expressed in fmoles/mgtissue.

GABABR1 RECEPTORS IN THE RAT BRAIN 3

surface of the cell. The values of 4–6 different fields of 1–3sections (depending of the density of cells) were pooled andsubmitted to a statistical analysis. Data reported in Table1 represent the mean of 100–250 cells 6 S.D. of the surfaceof grains/surface of cells 3 100 for each riboprobe. In caseof clearcut differences between cells, e.g., cells devoid ofsignal vs. cells with substantial expression, the quantifica-tion was then performed for each group of cells andspecified in the text (as an example, the granule cells andBlanes cells of the olfactory bulb). To compare the relativelevels of expression, a rating scale was defined in whichthe level of hybridization signal was ranked as very weak,weak, moderate, high, and very high (Table 2). Thisthorough quantitative analysis was performed on sectionsfrom one or two rats, but a similar general distributionpatterns of the three riboprobes has been observed in 15additional rats and mice. Therefore, the present study ismore of indicative than of mathematical value. Photomicro-graphs (Figs. 4 and 5) were generated from representativecoronal sections that have been used for quantitativeanalysis. Sections were placed under the light microscopeat low or high magnification and bright- or darkfieldillumination (stereomicroscope Stemi SV 11 or Axioplan,Zeiss). Digitized images were acquired via the CCD-camera by the PC. Colors were adjusted by means of theKS400 software to improve the resolution, limit the loss ofdetails, and avoid alterations. Images stored as Tiff fileswere then mounted by using the graphic software Micro-soft PowerPoint 4.0 before printing on high quality glossypaper at 720 dpi.

Quantitative autoradiography

The slides were kept at room temperature for 1–2 hoursand then preincubated for 15 minutes in Krebs-Henseleitbuffer (containing 120 mM NaCl, 6 mM glucose, 20 mMTris, 4.7 mM KCl, 1.8 mM CaCl2 2H2O, 1.2 mM KH2PO4,1.2 mM MgSO4, pH 7.4). Total binding was performed byincubating [3H]CGP54626 (2 nM) for 2 hours at roomtemperature in Krebs-Henseleit buffer with the appropri-ate tissue sections. The slides were washed twice for 8seconds in Krebs-Henseleit buffer at 4°C, dipped intoice-cold water, dried in the cold for 30 minutes, andexposed to tritium-sensitive Ultrofilm-3H (Leica, Switzer-land) for 10 days at room temperature. Nonspecific bindingwas defined in the presence of 10-5 M baclofen. Quantita-tive analysis was performed using the MCID image process-ing system (Imaging Research, St. Catherines, Ontario,Canada). Optical densities of gray values on the film wereconverted into bound radioactivity with a polynomialregression curve derived from autoradiographic [3H]micro-scales (RPA 510, Amersham) used as calibration markers.Data were expressed in fmoles/mg tissue. For a bettervisualization, the gray values were converted into pseudo-colors representing the different binding densities (Fig.1B).

In vitro [3H]CGP54626 binding assay

Rat cerebral cortices were homogenized in 10 volumes ofice-cold 0.32 M sucrose containing 1 mM MgCl2 and 1 mMKH2PO4. The membranes were centrifuged at 1,000 3 g for15 minutes, the pellet resuspended and the centrifugationrepeated. The supernatants were pooled and centrifugedat 18,000 3 g for 15 minutes. The pellet was osmoticallyshocked in 5 ml H2O and kept on ice for 30 minutes. Thesuspension was centrifuged at 39,000 3 g for 15 minutes,

resuspended in Krebs-Henseleit buffer, pH 7.4, and keptfor 2 days at 220°C. The membrane preparations werethawed at 20–23°C, washed three times with Krebs-Henseleit buffer by centrifugation at 18,000 3 g for 15minutes, left overnight at 4°C and washed three times.The final pellet was resuspended in 20 ml of buffer; 2 mlaliquots were frozen and stored in liquid nitrogen. For thebinding assays the membranes were thawed in a waterbath at 37°C and washed three times by centrifugation at18,000 3 g for 15 minutes. Radioligand binding at 20–23°Cfor 20 minutes was performed in 1 ml Krebs-Henseleitbuffer, pH 7.4, including 50–100 µg membrane protein, 1nM [3H]CGP54626 and the compounds to be tested. Theincubation was terminated by rapid filtration throughUniFilterTM GF/C 96-well plates (Packard) rinsing twicewith ice-cold buffer. Filterbound radioactivity was countedin Microscint 20 in a Topcount scintillation counter (Pack-ard). Incubations were performed in triplicate. Nonspecificbinding was determined in the presence of 10 µM (-)ba-clofen. Both the in situ hybridization and autoradiographyexperiments have been repeated 3–5 times, using differentrat brain preparations. Similar patterns of GABABR1transcript and binding site distribution were obtained.Brain sections of C57BL/6 and Tif (MAG [Spf]) miceconfirmed the data obtained with horizontal sections of Tif(RAI [Spf]) rats, suggesting that no major species/straindifferences occur.

Drugs

All GABAB receptor agonists and antagonists havebeen synthesized in house and are referred to as CGPcompounds (Novartis, former Ciba-Geigy Products).[3H]CGP54626 (3-N[1(s)-3,4-dichlorophenyl-ethylamino]-2-(s)hydroxypropylcyclo-hexylmethyl phosphinic acid) wastritiated at a specific activity of 60 Ci/mmol (ANAWA,Wangen, Switzerland).

RESULTS

General aspects of the in situ hybridizationhistochemistry

The sequences of the templates used for the antisenseriboprobe synthesis were chosen such as to avoid a possiblecross-hybridization between R1a and R1b. Sense probesdid not specifically label any brain areas, as illustratedwith the R1 pan probe (Fig. 1). The R1a and R1b antisenseprobes detected distinct mRNA expression patterns, indi-cating specificity. The R1 pan antisense probe, derivedfrom sequences common to R1a and R1b, yielded anexpression pattern that corresponds to the superimposedpattern of the R1a and R1b probes, as shown for thecerebellum (Fig. 5A–C). The expression levels in brainlayers, nuclei, or neuronal populations were assessedusing a computer-assisted quantitative method. Data ob-tained with the R1a, R1b, and R1 pan in situ hybridizationprobes were compared with the density of [3H]CGP54626-labeled binding sites (Table 1). Three general observationswere made. Firstly, R1a and R1b transcripts are detectablein neuronal but not in glial cells (measured directly in thewhite matter of fiber tracts such as the corpus callosum,the fornix, the anterior commissura, or the lateral olfac-tory tract). However, glial expression cannot completely beruled out, as the R1 pan probe detected expression levels inglial cells slightly above background (9–13% of the surfaceof the cells is covered with grains as compared to 2–5%


measured with the R1a and R1b probes). Secondly,GABABR1 receptors are expressed in most neurons of thebrain. The level of expression differs between anatomicalentities, ranging from 8% in the molecular layer of thecerebellum to 70% in the dorsolateral geniculate nucleus.The highest densities of GABABR1 mRNA are found in thecerebellum, the hippocampus, and the thalamus. Thirdly,the distribution patterns of the R1a and R1b mRNAsreveal marked differences. The absolute levels of expres-sion of Table 1 cannot be compared directly, as the ribo-

