differential expression of ?-aminobutyric acid type b receptor-1a and -1b mrna variants in gaba and...

Differential Expression ofg-Aminobutyric Acid Type B Receptor-1a

and -1b mRNA Variants in GABA andnon-GABAergic Neurons of the Rat Brain

FENGYI LIANG,1* YUMIKO HATANAKA,2 HARUMI SAITO,1 TETSUO YAMAMORI,3

AND TSUTOMU HASHIKAWA1

1Laboratory for Neural Architecture, Brain Science Institute, Riken, Wako,Saitama 351–0198, Japan

2Laboratory for Neuronal Recognition Molecules, Brain Science Institute, Riken, Wako,Saitama 351–0198, Japan

3Division of Speciation Mechanisms 1, National Institute for Basic Biology, Okazaki,Aichi 444–8585, Japan

ABSTRACTTo understand the heterogeneity of g-aminobutyric acid type B receptor (GABABR)-

mediated events, we investigated expression of GABABR1a and 1b mRNA variants in GABAand non-GABAergic neurons of the rat central nervous system (CNS), by using nonradioactivein situ hybridization histochemistry and, in combination with GABA immunocytochemistry,double labeling. In situ hybridization with a pan probe, which recognizes a common sequenceof both GABABR1a and GABABR1b mRNA variants, demonstrated widespread expression ofGABABR1 mRNA at various levels in the CNS. Both GABABR1a and GABABR1b wereexpressed in the neocortex, hippocampus, dorsal thalamus, habenula, and septum, but onlyGABABR1a was detected in cerebellar granule cells, in caudate putamen, and most hindbrainstructures. A majority of GABA neurons in cerebral cortex showed hybridization signals forboth GABABR1a and GABABR1b, whereas those in most subcortical structures expressedeither or neither of the two. GABA neurons in thalamic reticular nucleus and caudateputamen hybridized primarily for GABABR1a. Purkinje cells in the cerebellar cortexexpressed predominantly GABABR1b. GABA neurons in dorsal lateral geniculate nucleus didnot display significant levels of either GABABR1a or GABABR1b mRNAs. These datasuggested widespread availability of GABABR-mediated inhibition in the CNS. The differen-tial but overlapping expression of GABABR1 mRNA variants in different neurons and brainstructures may contribute to the heterogeneity of GABABR-mediated inhibition. Some GABAneurons possessed, but others might lack the molecular machinery for GABABR-mediateddisinhibition, autoinhibition, or both. J. Comp. Neurol. 416:475–495, 2000. r 2000 Wiley-Liss, Inc.

Indexing terms: GABAB receptor; double labeling; immunocytochemistry; in situ hybridization;

forebrain; cerebellum

As the main inhibitory neurotransmitter in the centralnervous system (CNS), g-aminobutyric acid (GABA) playsimportant roles in regulating neuronal activity, plasticity,and pathogenesis. Its action is mediated through distincttype A, B, or C (GABAA, GABAB, or GABAC) receptors. Incontrast to the ionotropic GABAA and GABAC receptorsthat mediate fast inhibitory postsynaptic potentials (IPSP),the metabotropic GABAB receptor (GABABR) mediatesslow, long-lasting inhibitory neuronal responses that havebeen implicated in many important physiological func-tions and pathologic alterations in the brain (Bowery et al.,1990; Soltesz and Crunelli, 1992; Bonanno and Raiteri,1993b; Mott and Lewis, 1994; Misgeld et al., 1995; Bowery,

1997; Deisz, 1997; Bettler et al., 1998). GABABRs havebeen demonstrated in both pre- and postsynaptic compo-nents of both excitatory and inhibitory neurons in the

Grant sponsor: Riken BSI; Grant number: 57911; Grant sponsor: NIBBCooperative Research Program; Grant number: 98–173.

Yumiko Hatanaka’s current address is: Division of Behavior and Neurobi-ology, National Institute for Basic Biology, Myodaiji, Okazaki, Aichi 444–8585, Japan.

*Correspondence to: Dr. Fengyi Liang, Laboratory for Neural Architec-ture, Brain Science Institute, Riken, 2–1 Hirosawa, Wako, Saitama 351–0198, Japan. E-mail: [email protected]

Received 26 March 1999; Revised 4 October 1999;Accepted 5 October 1999

THE JOURNAL OF COMPARATIVE NEUROLOGY 416:475–495 (2000)

r 2000 WILEY-LISS, INC.

central nervous system. Their existence in glia cells hasalso been reported (Hosli and Hosli, 1990; Hosli et al.,1990; Fraser et al., 1994). Presynaptic GABABR-mediatedauto-/heteroinhibition represents a classic, well-knownmodel for regulation of neurotransmitter release and hasbeen subjected to extensive pharmacologic and electro-physiological studies in a variety of brain structures.Postsynaptic GABABRs mediate a late IPSP that mayresults in long-term changes in neurotransmission anddynamic response behavior of neurons (Misgeld et al.,1995; Davies and Collingridge, 1996; Mouginot andGahwiler, 1996; Bowery, 1997; Deisz, 1997; Isaacson andHille, 1997; Bettler et al., 1998).

Among other findings, previous pharmacologic and elec-trophysiological studies have suggested the existence ofdifferent subtypes of GABABRs at pre- or postsynapticsites and in different cell types and brain structures (Dutarand Nicoll, 1988; Bonanno and Raiteri, 1993b; Fukuda etal., 1993; Pitler and Alger, 1994; Bowery, 1997; Deisz et al.,1997; Bettler et al., 1998). The distribution of GABABR inthe CNS has been demonstrated with receptor bindingautoradiography (Bowery et al., 1987; Chu et al., 1990;Knott et al., 1993; Turgeon and Albin, 1993). Expression ofGABABR1 mRNA in the rat brain and in the monkeythalamus has been shown by using radioisotope labeledriboprobes that recognize both of the two major GABABR1mRNA variants, GABABR1a and GABABR1b (Kaupmannet al., 1997; Munoz et al., 1998; Lu et al., 1999). Inaddition, GABABR1 immunoreactivity in the rat brain andretina and GABABR1a and GABABR1b mRNA variantdistribution in the cerebellar cortex and mouse retina havebeen reported (Kaupmann et al., 1998b; Koulen et al.,1998; Zhang et al., 1998; Fritschy et al., 1999; Margeta-Mitrovic et al., 1999). However, it is still unclear how thetwo major GABABR1 mRNA variants are distributed indifferent neuronal cell types in the CNS and how thepossible differences in their distribution may contribute tothe heterogeneity of GABABR-mediated late IPSP andhetero-/autoinhibition of neurotransmitter release in vari-ous CNS structures. To address these issues, we mapped,at cellular level, the expression of GABABR1 gene and itstwo major mRNA variants, GABABR1a and GABABR1b, inthe rat brain by using nonradioactive in situ hybridizationhistochemistry.