TABLE 1. Levels of Expression of mRNAs and Density of Binding Sites ina Sample of Nuclei Representative of Main Neuroanatomical Entities1

Brain areas, nucleior cell types

R1aprobe

R1bprobe

Panprobe

[3H]CGP54626

Main olfactory bulbPeriglomerular cells 7 6 6 vl2 17 6 10 l 25 6 12 l 1,034Mitral cells 19 6 9 m 12 6 5 l 32 6 10 m 369Granule cells 3 6 2.7 vl 4.8 6 4.9 vl 16 6 10 l 425Tufted cells 14 6 6 l 15 6 7 l 29 6 8 m 248

Basal forebrainCaudate putamen 20 6 9 m 22 6 8 l 19 6 9 l 494Globus pallidus 22 6 9 m 12 6 8 l 34 6 12 m 290Nucleus accumbens 12 6 6 l 25 6 9 m 34 6 11 m 567Olfactory tubercle 8 6 6 l 19 6 7 l 17 6 9 l 485Islands of Calleja 4 6 3 vl 17 6 9 l 29 6 14 m 337Ventral pallidum 9 6 7 l 24 6 8 m 44 6 16 h 442

Amygdaloid nucleiBasolateral nuclei

Anterior 23 6 10 m 21 6 10 l 42 6 17 h 764Posterior 30 6 9 h 16 6 10 l 40 6 15 m 1,006

Basomedial nuclei 18 6 9 m 16 6 9 l 45 6 17 h 922Central nucleus, lateral 27 6 15 h 27 6 15 m 51 6 13 h 812Medial nucleus

Posteroventral 24 6 9 h 27 6 14 m 41 6 17 m 918Posterodorsal 25 6 15 h 23 6 10 m 31 6 15 m 804

Lateral nucleusVentromedian/ventrolateral 30 6 11 h 17 6 9 l 32 6 13 m 1,052Dorsolateral 23 6 9 m 17 6 12 l 45 6 16 h 836

Posteromedial cortical amygnuc. 31 6 12 h 14 6 8 l 46 6 14 h 1,334

Septal areaLateral septum 16 6 9 m 31 6 10 m 34 6 11 m 944Medial septum 9 6 6 l 32 6 11 m 26 6 13 l 679Bed nucleus stria terminalis 12 6 7 l 31 6 10 m 23 6 9 l 607

Hippocampal formationPyramidal layer CA1 34 6 15 vh 43 6 16 h 62 6 17 vh 767Pyramidal layer CA3 35 6 13 vh 31 6 9 m 51 6 14 h 803Dentate gyrus, granular layer 18 6 10 m 19 6 8 l 32 6 13 m 683Hilar cells 31 6 10 h 26 6 10 m 49 6 17 h 1,161Other nonprincipal cells 28 6 9 h 29 6 11 m 47 6 17 hSubiculum dorsal 29 6 10 h 32 6 9 m 57 6 18 vh 961

ThalamusDorsal thalamus

Anterior groupLaterodorsal, ventrolat-

eral (LDVL) 20 6 9 m 48 6 18 vh 51 6 14 h 1,672Laterodorsal, dorsome-

dial (LDDM) 21 6 8 m 51 6 13 vh 46 6 18 h 1,484Dorsal group (IMD) 18 6 9 m 30 6 11 m 46 6 17 h 1,772Lateral group

Lateral posteriormediorostral (LPMR) 18 6 7 m 44 6 14 h 48 6 15 h 1,788

Medial groupParaventricular (PVP) 21 6 8 m 14 6 8 l 19 6 10 l 1,696Mediodorsal (MDM),

medial 19 6 7 m 47 6 14 h 34 6 17 m 1,664Mediodorsal (MDC), cen-

trolateral 22 6 8 m 39 6 11 h 40 6 15 m 1,664Ventral group

Ventral medial (VM) 12 6 6 l 41 6 14 h 54 6 18 h 1,342Ventral posteromedial

(VPM) 17 6 7 m 46 6 13 h 47 6 18 h 1,357Ventral posterolateral

(VPL) 17 6 7 m 43 6 15 h 56 6 18 h 1,074Posterior group 21 6 8 m 46 6 15 h 43 6 16 h 1,452Reuniens 20 6 7 m 17 6 12 l 46 6 15 h 1,782Geniculate group

Medial geniculate nuclei 35 6 12 vh 39 6 14 h 65 6 17 vh 1,548Lateral geniculate nuclei,

dorsal 22 6 8 m 46 6 11 h 70 6 12 vh 1,579Lateral geniculate nuclei,

ventral 32 6 10 vh 26 6 9 m 39 6 14 m 670Epithalamus

Medial habenula 13 6 7 l 59 6 14 vh 44 6 13 h 1,736Lateral habenula 18 6 9 m 45 6 16 l 49 6 17 h 493

Ventral thalamusZona incerta 26 6 11 h 45 6 13 h 50 6 19 h 1,457Reticular thalamic

nucleus 22 6 9 m 9 6 6 vl 37 6 12 m 347Subthalamic nucleus 21 6 9 m 10 6 5 vl 36 6 16 m 273

HypothalamusMedian preoptic nucleus 23 6 10 m 21 6 9 l 29 6 14 m 524Periventricular nucleus 18 6 11 m 14 6 7 l 23 6 14 l 487Medial preoptic area 19 6 8 m 27 6 9 m 37 6 14 m 553Lateral preoptic area 19 6 8 m 24 6 8 m 33 6 15 m 513Anteroventral preoptic

nucleus 15 6 7 l 23 6 9 l 37 6 13 m 487Anterior medial preoptic

nucleus 23 6 11 m 15 6 8 l 27 6 10 l 582Nucleus of the diagonal band 23 6 10 m 24 6 9 m 35 6 18 m 334