Of particular interests and obvious functional roles areGABABRs on GABA neurons. GABABRs on somata/dendrites of GABA neurons may serve as a mechanism toreduce GABA neuronal activity (disinhibition) by recur-rent collaterals or by GABAergic input from other GABAneurons. GABABRs on GABAergic axon terminals (autore-ceptors) play important roles in regulating GABA releasein a frequency-dependent manner. Both processes may actas gating mechanisms for the generation of long-termpotentiation (LTP) and other types of use-dependent modi-fication of neuronal excitability (Floran et al., 1988; Deiszand Prince, 1989; Giralt et al., 1990; Davies et al., 1991;Mott and Lewis, 1991; Metherate and Ashe, 1994; Doze etal., 1995; Hosford et al., 1995; Morishita and Sastry, 1995;Brucato et al., 1996; Caddick and Hosford, 1996; Bonannoet al., 1997). Although GABAB receptors on GABAergicaxon terminals or somata have been demonstrated pharma-cologically and electrophysiologically in a variety of brainstructures, further clarification of their distribution andheterogeneity at molecular level would not only advance

our understanding of the function of this important class ofinhibitory receptor, but also facilitate the search for moreselective GABABR agonists/antagonists (Floran et al.,1988; Harrison et al., 1988; Giralt et al., 1990; Seabrook etal., 1991; Davies and Collingridge, 1993; Lambert andWilson, 1993; Doze et al., 1995; Ulrich and Huguenard,1996; Jarolimek and Misgeld, 1997; Kim et al., 1997; LeFeuvre et al., 1997; Vigot and Batini, 1997; Mouginot et al.,1998; Koulen et al., 1998). Therefore, the second aim of thepresent study was to investigate the expression ofGABABR1a and 1b mRNA variants in different types ofGABA neurons of the rat brain by using immunocytochem-istry in situ hybridization histochemistry double-labelingtechniques. Our results indicated that the distribution ofGABABR1a in the CNS markedly differed from that ofGABABR1b in different GABA and non-GABAergic neu-rons. Preliminary results have been reported in abstractform (Liang et al., 1998).

MATERIALS AND METHODS

cDNA cloning and in vitro transcription

Rat brain mRNA was isolated from a 12-day-old rat pup(Wistar strain). First strand cDNA was synthesized byreverse transcription by using SuperScript II kit (Gibco-BRL, Grand Island, NY). GABABR1 cDNA fragments wereamplified by polymerase chain reaction and isolated byelectrophoresis on agarose gel. They were then subclonedinto the EcoRV site of pBluescript II SK vector (Strata-gene, La Jolla, CA). JM109 competent cells (Stratagene)were transformed by the respective recombinant plasmidsand cultured on LB plates containing ampicillin, isopropyl-1-thio-b-D-galactoside (IPTG) and 5-bromo-4-chloro-3-indolyl-b-D-galactoside (Xgal). White colonies were pickedup from the plate and cultured in 23 TY medium contain-ing ampicillin. Recombinant plasmids were purified fromthe culture and the inserts were verified by sequenceanalysis by using an ABI 310 or ABI 377 DNA sequencer(Perkin-Elmer, Foster, CA).

Three cDNA fragments were cloned. GABABR1a frag-ment is specific for GABABR1a variant, has a C1G ratio of61.78% and corresponds to nucleotides 1 to 437 of ratGABABR1a gene (EMBL accession number Y10369).GABABR1b is specific for GABABR1b variant, has a C1Gratio of 72.73% and corresponds to nucleotides 21 to 141 ofrat GABABR1b gene (EMBL accession number Y10370).GABABR1p recognizes both GABABR1a and GABABR1bvariants, has a C1G ratio of 57.60% and corresponds tonucleotides 2408–2841 (2060–2493) of rat GABABR1a(GABABR1b) gene.

The purified plasmids were linearized and then used astemplates for in vitro transcription of digoxigenin-labeledantisense (or sense control) RNA probes with T3 (or T7)RNA polymerase. The nucleoside triphosphate mix for invitro transcription consists of 10 mM ATP, 10 mM CTP, 10mM GTP, 6.5 mM UTP, and 3.5 mM digoxigenin-11-UTP.According to the supplier (Boehringer Mannheim, Mann-heim, Germany) average digoxigenin-11-UTP incorpora-tion into transcribed RNA is about 4–5% of all nucleotides.

In situ hybridization

Sixteen adult rats (Wistar strain) of either sex, ages 10to 14 weeks and weighing 160–240 grams, were deeplyanesthetized with Nembutal (60 mg/kg, i.p.) and transcar-dially perfused with physiological saline (about 50 ml)

476 F. LIANG ET AL.

followed by 4% paraformaldehyde in 0.1 M phosphatebuffer (800 ml, pH 7.4). The brain and spinal cord wereremoved and postfixed in the same fixative for 4–6 hours at4°C. They were then cryoprotected in 30% sucrose at 4°Cfor 24–48 hours. Thirty-five-micrometer frozen sectionswere cut on a freezing microtome. All procedures involvingexperimental animals were approved by Riken AnimalCare Committee.

In situ hybridization histochemistry followed a protocolmodified from previous descriptions (Liang and Jones,1997; Hatanaka and Jones, 1998). Free-floating sectionswere rinsed twice in phosphate buffered saline (pH 7.4)and then treated sequentially with 0.75% glycine (15minutes 3 2), 0.3% Triton X-100 (10 minutes), 1 µg/mlproteinase K (15 minutes) and 0.25% acetic anhydride in0.1 M triethanolamine (15 minutes). After two washes in23 saline sodium citrate (SSC; 13 SSC 5 0.15 M NaCl,0.015 M Na3C6H5O7 ? 2H2O) and 1 hour prehybridizationat 60°C in a hybridization buffer (pH 7.0) without probe,sections were hybridized at 60°C for approximately 14–16hours in a new hybridization buffer (pH 7.0) that containedone of the digoxigenin-labeled RNA probes. The hybridiza-tion buffer consisted of 2–53 SSC (see below), 2% blockingreagent (Boehringer Mannheim), 50% deionized for-mamide, and 0.1% N-lauroylsarcosine. SSC concentrationin the hybridization buffer was varied between 23 and 53to offset differences in melting temperature derived fromvariations in the length and G1C content of each probes.Probe concentrations of approximately 0.07–0.18 µg/ml forGABABR1a, GABABR1p and 0.25 µg/ml for GABABR1bwere used.

At the end of hybridization, sections were sequentiallytreated in the following solutions (except for ribonucleaseA, all containing 0.1% N-lauroylsarcosine): 23 SSC plus50% deionized formamide, 60°C, twice, 20 minutes each;ribonuclease A buffer (0.01 M Tris-HCl buffer, pH 8.0, plus1 mM ethylenediaminetetraacetic acid and 0.5 M NaCl),room temperature, 10 minutes; ribonuclease A (0.02 mg/mlin ribonuclease A buffer), 37°C, 30 minutes; 23 SSC, 60°C,twice, 20 minutes each; 0.23 SSC, room temperature andthen 60°C, 20 minutes each.

To visualize hybridization signals by immunoalkalinephosphatase histochemistry, sections were rinsed in 0.1 MTris-HCl buffered saline (TBS, pH 7.5, 10 minutes), incu-bated in 1% blocking reagent (in TBS, 60 minutes), andthen in alkaline phosphatase-conjugated sheep anti-digoxigenin antibody (Fab fragments, Boehringer Mann-heim, 1:1,500 dilution in TBS, pH7.5, plus 1% blockingreagent, 60 minutes). After three washes in TBS plus 0.1%Tween 20, alkaline phosphatase (AP) activity was visual-ized either by nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) or by 2-hydroxy-3-naphtoic acid-28-phenylanilide phosphate and fast red TR.The former reaction gave rise to a blue precipitate inhybridization-positive cells, whereas the latter produced ared fluorescent deposit with a maximum absorbance at 550nm and maximum emission at 562 nm. The NBT-BCIPreaction lasted for 6–18 hours at room temperature in asubstrate solution consisting of 0.033% NBT, 0.017% BCIP,0.001 M levamisole, 0.1 M NaCl, and 0.05 M MgCl2 ? 6H2Oin 0.1 M Tris-HCl buffer, pH 9.5. The fluorescent APreaction lasted for 0.5–2 hours at room temperature in asubstrate solution consisting of 0.01% 2-hydroxy-3-naph-toic acid-28-phenylanilide phosphate, 0.025% fast red TR,0.001 M levamisole, 0.1 M NaCl, and 0.01 M MgCl2 in 0.1

M Tris-HCl buffer (pH 8.0). Sections were then eitherprocessed further for GABA immunocytochemistry fordouble labeling (see below) or mounted on chrome alum-gelatin–coated glass slides, air-dried, and cover-slipped byusing an aqueous mounting medium.

Negative control experiments included hybridizationthat used sense riboprobes and ribonuclease A treatmentafter hybridization in 0.01 M Tris-HCl buffer, pH 8.0, and 1mM ethylenediaminetetraacetic acid without NaCl. Posi-tive controls consisted of in situ hybridization with anti-sense riboprobes for glutamic acid decarboxylase-67(GAD67) and brain-derived neurotrophic factor (BDNF).For each animal, a series of sections was also stained withthionin for cytoarchitecture.

GABA immunocytochemistry

For GABA immunocytochemistry or double labeling ofGABA immunoreactivity and GABABR1 mRNA, sectionswere rinsed twice in 0.01 M phosphate buffered saline (pH7.4) before being placed in 5% normal goat serum plus0.3% Triton X-100 in 0.01 M phosphate buffered saline (pH7.4) for 1 hour at room temperature. They were thenincubated for 12 hours at room temperature in an affinity-purified, rabbit anti-GABA serum (Sigma, St. Louis, MO;1:2,000 dilution) and washed in 0.01 M phosphate bufferedsaline (pH 7.4). For GABA immunoperoxidase, subsequentprocesses followed instructions supplied with the avidinbiotinylated-peroxidase complex (ABC) kit from VectorLaboratories (Burlingame, CA). Peroxidase activity wasvisualized by H2O2 (0.01%) and diaminobenzidine tetrahy-drochloride (DAB·4HCl, 0.05%) that gave rise to a brown-ish deposit at the site of GABA immunoreactivity. ForGABA immunofluorescence, bound antibody was revealedby goat anti-rabbit IgG secondary antibody conjugated tofluorescein (Vector Laboratories; 1:100) that yielded greenfluorescence in GABA positive cells. For control reactionsof GABA immunocytochemistry, the primary antibody wasreplaced by equal volume of normal rabbit serum.

Double labeling

For studying the distribution of GABABR1 mRNA signalin GABA neurons, a sequential double-labeling procedurewas carried out on the same histologic sections to visualizefirst GABABR1a, GABABR1b or GABABR1p mRNA signalsby in situ hybridization histochemistry and then GABAimmunoreactivity by immunocytochemistry. As with anydouble- or multiple-labeling techniques, there exists thepossibility of masking of one reaction by the other(s) andthere were those cells that were marginally (ambiguously)positive for one marker or the other; therefore, theiridentity as single- or double-labeled cells could not bedefinitely resolved. These issues were dealt with in thepresent study as follows: First, in addition to a nonfluores-cent double-labeling technique that used NBT-BCIP forrevelation of GABABR1 mRNAs and DAB/H2O2 for GABAimmunoreactivity, a fluorescent double-labeling procedurewas carried out by using the red fluorescent AP reaction forvisualization of GABABR1 mRNAs and then immunofluo-rescence for GABA. Preparations from fluorescent double-labeling experiments were compared with those fromnonfluorescent double labeling to take the advantages ofboth methods and to cross-verify the results. Second, inaddition to conventional microscope, Bio-Rad MRC600laser scanning confocal microscope (Bio-Rad, Hercules,CA) was used to analyze fluorescent double-labeled materi-

EXPRESSION OF GABAB RECEPTOR-1 mRNA VARIANTS IN RAT CNS 477

als to ascertain the identity of individual cells. Finally,only those cells with discernible nuclei were considered fordrawing conclusions on the coexistence of GABA andGABABR1.

Data analysis

Both fluorescent and nonfluorescent histologic prepara-tions were observed under a Nikon Eclipse 800 microscope.Fluorescent histochemical materials were further ana-lyzed and imaged by a Bio-Rad MRC-600 laser scanningconfocal microscope. Levels of hybridization signals forGABABR1 mRNAs in various brain structures were esti-mated by intensity of NBT-BCIP reaction products orfluorescence. Hybridization signals with antisense probesin medial habenular nucleus or Purkinje cells (forGABABR1b and GABABR1p) were denoted as very strong(1111, very high levels) and background hybridizationsimilar to that with sense control probes as negative (-).Intermediate intensities were designated successively asstrong (111, high levels), medium (11, medium levels),weak (1, low levels), and very weak or marginal (6, verylow levels). Initial ratings of relative signal levels invarious structures were carried out independently by twoinvestigators and wherever disagreements arose, a thirdinvestigator was called upon to verify the results.

Low-magnification images of nonfluorescent prepara-tions were photomicrographed first and then digitizedwith a Nikon LS-4500AF film scanner from negative films.High magnification photomicrographs were grabbed di-rectly from the microscope to a Macintosh G3 computer bymeans of a digital color image acquisition system (SonyDXC-950 3CCD color video camera, Scion CG-7 colorimage grabber and Scion Image 1.62 software). Figureswere prepared from digitized images by using AdobePhotoshop 5.0 and printed on a full-color digital printer(Fujix Pictography 3000, Fuji Film, Tokyo, Japan). Forbest printouts, the original image resolutions were scaledto 320 or 400 pixels per inch with Adobe Photoshopsoftware.

RESULTS

All GABABR1a, GABABR1b, or GABABR1p mRNA-positive stainings were located in the cell bodies. Manycells in a majority of neural structures were GABABR1mRNA positive, but levels of mRNA signals in differentcells varied to a large extent. Neurons in the medialhabenular nucleus and cerebellar Purkinje cells showedthe strongest hybridization for GABABR1p riboprobe.Marked differences in the distributions of GABABR1a andGABABR1b mRNA variants were observed in many brainstructures, with some neuronal cell types expressing nei-ther and others expressing either or both. Different sub-types of GABA neurons were distinguished based on theirpossession or lack of GABABR1a and/or GABABR1b mRNAsignals. Table 1 summarizes relative levels of in situhybridization signals for GABABR1a, GABABR1b, orGABABR1p mRNA variant in major brain structures/celltypes. It should be noted that GABABR1b antisense probemight produce a hybridization signal of lower intensity byone order of magnitude in comparison with GABABR1aand GABABR1p because of its short length and extremelylow probability of incorporating digoxigenin-11-UTP (seeDiscussion section). Any correlation between staining in-

tensity and relative mRNA levels for GABABR1a/1p wasnot directly applicable to that for GABABR1b. Also for thisreason, the levels of GABABR1b mRNA expression in theCNS as presented in the present study may represent anunderestimation. Although intensity of hybridization sig-nals in sections from different animals slightly varied, itsrelative ratings among different brain structures in indi-vidual animals kept consistent.

GABABR1b sense control probe yielded a very weakhybridization signal in the pyramidal cell layer of hippo-campal CA1–3 and granule cell layer of the dentate gyrus(Fig. 1B). Other CNS structures did not show any detect-able hybridization other than a very faint backgroundstaining. Hybridization with sense control probes forGABABR1a and GABABR1p did not give rise to positivesignal above background in any CNS structures (Fig. 1A).Posthybridization treatment of sections with ribonucleaseA at low salt concentration also resulted in loss of positivehybridization signals.