TABLE 1. (continued)

Brain areas, nucleior cell types

R1aprobe

R1bprobe

Panprobe

[3H]CGP54626

Magnocellular preopticnucleus 24 6 13 h 29 6 10 m 34 6 13 m 425

Arcuate nucleus 20 6 10 m 12 6 6 l 21 6 12 l 931Dorsomedial hypothalamic

(DMD) 25 6 11 h 15 6 8 l 36 6 14 m 885Ventromedial hypothalamic

Dorsomedial (VMHDM) 27 6 11 h 16 6 9 l 44 6 14 h 765Ventrolateral (VMHVL) 24 6 11 h 16 6 8 l 38 6 14 m 631

Supraoptic nucleus 19 6 7 m 19 6 6 l 29 6 10 m 777Mammillary bodies 30 6 12 h 12 6 9 l 45 6 15 h 789Paraventricular hypothalamic

nucleus 14 6 6 l 18 6 7 l 24 6 8 l 857Midbrain/brainstem

Substantia nigraPars compacta 36 6 13 vh 23 6 10 l 64 6 18 vh 621Pars reticulata 33 6 13 vh 11 6 7 vl 47 6 15 h 544

Ventral tegmental area 36 6 15 vh 21 6 11 l 47 6 16 h 648Interpeduncular nucleus 37 6 13 vh 14 6 7 l 47 6 16 h 1,755Mesencephalic reticular

nucleus 34 6 14 vh 12 6 6 l 38 6 17 m 246Superior colliculi

Superficial gray layer 22 6 11 m 10 6 7 vl 35 6 15 m 1,423Optical layer 30 6 13 h 14 6 7 l 45 6 16 h 579

Periacqueductal gray 35 6 13 vh 24 6 7 m 51 6 14 h 808Ventral cochlear nucleus 29 6 9 h 19 6 8 l 39 6 12 m 225Locus coeruleus 32 6 12 vh 16 6 6 l 41 6 11 m 481Raphe nucleus dorsalis 34 6 12 vh 14 6 6 l 44 6 17 h 786

CortexFrontal

Layer I–II 20 6 8 m 19 6 7 l 35 6 13 m 1,379Layer III 20 6 8 m 14 6 7 l 32 6 14 m 1,303Layer V 17 6 7 m 20 6 8 l 34 6 13 m 1,107Layer VI 24 6 11 h 32 6 11 m 47 6 16 h 659

ParietalLayer I–II 30 6 12 h 23 6 10 l 44 6 16 h 1,387Layer III 31 6 11 h 19 6 7 l 41 6 12 m 1,283Layer IV 30 6 10 h 10 6 6 vl 33 6 12 m 1,093Layer V 25 6 10 h 24 6 8 m 36 6 14 m 898Layer VI 28 6 12 h 32 6 13 m 47 6 16 h 625

CerebellumMolecular layer 9 6 7 l 4.2 6 4.2 vl 8 6 7 vl 1,031Granular layer 16 6 10 m 7 6 12 vl 14 6 6 l 375Purkinje cells 5 6 3 vl 53 6 14 vh 60 6 21 vh 672Deep cerebellar nucleus (lat-

eral) 28 6 8 h 7 6 4 vl 42 6 12 h 231White matter areas (glial cells)

Anterior commissura 1.9 6 1.9 vl 2.1 6 3.6 vl 8.9 6 8.5 vl 145Fornix 7.4 6 6.5 vl 2.2 6 2.9 vl 13 6 12 vl 167Lateral olfactory tract 1.9 6 1.5 vl 2.6 6 4.1 vl 9.6 6 9.6 vl 154Corpus callosum 2.3 6 2.2 vl 4.7 6 5.2 vl 10 6 8.5 vl 152

1The levels of expression in individual brain regions, nuclei, or layers are expressed inthe percentage of the surface of the cells covered by the silver grains and represent themean 6 S.D. of the total number of counted cells. The densities of binding sites,expressed in fmoles/mg tissue, were calculated from the optical densities on the X-rayfilm using tritiated calibration markers.2vl (very low), l (low), m (moderate), h (high), and vh (very high) are the ratings ofexpression levels as defined in Table 2.

TABLE 2. Ratings of Expression Levels1

Probe Very low Low Moderate High Very high

R1a 0–7 8–15 16–23 24–31 32–39R1b 0–11 12–23 24–35 36–47 48–59pan 0–13 14–27 28–41 42–55 56–70[3H]CGP 54626 0–356 357–713 714–1,069 1,069–1,425 1,425–1,781

1The highest value of expression obtained with each riboprobe or of binding density wasdivided equally into five groups.


probes are of different lengths and might have differentnucleotide incorporation rates, and as variable exposuretimes on the nuclear emulsion were applied. For the samereasons, the intensity of the signals on the photomicro-graphs also can not be compared between R1a and R1b.However, comparisons were rendered possible by normaliz-ing each individual value in percent of the highest level ofexpression reached by the given riboprobe. Thus, whenR1a and R1b values are plotted against each other, nosignificant correlation of the expression levels was found(Fig. 2).

[3H]CGP54626 binding and autoradiography

The antagonist [3H]CGP54626 rather than the [3H]agonist ligands was used for autoradiography for two mainreasons. Firstly, with the much higher affinity for GABABreceptors we expected (and indeed reached) a better signal-to-noise ratio and subsequently a higher resolution of theautoradiograms. Secondly, the high selectivity of[3H]CGP54626 minimizes the risk of binding at GABAAreceptors. The binding pharmacology of [3H]CGP54626was determined by using a crude membrane preparationfrom rat cerebral cortex. As shown in saturation bindingexperiments (Fig. 3A,B), [3H]CGP54626 binds specificallyand with high affinity (KD 5 1.7 nM) to a large number of[3H]CGP54626 binding sites (Bmax: 3,003 fmoles/mg pro-tein). Scatchard transformation of the saturation curvesuggests a one-site receptor system. The binding is revers-ible with association and dissociation half times of 1.5 and5.3 minutes, respectively (Fig. 3C). GABA and baclofencompete with [3H]CGP54626 for binding with submicromo-lar IC50 values. Selective antagonists inhibit [3H]CGP54626binding with Hill coefficients close to 1 (Fig. 3D,E).CGP54626 binds recombinant R1a and R1b proteins withthe same affinity as GABAB receptors in cerebral cortex(Kaupmann et al., 1997). CGP54626 does not bind recombi-nant GABABR2 protein with measurable affinity (Kaup-

mann et al., 1998b). Thus, CGP54626 appears to be asuitable ligand for the selective detection of R1a and R1bproteins using autoradiography. The distribution of[3H]CGP54626 binding sites in the brain (Fig. 4A–D)parallels that reported for [3H]GABA (in the presence ofisoguvacine to occlude GABAA receptors) or [3H]baclofen(Bowery et al., 1984, 1987; Bowery and Pratt, 1992; Chu etal., 1990; Gehlert et al., 1985; Turgeon and Albin, 1993;Wilkin et al., 1981), supporting that R1a and R1b receptorsrepresent the most abundant GABAB receptors in thebrain (Bettler et al., 1998). In agreement with previousstudies, the highest density of [3H]CGP54626-labeled bind-ing sites appear in the glomerular layer of the olfactorybulb, the superficial layers of the cortex (I–III), the molecu-lar layer of the dentate gyrus, the medial habenula, thedorsal and medial thalamic nuclei, the lateral and medialgeniculate nuclei, the superficial gray layer of the superiorcolliculi, the interpeduncular nucleus, the pontine nucleus,and the molecular layer of the cerebellum. This distribu-tion pattern markedly differed from that of GABAA recep-tors (Bowery et al., 1987; Bowery and Pratt, 1992; Chu etal., 1990). In most of the areas, the densities of GABAAreceptors were 2–12 times lower. Only the geniculatenuclei and the superior colliculi bear equally high densi-ties of GABAA and GABAB receptors.

Regional distribution of mRNAsand binding sites

The sections on the photomicrographs (Fig. 4A–D) wereanalyzed for the density of [3H]CGP54626 binding incellular populations, nuclei, and layers of the major ana-tomical entities. The data from this study are summarizedin Table 1 and described below.

Olfactory system. The R1a, R1b, and R1 pan probessignificantly hybridized in both the main and accessoryolfactory bulb (Fig. 4A). Quantitative measurements atthe cellular level reveal that the GABABR1 receptor mRNAis heterogeneously expressed at a low level throughout theolfactory bulb. The highest levels of transcripts are foundin the mitral cells and correspond to R1a. In other celltypes, R1a and R1b mRNAs are rare. In the granular layer,R1a and R1b mRNA levels are close to background levels.Only some faintly counterstained cells that are larger thangranule cells and that may represent Blanes cells (Shipleyet al., 1995) exhibit slightly elevated levels of R1 pan-labeled mRNA. [3H]CGP54626 binding is weak in mostparts of the olfactory bulb, except along the glomerularlayer. Thus, R1a/R1b receptors appear to occupy a keyposition at the input interface between olfactory neuronsand the juxtaglomerular cells. It is unlikely that thesereceptors originate in the juxtaglomerular cells, given thelow abundance of mRNA in these cells. GABABR1 recep-tors appear less involved in the control of the output of theolfactory bulb than the input. In particular, the marginalbinding in the external plexiform layer suggests that theGABAergic modulation of the mitral and tufted cells bygranule neurons (Shipley et al., 1995) is probably notmediated by GABABR1 receptors. The moderate level ofR1a transcripts in mitral cells is not translated into asignificant number of [3H]CGP54626 binding in the peri-karya or the dendrites. Therefore, the corresponding recep-tors might be localized presynaptically in terminal fieldswithin the anterior olfactory nucleus, the olfactory tu-bercle, or the piriform cortex (Heimer, 1968).

Fig. 2. Statistical comparison of the levels of expression of R1a andR1b. All values of R1a and R1b, calculated in percent of the highestlevel of expression to each riboprobe, are plotted against each otherand submitted for regression analysis. No correlation at all is foundbetween both receptor variants. GABABR1a, GABABR1b, g-aminobu-tyric acid receptors.


Cortex. We examined the distribution of R1a, R1b,and R1 pan transcripts in all cortical areas but performedthe quantitative analysis in two typical areas of the cortex.The frontal cortex is an agranular isocortical area, repre-sentative of the primary motor cortex, whereas the pari-etal cortex has a clearly visible inner granular layer (layerIV) and represents the main somatosensory cortex (Zilles,1990). Layer I was excluded from the quantitative analysisbecause of the low density of neurons therein; however, thelevels of R1a, R1b, and R1 pan expression are roughlysimilar to those in layers II and III. R1a transcript levels

are moderate to high in various layers of the frontal cortexwith no clear variation. In all five layers of the parietalcortex examined, R1a transcripts are more abundant thanin the frontal cortex. R1b transcript expression in thefrontal and parietal cortex is more heterogeneous andgenerally weaker than for R1a. R1b transcripts are lowin the middle layer (layer III in frontal and granular layerIV in parietal cortex) and increase in abundance in thedeeper layers, with a maximal expression in layer VIb.The density of [3H]CGP54626 binding is globally high inthe most cortical areas. Marked differences occur be-

Fig. 3. Binding characteristics of [3H]CGP54626. Prior to theautoradiography, the binding characteristics of [3H]CGP54626 wereexamined on a membrane preparation of the rat cerebral cortex.A: Total specific and nonspecific binding curves as a function of theconcentration of radioligand. Whereas the curve of nonspecific bindingis linear, that of specific binding reaches a plateau around 5–10 nM,indicating that [3H]CGP54626 binding is saturable. B: Scatchardtransformation and analysis of this saturation curve. The linearity isindicative for a single-site binding with a KD of 1.7 nM and a Bmax of3,003 fmoles/mg protein. C: The dashed line represents specific[3H]CGP54626 binding measured at 25 time intervals from 1 to 60

minutes. It reveals an association rate (T1/2) of the radioligand to thebinding site of 1.5 minutes. At 60 minutes, an excess of L-baclofendissociated the radioligand from the binding sites in a time-dependentmanner with a half-life of 5.3 minutes. These association/dissociationcurves indicate that [3H]CGP54626 binding is reversible. D: Inhibitionof [3H]CGP54626 binding by agonists with the following IC50 values:g-aminobutyric acid (GABA; 3.1 10-7 M), L-baclofen (3.5 10-7 M),CGP47656 (5.3 10-7 M), CGP27492 (3.9 10-8 M). E: Inhibition byantagonists: CGP36742 (1.9 10-7 M), CGP35348 (1.8 10-5 M), CGP52432(8.2 10-8 M), CGP56999 (4.0 10-9 M), and CGP54626 (2.8 10-7 M).


tween individual layers, with a continual decrease in[3H]CGP54626 binding from the external rim of the cor-tex to layer VIb. Layer I exhibits a high density of[3H]CGP54626 binding, similar to layers II and III, despitethe low numbers of neurons present there.