Neocortex

Many neurons across all neocortical layers were GAB-ABR1 mRNA positive. Figure 2 compares the expressionpattern of GABABR1a (Fig. 2A) and GABABR1p (Fig. 2B)in the primary somatosensory cortex. Left panels in Figure3 illustrates GABABR1b hybridization in different layersof the motor cortex.

TABLE 1. Relative Levels of Hybridization Signals for GABABR1 mRNAVariants in Major CNS Structures1

Brainregions Structure/cells GABABR1a GABABR1b GABABR1p

Neocortex Layer V pyra-midal cells (11)–(111) (111) (11)–(111)

Other layers (1)–(111) (2)–(11) (1)–(111)Hippocampus Pyramidal cells (111) (11)–(111) (11)–(111)

Granule cells (11) (1) (11)Other neurons (1)–(11) (2)–(11) (1)–(11)

Septum LSD (11)–(111) (11)–(111) (11)–(111)Others (11) (11) (11)

Habenula Medial (11)–(111) (1111) (1111)Lateral (11) (6)–(11) (11)

Thalamus TRN/VLG (1)–(11) (2)–(6) (1)–(11)Other nuclei (11)–(1111) (11)–(1111) (11)–(1111)

Basal ganglia Caudateputamen (1)–(11) (2)–(6) (1)–(11)

Subthalamus (1)–(11) (2)–(6) (1)–(11)SNC (11) (1) (11)–(111)SNR (1)–(11) (2) (1)–(11)

Hypothalamus Supraopticnucleus (11)–(111) (2)–(6) (11)–(111)

Others (1)–(11) (2)–(6) (1)–(11)Cerebellum Basket/stallete

cells (1) (2) (6)Purkinje cells (2)–(6) (1111) (1111)Golgi cells (11)–(111) (2) (11)–(111)Granule cells (1)–(11) (2) (1)–(11)Deep cerebellar

nuclei (1)–(11) (2) (1)–(11)Brain stem Colliculus (1)–(11) (2)–(6) (1)–(11)

Periaquiductgray (1)–(111) (2)–(6) (1)–(111)

Pontine nuclei (11) (2)–(6) (11)Cranial nerve

nuclei (11)–(111) (2)–(1) (11)–(111)Others (1)–(11) (2)–(6) (1)–(11)

Spinal cord Motoneurons (11)–(111) (1) (11)–(111)Other laminae (1)–(11) (2)–(6) (1)–(11)White matter

glia cells (1) (2)–(6) (1)

1Some but not necessarily all neurons in specified structures were positive for themRNA. Hybridization signals: undetected (2), marginal/very weak (6), weak (1),medium (11), strong (111), very strong (1111). Ratings were not for directcomparison between GABABR1a/1p and GABABR1b signals for reasons stated in theDiscussion section. LSD, dorsal lateral septal nucleus; SNC, substantia nigra parscompacta; SNR, substantia nigra pars reticulata; TRN, thalamic reticular nucleus;VLG, ventral lateral geniculate nucleus.

478 F. LIANG ET AL.

In layer I, scattered neurons showed medium levels ofGABABR1a/1p mRNA, but only a few neurons in this layerwere weakly positive for GABABR1b mRNA. In layers IIand III, hybridization signals for GABABR1a/1p variantwere medium to high in many neurons of variable sizes,but those for GABABR1b were low to marginal. Amongdifferent neocortical areas, layers II-III neurons of thetemporal, occipital, and the barrel cortex showed more

pronounced hybridization than other areas. Hybridizationsignals in layer IV neurons were generally weak forGABABR1a/1p and very weak to unobservable forGABABR1b mRNA. In the barrel cortex, neurons insidethe barrels were visibly weaker in their hybridizationsignal for GABABR1a and GABABR1p (Fig. 2). Layer Vpyramidal neurons, especially those of the motor cortex,showed the strongest hybridization of all neocortical neu-

Fig. 1. Photomicrographs showing hybridization by g-aminobu-tyric acid (GABA)BR1a and GABABR1b sense control probes in thehippocampus. A: Hybridization with GABABR1a sense probe producedno hybridization signal above background. Gr, granule cell layer; Or,

stratum oriens; Py, stratum pyramidalis; Rad, stratum radiatum.B: Hybridization with GABABR1b sense probe gave rise to a very weakhybridization signal in pyramidal cells of CA1-CA3 and in granulecells of the dentate gyrus. Scale bar 5 200 µm in B (applies to A,B).


rons for GABABR1a, 1b, or 1p probe, but hybridizationsignals in neurons of smaller sizes in layer V were gener-ally medium to low for GABABR1a/1p and low to belowdetection for GABABR1b mRNA. mRNA levels in mostlayer VI neurons were low for GABABR1a/1p and low tomarginal for GABABR1b (Figs. 2A,B, 3A,C,E).

As revealed by fluorescent double labeling and illus-trated in Figure 4, a majority of GABA neurons in theneocortex expressed GABABR1a/1p. Many neocorticalGABA neurons, especially those in deep layers, also dis-played low to medium levels of GABABR1b mRNA (Fig.3A–F). In layer I, virtually all GABABR1a/1p-positiveneurons were GABAergic. Some GABA neurons in layer Ialso showed low levels of GABABR1b mRNA expression.GABA neurons in layers II–VI were scattered amongnon-GABAergic GABABR1 mRNA positive neurons. Thosein layers II to IV expressed variable levels of GABABR1a/1pand very low to indiscernible GABABR1b mRNA. MostGABA neurons in layers V and VI had weak to mediumGABABR1a, 1b, or 1p mRNA expression (Figs. 3A–F,4A–D). Same results were obtained from nonfluorescentdouble-labeling experiments (data not shown).

Hippocampus and septum

Pyramidal neurons of CA1–3 expressed strongGABABR1a and medium GABABR1b mRNA with rela-tively higher levels of expression for the latter variant inCA1 than in CA2–3. GABABR1p expression representedthe summation of GABABR1a and GABABR1b mRNAvariants in that pyramidal neurons in CA1 showed moreprominent hybridization signals than in CA2–3. Granulecells of the dentate gyrus expressed medium levels ofGABABR1a/1p and low levels of GABABR1b mRNA. Scat-tered neurons in strata oriens, radiatum, and lacunosummoleculare of CA1–3 and molecular, polymorphic layers ofthe dentate gyrus exhibited medium levels of GABABR1a/1pmRNA. Most of them were also weakly positive forGABABR1b mRNA (Fig. 5A–C).

GABA immunocytochemistry revealed a distinct band ofdense GABA axon terminals in stratum radiatum at theproximal dendritic segment of CA3 pyramidal cells (Fig.6A,B). On double-labeling preparations, most GABA neu-rons stained positive for GABABR1a/1p, but a few of themin strata oriens and pyramidalis displayed little

Fig. 2. Photomicrographs showing variable expression levels of g-aminobutyric acid (GABA)BR1a (A)or GABABR1p (B) mRNA in different layers of the primary somatosensory cortex of the rat brain. Corticallayers are indicated in A. Scale bar 5 200 µm in B (applies to A,B).

480 F. LIANG ET AL.

GABABR1a/1p hybridization signal. Both GABA andGABABR1a/1p mRNA levels varied to a certain extent inhippocampal GABA neurons (Fig. 6A–D). GABABR1b sig-nals were also observed in a large proportion of hippocam-pal GABA neurons (Fig. 7A–D).