Basal ganglia. The forebrain areas of the dorsal(caudate putamen and globus pallidus) and the ventral(nucleus accumbens, ventral pallidum) basal ganglia cir-cuitry exhibit modest R1 pan transcript levels and a lowdensity of [3H]CGP54626 binding. Differences occur be-tween the dorsal and the ventral circuits when comparingR1a and R1b transcript levels. In the dorsal structures,R1a mRNA expression is moderate, whereas R1b mRNAexpression is low. Ventral structures are particularly poorin R1a mRNA, with a low expression in the nucleusaccumbens and a very low expression in the olfactorytubercle and ventral pallidum. In the islands of Calleja,R1a mRNA levels are barely detectable. The density ofradioligand [3H]CGP54626 binding is low in all theseareas and very low in the globus pallidus because only fewneurons are present there.

Hippocampal formation. The hippocampal forma-tion exhibits a marked presence of GABAB receptors (Fig.4B,C). Strong hybridization signals are obtained in thepyramidal cell layer of the hippocampus proper as well asin the granular layer of the dentate gyrus. At the cellularlevel, a quantitative analysis performed on sections hybrid-ized with the R1 pan probe yielded levels of expressionranging from high to very high, with the exception of thedentate gyrus (Table 1). Similarly, hybridization with theR1a and R1b probes resulted in a dense spread of silvergrains across all neuronal populations in the hippocampalformation. All probes detected a 30–50% lower level oftranscript expression in the granular layer as compared tothe pyramidal cells. In pyramidal neurons, R1a mRNAexpression is rated very high in the CA1 and CA3 fields,whereas the R1b mRNA expression is slightly lower in theCA3 than in CA1 (Fig. 5D–G). Counterstaining with cresylviolet identified interneurons (mostly GABAergic) withinthe hilus of the dentate gyrus (mainly hilar/mossy cells)and the stratum oriens and radiatum of the CA1–CA3fields. Interneurons express R1a and R1b transcripts.Transcript levels are high for R1a and moderate for R1b. Asimilar distribution is found in the subiculum. The distri-bution of radioligand [3H]CGP54626 binding in the hippo-campal formation is more heterogeneous than that for thecorresponding mRNAs. The principal cell layers (pyrami-dal and granule) have the lowest density of [3H]CGP54626binding sites. A higher density of [3H]CGP54626 binding isfound in the stratum oriens and radiatum of the CA3 whencompared to the CA1 subfield (1,021–1,272 vs. 718–924fmoles/mg). A similarly high density of radioligand bindingsites as in CA3 was measured in the molecular layer (1,281fmoles/mg) and the hilus (1,161 fmoles/mg) of the dentategyrus.

Amygdala. The expression of R1 pan, R1a, and R1bhas been analyzed in all amygdaloid nuclei. R1 pantranscript expression levels are moderate in half of thenuclei and high in the other half. These differences do notreflect the anatomical subdivision referred to as the corti-comedial amygdala and the basolateral amygdala (seeMcDonald, 1992). R1a transcripts display high levels ofexpression in two-thirds of the nuclei, whereas R1b tran-scripts show a low to moderate (three nuclei) level ofexpression. The R1b mRNA is more abundant than R1a

mRNA in the bed nucleus of the stria terminalis, whichwas included in this anatomical entity as part of theextended amygdala (Alheid and Heimer, 1988). The den-sity of [3H]CGP54626-labeled binding sites is moderate inmost nuclei of the amygdaloid complex. Slightly higherlevels of [3H]CGP54626 binding are found in the bednucleus of the stria terminalis. The highest density in theamygdala occurs in the posteromedial cortical nucleus(1,334 fmoles/mg vs. an average of 858 fmoles/mg).

Hypothalamus. Fifteen areas and nuclei of the hypo-thalamus were quantitatively analyzed for mRNA levels.Measurements of sections hybridized with the R1 panprobe reveal a homogeneous expression across the entirehypothalamus. The expression of R1a is moderate in amajority of nuclei, whereas it reaches high levels in thenuclei of the medial hypothalamus and the mammillarybody (Fig. 4B,C). In contrast, a majority of the hypotha-lamic nuclei show a low level of R1b transcripts and noneof them reaches high rates. In general, R1a transcriptlevels are higher than R1b transcript levels. The mammil-lary body displays the largest difference between R1a(rated high) and R1b (rated low) transcript levels.[3H]CGP54626 binding sites are more homogeneouslydistributed than the mRNA transcripts throughout thehypothalamus. The overall density is rated as low tomoderate. The nuclei of the medial portion of the hypothala-mus tend to contain a higher density of binding sites thanthose nuclei of the preoptic region.

Thalamus. GABAB receptors are postulated to play arole in the synchronization of the rhythmic membranepotential oscillations in thalamocortical cells (see review,Crunelli and Leresche, 1991). Thus, activation of postsyn-aptic GABAB receptors generates alternating excitatorylow-threshold Ca21 potentials and late inhibitory postsyn-aptic potentials. Thalamocortical neurons represent theoutput neurons of the thalamic principal nuclei that relaysensory and motor information to discrete cortical areas.Their rhythmic activity represents the outcome of a com-plex neuronal network that involves afferents from sen-sory nerves or nuclei in the mesencephalon, GABAergicinterneurons and reciprocal connections with the GABAer-gic thalamic reticular nucleus (see Price, 1995). It istherefore not that surprising that high levels of R1 pantranscripts are detected in the thalamus. Some thalamicnuclei such as the medial and dorsolateral geniculatenuclei as well as most of the dorsal and medial thalamicnuclei contain the highest density of [3H]CGP54626 bind-ing that can be detected in the brain (Fig. 4B,C). GABABreceptors in this group of nuclei are mainly of the R1b type,with high to very high transcript expression levels in mostnuclei. R1a transcript levels are generally low to moder-ate, with the exception of the medial and ventrolateralgeniculate nuclei, which exhibit abundant transcript ex-pression levels. Some nuclei show marked differencesbetween R1a and R1b transcript expression. For example,the medial habenula contains very high amounts of R1btranscripts and low amounts of R1a transcripts (Fig.5H–K). Similarly, the nuclei of the dorsal aspect of thethalamus generally express a higher level of R1b than ofR1a mRNAs. Only in few cases the opposite expressionpattern can be detected: in the nucleus reuniens, thereticular thalamic nucleus, or the subthalamic nucleus,the expression level of the R1b transcripts is lower than forR1a transcripts. Finally, a further interesting mismatchbetween R1a and R1b occurs in the lateral geniculate


complex. Therein, R1b is expressed at much higher levelsin the dorsal aspect, whereas the R1b variant is expressedat very high levels in the ventral part compared to themoderate levels registered in the dorsal nucleus.[3H]CGP54626 binding sites are densely spread over thethalamic nuclei. The nuclei of the dorsal (medial habenula,laterodorsal thalamic nucleus, ventrolateral [LDVL], lat-erodorsal thalamic nucleus, dorsomedial [LDDM]), me-dial, (intermediodorsal thalamic nucleus [IMD], paraven-tricular thalamic nucleus, posterior [PVP], reuniens, zonaincerta) and caudal (dorsolateral and medial geniculatenuclei) aspects exhibit a high to very high density of[3H]CGP54626 binding sites. In contrast, the more lateraland ventral nuclei show a reduced density of radioligandbinding sites. The medial habenula exhibits among thehighest density of [3H]CGP54626 binding sites within thebrain (1,731 fmoles/mg), whereas the reticular thalamicnucleus exhibits very low levels (363 fmoles/mg).

Midbrain/brainstem. The distribution of R1a andR1b transcripts within the midbrain/brainstem was exam-ined in a rostral part. As found in other brain areas,virtually all cells that stain with cresyl violet exhibit R1pan transcripts. Again significant differences are seenbetween R1a and R1b transcript levels (Fig. 4C,D). R1atranscript expression levels range from moderate to veryhigh and in, e.g., the interpeduncular nucleus reach maxi-mal levels. By contrast, the R1b transcript expressionlevels are low to very low. A typical example of such adifferential expression pattern is the substantia nigra/ventral tegmental area (VTA) complex. Neurons in boththe pars compacta and pars reticulata of the substantianigra and in the VTA exhibit a maximal level of R1atranscripts. By contrast, the expression of R1b transcriptsis moderate in the pars compacta and low in the parsreticulata and in the VTA. A similar predominance of R1atranscripts is seen in the raphe nucleus and the locuscoeruleus. In the superior colliculi, the intensity of the R1ahybridization signal varies between layers. In the superfi-cial gray layer the level of expression is moderate, whereasit is high in the optical layer. This is explained by a highlevel of expression in a limited number of large, faintlycounterstained neurons. This contrasts with a low R1atranscript level in small, heavily counterstained cells. R1btranscript expression is low to very low in these cells, withvirtually no expression in the small cell types. The densityof [3H]CGP54626 binding sites varies considerably be-tween the midbrain and the brainstem. Some areas in themidbrain, such as the interpeduncular nucleus or thesuperficial gray layer of the superior colliculi, exhibit highto very high density of radioligand binding sites, whereasbinding within the brainstem appears very low. A fewnuclei such as the suprageniculate nucleus and the pon-tine central gray have a higher density of radioligandbinding sites (700 fmoles/mg). All nerve tracts includingthe vestibulocochlear nerve, the vestibular nerve, rubrospi-nal nerve, the facial nerve, and the pyramidal tract lackdetectable [3H]CGP54626 binding.

Cerebellum. The cerebellum is a brain area that isabundant in R1a/R1b receptors, both in terms of transcriptexpression levels and [3H]CGP54626 binding sites. At lowmagnification, the R1 pan probe yields a strong hybridiza-tion signal in Purkinje cells and the granule cell layer (Fig.4D). Transcript level quantification reveals differences inthe expression level. It is very weak in the molecular layer,weak in the granular layer, high in the deep cerebellar

lateral nucleus, and very high in the Purkinje cells (Table1; Figs. 5A–C, 6). This confirms previous observations inrat and human cerebellum (Bischoff et al., 1997b, 1998;Kaupmann et al., 1997, 1998a). R1a transcripts are weaklyexpressed in stellate and basket cells of the molecularlayer. In the granular layer, the expression of R1a ismoderate and appears as a diffuse and sparse spread ofsilver grains over the entire layer (Fig. 5B). This might beinterpreted as an expression in the Golgi rather than ingranule cells. However, an unequivocal granular expres-sion of R1a is demonstrated by a dramatic decrease ofmessage in the cerebellum of weaver mutant mice that arelacking granule cells (Bischoff et al., 1998). In Purkinjecells, R1a transcript expression is barely above back-ground level. The highest level of R1a expression is seen inthe deep cerebellar nuclei. R1b transcripts are very highlyand almost exclusively expressed in the Purkinje cells(Fig. 5C). As in previous autoradiographic studies usingtritiated agonists, we found a high density of [3H]CGP54626binding sites in the molecular layer of the cerebellum. Thisradioligand binding pattern has been observed with rats(Wilkin et al., 1981; Bowery et al., 1987; Chu et al., 1990;Bowery and Pratt, 1992; Turgeon and Albin, 1993), mice(Bischoff et al., 1998), guinea pigs (Juiz et al., 1994), andhumans (Albin and Gilman, 1990; Kaupmann et al.,1998a). [3H]CGP54626 binding is weak in the Purkinjeand granule cell layers as well as in the deep cerebellarnuclei. No [3H]CGP54626-binding can be detected in theinterposed nucleus.

DISCUSSION

Two GABAB receptor genes that encode the R1a, R1b,and R2 receptors have been identified (Kaupmann et al.,1997, 1998b). It is expected that the predominant nativeGABAB receptors form heteromeric assemblies of R1a/R2and R1b/R2 receptors, but homomeric receptors may occurin vivo as well. Significantly, R1a and R1b bind theantagonist CGP54626A, whereas R2 does not (Kaupmannet al., 1998b). In this report, we have characterized therelative abundance and spatial distribution of the R1a andR1b transcripts that derive from the same gene byN-terminal alternative splicing. In neurons, the sites oftranscript and receptor protein expression do not necessar-

Fig. 4. Photomicrographs of in situ hybridization and autoradio-grams. Low magnification (5–103) darkfield photomicrographs ofadjacent sections expressing the g-aminobutyric acid (GABA)B recep-tor (GABABR1) as assessed with the pan, R1a, and R1b riboprobes.The lowest row of sections illustrates the distribution of [3H]CGP54626 binding sites. Pseudocolors reflect densities of binding sites asdefined in Figure 1. A: From left, first two columns, two levels ofolfactory bulb (scale bar 5 1 mm); third column, cortex and anteriorolfactory nucleus (scale bar 5 1.3 mm); fourth column, caudateputamen and nucleus accumbens (scale bar 5 2 mm). B: Left column,level of caudate putamen and septum, with an indication of corticallayers I to VI on the autoradiogram; middle column, dorsal hippocam-pus and rostral thalamic nuclei; right column, dorsal hippocampusand caudal thalamic nuclei (scale bars 5 2.1 mm). C: Left column,level of ventral hippocampus and mammillary bodies; middle column,ventral hippocampus and substantia nigra/ ventral tegmental area(VTA) complex; right column, midbrain with raphe nucleus andinterpeduncular nucleus (scale bars 5 2.1 mm). D: Left column, levelof brainstem with pontine nucleus and periacqueductal gray (scalebar 5 2.3 mm); middle and right columns, two levels of cerebellum andmedulla oblongata (scale bars 5 1.6 and 2 mm). For abbreviations, seelist.