GABABR1a and GABABR1b coexpressed in the septum.Although there existed considerable variation of GAB-ABR1 mRNA staining intensity among individual neuronal

cells, neurons in the dorsal lateral septal nucleus showedstronger GABABR1a, GABABR1b as well as GABABR1pmRNA staining than those in ventral or medial septalnuclei (Fig. 8A). Panels B to G in Figure 8 show fluorescentdouble labeling of GABABR1 mRNA variants and GABAimmunoreactivity in the septum. All GABA neurons in theseptum appeared positive for GABABR1a, GABABR1b, andGABABR1p mRNA.

Fig. 3. Laser scanning confocal photomicrographs illustratingdouble labeling of g-aminobutyric acid (GABA)BR1b mRNA (leftpanels) and GABA immunoreactivity (right panels) on the samehistologic section of the rat motor cortex. Expression of GABABR1bmRNA in GABA and non-GABAergic neurons in layers I–III (A,B) wasvery weak or below detection. Many cells in layers V (C,D) and VI

(E,F) were moderately positive for GABABR1b. Note the relativelystrong hybridization in layer V pyramidal neurons. Arrows in the rightpanels point to examples of GABA neurons and corresponding arrowsat the same locations in the left panels indicate the GABABR1bexpression status of the same neurons. Scale bar 5 50 µm in F (appliesto A–F).


Thalamus and caudate putamen

Neurons in a majority of dorsal thalamic nuclei dis-played medium to high levels of hybridization signals forboth GABABR1a and GABABR1b mRNA. Thalamic reticu-lar nucleus and ventral lateral geniculate nucleus, how-ever, lacked significant hybridization signal for GABABR1bunder the present experimental condition. As a result,GABABR1p mRNA levels in these nuclei were markedlylower than those in other thalamic nuclei, although levelsof GABABR1a hybridization signals were comparable tothose in the latter. Among different nuclei in the dorsalthalamus, mRNA levels varied in a similar pattern forGABABR1a, 1b, and GABABR1p. Hybridization signals forall three cRNA probes were visibly stronger in neurons ofanterodorsal, mediodorsal, laterodorsal, medial genicu-late, and dorsal lateral geniculate nuclei than in otherthalamic subdivisions. Ventrolateral nucleus exhibitedslightly higher signal intensities than the neighboringventral posteromedial and ventral posterolateral nuclei.Neurons in intralaminar nuclei displayed weaker hybrid-ization signals (Fig. 9A–F, results from anterodorsal andmedial geniculate nuclei not illustrated).

Upper panels in Figure 10 show fluorescent doublelabeling of GABABR1p mRNA hybridization (A) and GABAimmunoreactivity (B) in thalamic reticular nucleus. All

neurons in this structure seemed GABAergic and ex-pressed low to medium levels of GABABR1a/1p mRNAwithout detectable GABABR1b (Figs. 9A–C, 10A,B). In thedorsal lateral geniculate nucleus, GABA neurons weresmaller in size than the surrounding non-GABAergicneurons, showed strong GABA immunoreactivity, but gaveonly marginal GABABR1a and no GABABR1b orGABABR1p mRNA signal (Fig. 11A–F). GABA neurons inventral lateral geniculate nucleus had medium levels ofhybridization signals for GABABR1a and GABABR1p (datanot shown). Most other thalamic nuclei lacked significantnumber of GABA neurons.

In the caudate putamen and globus pallidus, manyneurons expressed low to medium levels of GABABR1a andGABABR1p mRNA, but GABABR1b hybridization signalwas mostly not observed. Scattered GABA neurons in thecaudate putamen and globus pallidus contained weak tomoderate levels of GABABR1a/1p mRNA but expressed noapparent GABABR1b mRNA (Fig. 10C,D).

Cerebellum

Different cell types in the cerebellar cortex had theirunique patterns of expression of GABABR1 mRNA vari-ants. Stellate/basket cells in the molecular layer showedweak GABA immunoreactivity, weak GABABR1a, very

Fig. 4. Laser scanning confocal photomicrographs showing fluores-cent double labeling of g-aminobutyric acid (GABA)BR1a (A,C) andGABA (B,D) on the same histologic section of the rat motor cortex. AllGABA neurons in layers I, II (A,B), and a majority of GABA neurons inlayers V and VI (C,D) were GABABR1a mRNA positive. Arrows in the

right panels point to examples of GABA neurons, and correspondingarrows at the same locations in the left panels indicate the GABABR1aexpression status of the same neurons. Cortical layers are indicated onthe right in A and C. Scale bar 5 100 µm in D (applies to A–D).

482 F. LIANG ET AL.

Fig. 5. Photomicrographs showing g-aminobutyric acid(GABA)BR1a (A), GABABR1b (B), or GABABR1p (C) mRNA expressionin the hippocampus. Note the stronger hybridization signal in CA1

pyramidal neurons than in CA2–3 for GABABR1b and 1p. Gr, granulecell layer; Or, stratum oriens; Py, stratum pyramidalis; Rad, stratumradiatum. Scale bar 5 200 µm in C (applies to A–C).

weak GABABR1p and undetectable GABABR1b mRNAexpression. Purkinje cells were weakly GABAimmunoreac-tive, had strong signals for GABABR1b and GABABR1p,but very weak signals for GABABR1a mRNA. Both Golgiand granule cells showed no signals for GABABR1b mRNA.Golgi cells had medium levels of GABA immunoreactivityand GABABR1a/1p mRNAs. The granule cells were nega-tive for GABA, but weakly positive for GABABR1a/1pmRNA (Fig. 12A–F).

Although Purkinje cells showed very weak hybridizationsignal for GABABR1a at probe concentrations used in thepresent study, they did show medium to strong hybridiza-tion for GABABR1a at higher probe concentrations (.0.4µg/ml) as demonstrated in a series of pilot experimentsdesigned to test the suitable probe concentration and tooptimize the specificity and sensitivity of the technique.This positivity for GABABR1a at higher probe concentra-tions was arbitrarily defined as ‘‘false positive.’’ However,together with the observation that hybridization signal for

GABABR1a was stronger than that for GABABR1p in thestellate/basket cells, these phenomena might have re-sulted from the possible existence of truncated forms ofGABABR1a or other unidentified GABABR1 mRNA vari-ants/subunits in the cerebellar cortex.

In the deep cerebellar nuclei, two populations of neuronscould be distinguished according to their size, GABAimmunoreactivity, and GABABR1 mRNA signals. The rela-tively large neurons, which lacked GABA immunoreactiv-ity, were moderately GABABR1a and GABABR1p mRNApositive. The small GABAergic neurons were weaklyGABABR1a and GABABR1p mRNApositive. No GABABR1bmRNA-positive neurons were detected in the deep cerebel-lar nuclei (data not illustrated).

Other CNS structures

Low to medium levels of GABABR1a/1p hybridizationsignals were observed in various hypothalamic nuclei,with relatively strong signal in the supraoptic nucleus

Fig. 6. Laser scanning confocal photomicrographs showing doublelabeling of g-aminobutyric acid (GABA)BR1 mRNA and GABA immu-noreactivity in the hippocampus. A,B: GABABR1p mRNA and GABAimmunoreactivity in CA2–3. Note a band of GABAergic axon termi-

nals in stratum radiatum in CA3. C,D: GABABR1a mRNA expressionin pyramidal neurons and GABAergic interneurons in CA2–3. Scalebars 5 200 µm in B (applies to A,B); 100 µm in D (applies to C,D).

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(Fig. 13A,B). GABABR1b hybridization in most cases couldnot be ascertained in the hypothalamus because of lowsignal. GABA neurons in the hypothalamus showed low tomedium levels of GABABR1a/1p mRNA.