Figure 4

Figure 4 (Continued)

Fig. 5. Photomicrographs at high magnification. A–C: Darkfieldrepresentation of the detail of a cerebellar lobule showing the expres-sion pattern with the pan (A), R1a (B), and R1b riboprobes (C).D,E, F,G: Expression of R1a and R1b, respectively, in the hippocam-pus. Some pyramidal cells are labeled with arrows, some interneuronsare labeled with arrowheads. D: Expression of R1a in the pyramidalcells (arrow) and in interneurons of the stratum oriens (arrowheads) ofthe CA1 field (at brightfield illumination). E: Darkfield representation

of the expression of R1a in the pyramidal cell layer of the CA3 field andin interneurons of the stratum lucidum and radiatum (arrowheads). Fand G illustrate the corresponding expression pattern of R1b in CA1and CA3. H,I, J,K: Expression of R1a and R1b, respectively, in themedial habenula. L, M: Cellular expression of R1a in the thalamicreticular nucleus at brightfield (L) and darkfield illumination (M) withsome of the positive cells (arrows). For abbreviations, see list. Scalebars 5 60µm for A–D,F,I,K,M; 2 mm for E,G; 10µm for H,J,L.

ily match. We therefore have examined, on sections adja-cent to the ones used for in situ hybridization, the autora-diographic localization of [3H]CGP54626 binding sites thatcorrespond to receptors that incorporate R1a and R1b. Aswe expect the predominant GABAB receptors in the brainto incorporate R1a and R1b, [3H]CGP54626 binding shouldreveal the majority of native GABAB receptors. This issupported by the fact that the [3H]CGP54626 and[3H]GABA (in the presence of isoguvacine) binding pat-terns are very similar. A direct comparison of the tran-script expression levels with binding site densities inidentified cell groups sometimes helps to clarify the cellu-lar and subcellular localization of GABAB receptor pro-teins. With that respect, several conclusions can be drawnfrom our study. Firstly, R1a and R1b transcripts areabundant in neuronal populations throughout the ratbrain. Secondly, the R1a and R1b transcripts display

marked differences in the regional distribution patternand relative abundance. Some of these disparities mayindicate a differential localization of the receptor proteinson somatodendritic processes versus axon terminals. Thiscould reflect a coupling to distinct effector systems. Anexample of an apparent mismatch between the cellularlocalization of R1a and R1b transcripts and [3H]CGP54626binding sites is the cerebellum (Fig.7). High levels of R1btranscripts are present in the Purkinje cells, whereas ahigh density of [3H]CGP54626 binding is found in themolecular layer. Low [3H]CGP54626 binding is found inthe granule and Purkinje cell layers and in deep cerebellarnuclei (the projection area). Altogether, this suggests thatR1b proteins are located mainly on the dendritic arboriza-tion of Purkinje cells in the molecular layer. A postsynapticR1b receptor is expected to regulate excitatory neurotrans-mission at parallel fiber-Purkinje spine synapses and/or

Fig. 6. Quantitation of the expression of the g-aminobutyric acid(GABA)B receptor (GABABR1) in the cerebellum. Levels of expressionwere measured in individual cells and qualified as the relative grainarea. In case of the granular layer, the density of silver grains wasquantified in arbitrary defined fields, as the density of cells is too high.They represent the percentage of the surface of the cells occupied bythe silver grains (see Materials and Methods). The histograms are

constructed from the computer outprints that show bars of 5%intervals of the relative grain area on the abcissa and the number ofcells/fields that display this level on the ordinate. The gaussiandistribution was automatically examined, and the statistical dataexpressed as mean values of the total number of cells/fields 6 S.D. arereported in Table 1.


convey the inhibitory signals provided by GABA releasedfrom the stellate and basket interneuron terminals. Such ahypothesis is supported by immunohistochemistry dataand electron microscopic analysis of Purkinje cell spineswith antibodies directed at receptor variants (Fritschy etal., 1999; Kaupmann et al., 1998; Kulik et al., 1998;Shigemoto et al., 1998) or antibodies directed at baclofen(Martinelli et al., 1992). Together, these findings confirmthe first report of a localization of GABAB receptors inPurkinje cells provided by binding studies in lesioned rats(Bowery et al., 1983). In the granular layer, the moderateexpression of R1a transcripts, together with a weak bind-ing of [3H]CGP54626, suggest that R1a protein is localizedat the terminals of the parallel fiber rather than on thesoma of granule cells. Such a claim is supported by thedemonstration that immunogold particles are present ataxonal sites and in presynaptic membrane specializationof parallel fibers (Kulik et al., 1998). The R1a receptor mayfunction as a presynaptic heteroreceptor and regulate therelease of glutamate from the granule cells.

Other examples of disparity between transcript expres-sion sites and [3H]CGP54626 binding support a pre- versuspostsynaptic localization of R1a and R1b, respectively, atselect neuronal sites. In the glomerular layer of theolfactory bulb, the weak expression of receptor transcriptsis unlikely to be responsible for the strong [3H]CGP54626

binding observed. Possibly slightly higher expression lev-els for R1b mRNA in the juxtaglomerular cells mayaccount for part of the [3H]CGP54626 binding.Apostsynap-tic R1b receptor may be responsible for conveying theGABAergic input originating in the nucleus of the diago-nal band to the olfactory bulb (Zaborszky et al., 1986).Most of the receptor proteins that bind [3H]CGP54626binding in the glomerular layer are likely to be localized onnerve terminals of afferent neurons that synapse onto orclose to juxtaglomerular neurons. Partly, GABAB receptorsmay also be located on nerve terminals of glutamatergicolfactory nerves synapsing onto apical dendrites of mitraland tufted cells within the glomeruli. They may representGABAB heteroreceptors that cause a profound presynapticinhibition of olfactory nerves (Keller et al., 1998; Nickell etal., 1994). Additionally GABAB receptors in the glomerularlayer may localize to prominent serotonergic projectionsfrom the raphe nuclei (McLean and Shipley, 1987) and tonoradrenergic projections arising from the locus coeruleus(Shipley et al., 1985), both of which predominantly expressR1a.