Neurons in both the pars compacta and reticulata ofsubstantia nigra hybridized at variable signal intensitiesfor GABABR1a and GABABR1p (Fig. 13C). Weak expres-sion of GABABR1b mRNA was only detected in substantianigra pars compacta. Many neurons in various brainstemstructures and all laminae of the spinal cord gray mattershowed low to medium levels of GABABR1a/GABABR1pmRNA. Some cells in the spinal cord white matter alsodisplayed weak GABABR1a/1p hybridization signal. Fig-ure 13D shows the superior colliculus hybridized forGABABR1a mRNA. Although detected at low levels in themotoneurons of various cranial nerve nuclei and of laminaIX of spinal ventral horn, GABABR1b mRNA expression inmost other structures of the brainstem and spinal cord

could not be definitely established due to poor hybridiza-tion signal.

Low to medium levels of GABABR1a/1p hybridizationsignal were detected in many GABA neurons of the brainstem and spinal cord structures including the superior andinferior colliculi, nucleus of the solitary tract, the superfi-cial laminae of the spinal trigeminal nucleus and laminaeII-III of the spinal cord dorsal horn. Figure 13E and Fillustrate such an example of GABABR1p expression inGABA neurons in laminae II-III of the spinal cord dorsalhorn.

DISCUSSION

The present study demonstrated widespread expressionof GABABR1 receptor gene but differential distributions ofthe 1a and 1b mRNA variants in a variety of brainstructures and neuronal cell types. GABA neurons of

Fig. 7. Laser scanning confocal photomicrographs showing double labeling of g-aminobutyric acid(GABA)BR1b mRNA (A,C) and GABA immunoreactivity (B,D) in CA1 strata oriens, pyramidalis, andradiatum (upper panels) or in stratum radiatum and lacunosum moleculare (lower panels) of thehippocampus. Scale bar 5 50 µm in D (applies to A–D).


different areal origins and cell types expressed very differ-ently the GABABR1a and GABABR1b variants.

Widespread availability of GABABR-mediatedinhibition?

GABABR1 mRNAs were expressed by surprisingly nu-merous neuronal cell types throughout the CNS. Althoughtheir levels varied considerably in different cell types andCNS structures, these results may still suggest that theGABAB receptor-mediated mechanisms are widely dispos-able to a variety of neuronal cell types and CNS structures.This corroborated previous data from molecular biological,electrophysiological, pharmacologic, receptor binding auto-radiographic as well as recent immunocytochemical workson GABABRs in a variety of brain structures and cell types.The present results of strong GABAB1 receptor geneexpression in the medial habenular nucleus, dorsal thala-mus, and cerebellar Purkinje cells conform to previousresults by receptor binding, in situ hybridization, andimmunocytochemistry (Bowery et al., 1987; Chu et al.,1990; Knott et al., 1993; Turgeon and Albin, 1993; Kaup-mann et al., 1997, 1998b; Munoz et al., 1998; Margeta-Mitrovic et al., 1999; Fritschy et al., 1999; Lu et al., 1999).GABABR-mediated physiological and pharmacologic ef-

fects have been described in many neural structures andcell types in spinal dorsal horn, cerebellum, substantianigra, thalamus, hypothalamus, hippocampus, and neocor-tex, in line with the present findings of widespread expres-sion of GABABR1 mRNA variants in the CNS (Bowery etal., 1980; Newberry and Nicoll, 1984, 1985; Andrade et al.,1986; Howe et al., 1987; Connors et al., 1988; Soltesz et al.,1989a; Hausser and Yung, 1994; Misgeld et al., 1995;Davies and Collingridge, 1996; Mouginot and Gahwiler,1996; Deisz, 1997; Isaacson and Hille, 1997; Bettler et al.,1998). However, it has been shown that in heterologousmammalian expression systems GABABR1 subunit alonecould not fully function as a mature receptor. A GABABR2receptor subunit has been cloned recently, and heterodimer-ization of the GABABR1 and GABABR2 subunits appearsto be necessary for the assembly of a fully functionalGABAB receptor (Jones et al., 1998; Kaupmann et al.,1998a; White et al., 1998; Kuner et al., 1999). Low-levelexpression of GABABR1 mRNA has also been detected inperipheral organs (Castelli et al., 1999). It remains to beseen whether expression products of the GABABR1 vari-ants serve functions other than mediating late IPSP andauto-/heteroinhibition of transmitter release.

Fig. 8. A: Photomicrograph showing g-aminobutyric acid(GABA)BR1p mRNA expression in the septum. B–G: Laser scanningconfocal photomicrographs showing double labeling of GABA (C, E, G)and GABABR1a (B), GABABR1b (D), or GABABR1p (F) mRNA inlateral septal nucleus. Middle column depicts GABABR1 mRNAhybridization and right column illustrates GABA immunoreactivity in

the same fields. Arrows in the right panels point to some of the GABAneurons and corresponding arrows at the same locations in the middlepanels indicate the expression status of GABABR1 mRNA variants inthe same neurons. Scale bars 5 100 µm in A,G (apply to A and B–G,respectively).

486 F. LIANG ET AL.

Fig. 9. Photomicrographs showing expression of g-aminobutyricacid (GABA)BR1 mRNA variants in the thalamus. A–C: Expression ofGABABR1a (A), GABABR1b (B), or GABABR1p mRNA (C), in differentnuclei of the thalamus. D–F: Differential expression of GABABR1a (D),GABABR1b (E), or GABABR1p (F) mRNA in the dorsal and ventrallateral geniculate nuclei (DLG and VLG). CL, centrolateral thalamic

nucleus; LD, laterodorsal thalamic nucleus; MD, mediodorsal tha-lamic nucleus; R, thalamic reticular nucleus; VL, ventrolateral nucleus;VPL, ventral posterolateral thalamic nucleus; VPM, ventral postero-medial thalamic nucleus. Scale bars 5 400 µm in C (applies to A–C);200 µm in F (applies to D–F).

In general, the present results on the distribution ofGABABR1 mRNAs agree well with that of GABABR1immunoreactivities (Koulen et al., 1998; Malitschek et al.,1998; Fritschy et al., 1999; Margeta-Mitrovic et al., 1999).The lack of detectable GABABR1b mRNA expression in thecaudate putamen, thalamic reticular nucleus, and mostbrain stem structures, as shown in the present study, isreflected by a negative immunostaining thereof (Fritschyet al., 1999). However, in contrast to the present results ofstronger GABABR1a hybridization signals in most CNSstructures, Western blotting results suggest GABABR1b asa dominant variant in the adult rat brain (Malitschek etal., 1998; Fritschy et al., 1999; Margeta-Mitrovic et al.,1999). This discrepancy may arise from the fact thatmRNA levels do not always reflect the synthesis of proteinor the functional relevance of the gene (Tecott et al., 1994;see Lu et al., 1999 for further discussions). More impor-tantly, it should be noted that sequence information spe-cific for GABABR1b is only 140 bp nucleotides. The relativelow GABABR1b signal levels were likely to be attributed to

the short length and very low U content of GABABR1bantisense probe (U contents of GABABR1a, 1p, and 1bantisense probes were 76, 98, and 8, respectively). Thus, ifone digoxigenin-11-UTP is incorporated out of every fiveprobe uracil bases, each copy of GABABR1b antisenseprobe had on average 1.6 digoxigenin molecules, whereaseach copy of GABABR1a and GABABR1p antisense probemight include 15.2 and 19.6 digoxigenin molecules, respec-tively. Considering the amplifying effect by anti-digoxi-genin antibody, the difference could be even bigger innumber of bound alkaline phosphatase molecules permRNA copy between GABABR1a/1p and GABABR1b. Fur-ther complicated by a nonlinear correlation between inten-sity of AP reaction deposits and the number of boundenzyme molecules or the duration of AP reaction, it may bemisleading to directly compare the hybridization signalintensity for GABABR1b variant with those for theGABABR1a or 1p probe. Also for the same reason, low-levelexpression of GABABR1b mRNA in certain structuresmight have escaped detection.