In the reticular thalamic nucleus, the low density of[3H]CGP54626 binding contrasts with a moderate expres-sion of R1a mRNA (Fig. 5L,M) but not R1b mRNA. As mostof the other thalamic nuclei are under the inhibitorycontrol of GABAergic reticular projections, it might be

Fig. 7. Model of the possible localization of the R1a and R1breceptor variants on the cerebellar circuitry. This hypothetical modelis based on our data on the localization of the mRNA transcripts, theautoradiographic distribution of [3H]CGP 54626-labeled binding sites,and the first immunohistochemical data with ultrastructural localiza-tion of antibodies (Kulik et al., 1998; Shigemoto et al., 1998) in themolecular (Mol), Purkinje cell (Pl), and granular layers (Gr) of thecerebellum. The R1b variant, which is essentially expressed in Pur-kinje cells, might play a key role in the inhibitory output of the

cerebellum. The R1a variant is expected to be located on presynapticterminals of the glutamate-containing (Glu) parallel fibers (PF), theexcitatory climbing fibers (CF) of glutamate and/or aspartate nature(Glu/Asp) and the serotonin/noradrenaline modulatory afferents fromthe raphe nucleus (RN) and locus coeruleus (LC). In view of the lowdensity of binding sites in the granular layer, few R1a receptors mightbe localized in the granule or Golgi cells or on the terminals of themossy fibers (MF).


postulated that the R1a mRNA encodes presynaptic auto-receptors that localize to synaptic sites of these projec-tions. However, thus far, no GABAergic autoreceptor activ-ity has been demonstrated (Crunelli and Leresche, 1991)and no GABAB receptors have been observed using elec-tron microscopy at GABAergic presynaptic nerve termi-nals within the thalamus (Shigemoto et al., 1998).

In the mesencephalon, the substantia nigra/VTA com-plex, the raphe nuclei or locus coeruleus which are thesources of the major ascending dopaminergic, serotoner-gic, and noradrenergic projections, respectively, all containhigh to very high densities of R1a mRNA when comparedto R1b. Because a primary role of GABAB receptors is theregulation of monoaminergic neurotransmitter release viapresynaptic heteroreceptors, the baclofen-induced inhibi-tion of the synaptic release of dopamine, serotonin, andnoradrenaline (see reviews, Bowery and Pratt, 1992; Mis-geld et al., 1995; Mott and Lewis, 1994) could be mediatedby R1a.

The medial habenula might be one example that contra-dicts the hypothesis of a pre- versus postsynaptic localiza-tion of R1a and R1b, respectively. This epithalamic nucleuspredominantly expresses R1b transcripts and exhibits avery high density of [3H]CGP54626 binding sites. Themedial habenula projects almost exclusively to the interpe-duncular nucleus in the midbrain (Herkenham and Nauta,1979). Ablation of the medial habenula causes 85% de-crease of GABAB binding in the interpeduncular nucleus(Price et al., 1984). This suggests that in the medialhabenula, R1b encodes presynaptic receptors in the inter-peduncular nucleus. Interestingly, a high density of[3H]CGP54626 binding and R1b immunoreactivity (Shige-moto, unpublished observation) were detected all alongthe fasciculus retroflexus, reflecting a rapid receptor turn-over in the habenula and a massive transport to theinterpeduncular nucleus.

The importance of the GABAB receptor in the brain,from a functional and pathophysiological point of view, hasbeen controversial (Mohler, 1992). The data presented inthis study show that GABAB receptors are abundant in allbrain areas. At least one of the R1a and R1b variants ispresent in most brain neurons, suggesting that GABABreceptors are pivotal for inhibitory neurotransmission.The cloned receptors are involved in the presynapticcontrol of neurotransmitter release (presumably in mostcases through R1a). In addition, the cloned GABAB recep-tor genes appear also to encode postsynaptic receptors thatmay play a crucial role in the outcome of several neuroana-tomical circuits. For example, the predominant expressionof R1b in the dendritic spines of the Purkinje cells, ascompared to R1a but also to GABAA receptors, suggeststhat R1b contributes to the final output of the cerebellum.Similarly, the abundance of R1b transcripts in CA1 pyrami-dal neurons or in thalamocortical cells points to an involve-ment of R1b in the output activity of the hippocampalformation or in relaying basal ganglia signals to the cortex.Altogether, this suggests a role for R1a and R1b in manyphysiological processes, and, in turn, may also point to acritical involvement in pathologies. Possible neurologicaldisorders with a defective GABAB receptor circuitry are,e.g.: (1) olivopontocerebellar atrophy, linked with death ofPurkinje cells (Albin and Gilman, 1990); (2) Parkinson’sdisease linked to a degeneration of nigral dopamine neu-rons; and (3) hemiballism and Huntington’s disease thatare associated with a disruption of the basal ganglia

circuitry at the level of the globus pallidus and thesubthalamic nucleus (Albin et al., 1989; Chesselet andDelfs, 1996; Crossman, 1987). Finally, the presence ofGABAB receptors in some thalamic nuclei and the dose-dependent suppression or activation of nonconvulsive ab-sences seizures (Vergnes et al., 1997) strongly suggest animplication of these receptors in the development and/orpropagation of some types of epileptic seizures. Theseconsiderations stress the potential therapeutic value ofdrugs acting at GABAB receptors. In this respect, ourfinding that R1a and R1b have a different distributionpattern may stimulate the search for compounds thatselectively target receptor variants.

ACKNOWLEDGMENTS

We thank W. Froestl and S.J. Mickel for synthesis ofGABAB ligands, and H. van der Putten for helpful discus-sions. Many thanks to S. Pfister (former graduate studentin our group), and P. Dreja, M. Kohler, P. Schwarb, P.Kaindl (Carl Zeiss A.G., Zurich, Switzerland) for their helpin the design of computer macros for the quantitativeanalysis of in situ hybridization sections. The criticalreview of the manuscript by J. Mosbacher (NovartisPharma, TA Nervous System) and J. Koenig (MarylandPsychiatric Research Center, Baltimore, MD) are grate-fully acknowledged.

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Spatial distribution of GABABR1 receptor mRNA and binding sites in the rat brain