Fig. 10. Laser scanning confocal photomicrographs showing expres-sion of g-aminobutyric acid (GABA)BR1p mRNA in GABA and non-GABAergic neurons in thalamic reticular nucleus and ventral postero-lateral nucleus (A,B) and in the caudate putamen (C,D). Left columnillustrates expression of GABABR1p mRNA and right column, GABA

immunoreactivity. Arrows in the right panels point to some of theGABA neurons, and corresponding arrows at the same locations in theleft panels indicate the GABABR1p expression status of the sameneurons. For abbreviations, see Figure 9. Scale bar 5 100 µm in D(applies to A–D).

488 F. LIANG ET AL.

In addition to neurons, glia-like cells in the spinal whitematter near the pia mater showed weak GABABR1a/1pmRNA expression. This result supports previous findings

of GABABR-mediated effects in glial cells of the CNS (Hosliand Hosli, 1990; Hosli et al., 1990; Fraser et al., 1994).Expression of GABABR1 mRNA in glial cells, however,

Fig. 11. Laser scanning confocal photomicrographs comparingg-aminobutyric acid (GABA) immunoreactivity (right column) andGABABR1 mRNA expression (left column) in dorsal lateral geniculatenucleus. Many non-GABAergic neurons stained positive for GABABR1a(A), GABABR1b (C) or GABABR1p (E) mRNA. However, GABA neu-rons in the same structure, as revealed by simultaneous staining ofGABA immunoreactivity on the same histologic sections and illus-

trated in B, D, and F, respectively, were only marginally positive forGABABR1a and mostly devoid of GABABR1b or GABABR1p mRNAexpression. Arrows in the right panels point to some of the GABAneurons, and corresponding arrows at the same locations in the leftpanels indicate the expression status of GABABR1 mRNA variants inthe same neurons. Scale bar 5 50 µm in F (applies to A–F).


Fig. 12. Photomicrographs showing expression of g-aminobutyricacid (GABA)BR1a (A,B), GABABR1b (C), and GABABR1p (E,F) mRNAexpression in the cerebellar cortex. A, C, and E depict in situhybridization results. B and F show non-fluorescent double labeling ofGABABR1a or GABABR1p mRNA variants (blue) and GABA immuno-

reactivity (brown) on the same histologic sections. D: Distribution ofGABA-immunoreactive cells in the cerebellar cortex. G, Golgi cell; gr,granule cell layer; P, Purkinje cell; s/b, stellate/basket cell. Scale bar 5100 µm in F (applies to A–F).

490 F. LIANG ET AL.

Fig. 13. Photomicrographs showing expression of g-aminobutyricacid (GABA)BR1p mRNA in the hypothalamus (A,B), GABABR1amRNA in the substantia nigra (C) and superior colliculus (D), andGABABR1p mRNA in spinal cord dorsal horn (E). F: GABA-immunoreactive neurons in the same field as E. GABA neurons wereGABABR1p mRNA positive in laminae II-III of the gray matter of the

spinal dorsal horn. InG, intermediate gray layer of superior colliculus;LA, lateral anterior hypothalamic nucleus; LH, lateral hypothalamicarea; SCh, suprachiasmatic nucleus; SNC, pars compacta of substan-tia nigra; SNR, pars reticulata of substantia nigra; SO, supraopticnucleus; SuG, superficial gray layer of superior colliculus. Scale bars 5200 µm in D (applies to A–D), 100 µm in F (applies to E,F).


may be restricted to certain regions of the CNS and at alevel much lower than in neurons because a previous studydid not find significant GABABR1 gene expression in glialcells of hippocampal CA3 region (Kaupmann et al., 1997).The present study, as well, did not detect significant levelsof GABABR1a, 1b, or 1p hybridization signal in glia cells ina majority of rat CNS structures other than the spinalwhite matter.

It is unlikely that the widespread distribution ofGABABR1a and 1p was an artifact caused by nonspecifichybridization or cross-hybridization of the probes withother types of mRNA. In a series of pilot experiments,probe concentrations ranging from 0.07 to 0.7 µg/ml forGABABR1a and GABABR1p were tested. Although probeconcentrations above 0.4 µg/ml gave rise to certain back-ground staining for GABABR1p and strong Purkinje cellpositivity for GABABR1a hybridization, probe concentra-tions ranging from 0.07–0.3 µg/ml produced consistenthybridization patterns for different brain structures andcell types. In addition, hybridization with sense controlprobes gave no positive signal except for the very weakhybridization for GABABR1b sense probe in the hippocam-pus. Finally, the present results on the distribution of thetwo mRNA variants in the cerebellar cortex agree withrecently published data in this structure (Kaupmann etal., 1998b).

Differential expression of GABABR1 mRNAvariants in GABA neurons

As illustrated schematically in Figure 14, four expres-sion profiles of GABABR1a and/or GABABR1b mRNAvariants were distinguished in different GABA neurons.Many GABA neurons in the neocortex, hippocampus and

septum expressed both GABABR1a and GABABR1b vari-ants. This finding is in strong contrast to GABA neurons inmost subcortical structures. GABA neurons in thalamicreticular nucleus, ventral lateral geniculate nucleus, cau-date putamen, and GABAergic stellate/basket cells in thecerebellar cortex hybridized mainly for GABABR1a anti-sense probe without detectable GABABR1b mRNA signal.Cerebellar Purkinje cells showed mainly GABABR1b mRNAexpression. Finally, GABA neurons in the dorsal lateralgeniculate nucleus showed little expression of GABABR1mRNA variants.

Presynaptic GABABR-mediated autoinhibition of GABArelease has been extensively studied and many importantneurophysiological and pathologic processes such as noci-ception, use-dependent depression of inhibition, LTP, andepileptogenesis have been associated with this self-controlling mechanism (Floran et al., 1988; Giralt et al.,1990; Seabrook et al., 1991; Morishita and Sastry, 1995;Bonanno et al., 1997; for reviews, see Bowery, 1997; Deisz,1997; Bettler et al., 1998). GABABR-mediated postsynap-tic responses in GABA neurons have been less well charac-terized. Nevertheless, this disinhibitory mechanism byother GABA neurons or by recurrent collaterals of theGABA neurons themselves would also reduce GABAergicinhibitory output. Thus, activation of GABABR-mediatedevents in GABA neurons, either pre- or postsynaptic,would cause increased excitation of the integrated net-work, contrary to the effects of GABABR activation inexcitatory neurons.

The present findings suggest that the GABABR-medi-ated late IPSP and presynaptic autoinhibition of GABArelease are differentially disposable to different GABAneurons. Those GABA neurons in the dorsal lateral genicu-

Fig. 14. Schematic diagrams showing the four expression profilesof g-aminobutyric acid (GABA)BR1a and/or GABABR1b mRNA vari-ants in different GABA neurons of the rat brain. The possiblydifferential targeting of GABABR1a and GABABR1b variants into pre-and postsynaptic GABABR receptors is according to a recent hypoth-esis, which remains unconfirmed (Bettler et al., 1998; Kaupmann etal., 1998b; Zhang et al., 1998). I: Most GABA neurons in the neocortex,hippocampus, and septum expressed both GABABR1a and GABABR1b.II: GABA neurons in thalamic reticular nucleus, caudate putamen,

and in stellate/basket cells of the cerebellum showed mainly GABABR1amRNA signal. III: As exemplified by Purkinje cells in the cerebellarcortex, this group of GABA neurons expressed predominantlyGABABR1b mRNA variant. IV: GABA neurons in the dorsal lateralgeniculate nucleus lacked significant expression of both GABABR1aand GABABR1b mRNA variants. GABABR(1a12), GABAB receptorheterodimer formed by GABABR1a and GABABR2 subunits.GABABR(1b12), GABAB receptor heterodimer formed by GABABR1band GABABR2 subunits.

492 F. LIANG ET AL.

late nucleus seemed lacking both mechanisms, in sharpcontrast to most GABA neurons in the neocortex andhippocampus. In the context of these results, it is interest-ing to note that little GABABR-mediated responses havebeen detected in the intrinsic GABAergic interneurons ofdorsal lateral geniculate nucleus of both cats and rats(Pape and McCormick, 1995; Williams et al., 1996). Theseinterneurons, capable of firing trains of action potentialsat a frequency exceeding 500 Hz in the cat, seemingly usealternative mechanisms, such as muscarinic acetylcholinereceptors, for modulating their capability to gate thetransmission of visual information from retina to visualcortex (McCormick and Pape, 1988; Pape and McCormick,1995).

The GABABR1a-positive GABA neurons such as those inthe striatum, thalamic reticular nucleus, and cerebellarstellate/basket cells may also have physiological and phar-macologic properties very different from the GABA neu-rons in the cerebral cortex that expressed both GABABR1aand GABABR1b, or from cerebellar Purkinje cells thatmainly expressed GABABR1b. It has been shown previ-ously that GABAB autoreceptors in the cerebral cortex andspinal cord represent pharmacologically distinct receptorsubtypes (Bonanno and Raiteri, 1993a). In view of theproposition that the two variants might be differentiallylocated to axon terminals and somatodendritic domains ofneurons (see below), it remains to be determined how thesefindings relate to the functions of individual GABA neuroncell types and to their positioning in the integrated neuralnetwork.

Margeta-Mitrovic and coworkers (1999) have found veryfew neurons that coexpressed GABABR1 immunoreactiv-ity and glutamic acid decarboxylase-67, in contrast to thepresent results showing expression of either or both of theGABABR1a and 1b mRNA variants in GABA neurons ofmost CNS structures. It remains unknown whether thisdiscrepancy with the present results is caused by ex-tremely low level of GABABR1 mRNA translation, limitedsensitivity of the antibody, or unrecognized GABABR vari-ants/subunits in GABA neurons. Of particular relevance tothe last point, for example, the antibody used by Margeta-Mitrovic et al. (1999) would not be able to detect therecently cloned GABABR1d (Isomoto et al., 1998). Thepresent GABABR1b antisense probe, in contrast, wouldalso hybridize with GABABR1c and 1d mRNA variants asthe two share the same 58 end with GABABR1b (Isomoto etal., 1998).

mRNA variants for receptor subtypes?

The differential but partly overlapping expression of thetwo mRNA variants in various neuronal cell types andbrain structures as shown in the present study, maycontribute to the production of diverse subtypes of GABABreceptors. In line with this molecular diversity, variablephysiological and pharmacologic profiles of GABABR ago-nist and antagonist actions have been demonstrated previ-ously in different CNS structures and cell types (forreviews see, Bowery, 1997; Deisz, 1997; Bettler et al.,1998). However, the differential and variable expressionpatterns of GABABR1a and GABABR1b mRNA variants inboth GABA and non-GABA neurons deny the possibility ofseparation of each variant into either GABA or non-GABAcategory. Differences in intracellular signal transductioncascades and effector systems may greatly enhance thefunctional heterogeneity and specificity derived from varia-

tions in GABABR variant expression in different neuronalcell types.

It has been recently proposed for the cerebellar cortexand retina that GABABR1a and GABABR1b variants mighttarget pre- and postsynaptic GABABR subtypes, respec-tively (Bettler et al., 1998; Kaupmann et al., 1998b; Zhanget al., 1998). Although the present study showed diversepatterns of expression of the two variants in the centralnervous system, a detailed correlation of the present datawith previous electrophysiological and pharmacologic stud-ies seems to extend the postulation to several other brainregions. The present study demonstrated, for example, thepredominant GABABR1a expression in the striatum, ven-tral lateral geniculate nucleus, and the thalamic reticularnucleus. In the same brain regions, electrophysiologicalstudies have failed to detect significant late IPSPs medi-ated by postsynaptic GABABRs (Soltesz et al., 1989b;Calabresi et al., 1991; Crunelli and Leresche, 1991; Nisen-baum et al., 1993; Ulrich and Huguenard, 1996; Sanchez-Vives and McCormick, 1997). Thus, it seemed conceivablethat the mature translation product of GABABR1a mRNAin neurons of these structures was mainly for locations atpresynaptic axon terminals. Both pre- and postsynapticGABABR-mediated events have been demonstrated for theneocortex, hippocampus, and most dorsal thalamic nuclei(Bowery, 1997; Deisz, 1997; Bettler et al., 1998). Again,this agrees with the present findings of coexpression of thetwo variants by these structures. In accordance with thepresent results showing abundant expression of GABABR1band very weak expression of GABABR1a mRNA by cerebel-lar Purkinje cells, previous studies have reported highlevels of postsynaptic GABABRs on Purkinje cell dendritesin molecular layer of the cerebellar cortex and low levels ofpresynaptic GABABRs on Purkinje axon terminals in deepcerebellar nuclei (Bowery et al., 1987; Chu et al., 1990;Turgeon and Albin, 1993; Morishita and Sastry, 1995;Mouginot et al., 1998). Likewise, it may be deduced thatmost brain stem and spinal neuronal cell types, whichmainly expressed the GABABR1a variant as shown in thepresent study, would manifest primarily a presynapticGABABR-mediated effect.

Except in the retina, no definite evidence has beenpresented for presynaptic GABABR1 immunoreactivity,either on GABA or non-GABAergic axon terminals, despitethe vast number of electrophysiological reports on thematter. A direct immunohistochemical demonstration ofGABABR1a peptide distribution in the CNS, for example,has not been possible because of the lack of suitablespecific antibodies (Koulen et al., 1998; Margeta-Mitrovicet al., 1999; Fritschy et al., 1999). In view of the recentcloning of GABABR1c/1d and the possibility of other uniden-tified GABABR1 variants, the subtraction strategy(GABABR1p 2 GABABR1b 5 GABABR1a distribution) asused by Fritschy and coworkers (1999) is no longer ten-able. Because peptides are mostly synthesized in the cellbodies and subsequently transported to their final destina-tions, immunoreactivity detected in the cell bodies andproximal dendrites might represent immature, pre-assembling forms of the receptor subunit and, therefore,could not rule out that the functioning form is located ataxon terminals. It awaits further studies, especially thoseat electron microscopic level by using variant-specificantibodies, to resolve the veracity of the postulation.


ACKNOWLEDGMENTS

We thank Dr. Katsuyoshi Ishii for many helpful discus-sions and Kiseko Shionoya, Takumi Agaki, YasuhiroYamazaki, Chishiro Wakabayashi, and Yuki Hasegawa fortechnical assistance.

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differential expression of ?-aminobutyric acid type b receptor-1a and -1b mrna variants in gaba and...

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