Cellular/Molecular

Redistribution of GABAB(1) Protein and Atypical GABAB Responses in GABAB(2)-Deficient Mice

Martin Gassmann, Hamdy Shaban, Réjan Vigot, Gilles Sansig, Corinne Haller, Samuel Barbieri, Yann Humeau, Valérie Schuler, Matthias Müller, Bernd Kinzel, Klaus Klebs, Markus Schmutz, Wolfgang Froestl, Jakob Heid, Peter H. Kelly, Clive Gentry, Anne-Lise Jaton, Herman Van der Putten, Cédric Mombereau, Lucas Lecourtier, Johannes Mosbacher, John F. Cryan, Jean-Marc Fritschy, Andreas Lüthi, Klemens Kaupmann and Bernhard Bettler
Journal of Neuroscience 7 July 2004, 24 (27) 6086-6097; https://doi.org/10.1523/JNEUROSCI.5635-03.2004

Abstract

GABAB receptors mediate slow synaptic inhibition in the nervous system. In transfected cells, functional GABAB receptors are usually only observed after coexpression of GABAB(1) and GABAB(2) subunits, which established the concept of heteromerization for G-protein-coupled receptors. In the heteromeric receptor, GABAB(1) is responsible for binding of GABA, whereas GABAB(2) is necessary for surface trafficking and G-protein coupling. Consistent with these in vitro observations, the GABAB(1) subunit is also essential for all GABAB signaling in vivo. Mice lacking the GABAB(1) subunit do not exhibit detectable electrophysiological, biochemical, or behavioral responses to GABAB agonists. However, GABAB(1) exhibits a broader cellular expression pattern than GABAB(2), suggesting that GABAB(1) could be functional in the absence of GABAB(2). We now generated GABAB(2)-deficient mice to analyze whether GABAB(1) has the potential to signal without GABAB(2) in neurons. We show that GABAB(2)-/- mice suffer from spontaneous seizures, hyperalgesia, hyperlocomotor activity, and severe memory impairment, analogous to GABAB(1)-/- mice. This clearly demonstrates that the lack of heteromeric GABAB(1,2) receptors underlies these phenotypes. To our surprise and in contrast to GABAB(1)-/- mice, we still detect atypical electrophysiological GABAB responses in hippocampal slices of GABAB(2)-/- mice. Furthermore, in the absence of GABAB(2), the GABAB(1) protein relocates from distal neuronal sites to the soma and proximal dendrites. Our data suggest that association of GABAB(2) with GABAB(1) is essential for receptor localization in distal processes but is not absolutely necessary for signaling. It is therefore possible that functional GABAB receptors exist in neurons that naturally lack GABAB(2) subunits.

Introduction

GABA, the predominant inhibitory neurotransmitter in the mammalian nervous system, signals through ionotropic GABAA and metabotropic GABAB receptors. GABAB receptors are coupled to G-proteins and modulate synaptic transmission by activating postsynaptic inwardly rectifying Kir3-type K+ channels and by controlling neurotransmitter release (Bowery et al., 2002; Calver et al., 2002; Bettler et al., 2004).

Molecular studies on GABAB receptors provide compelling evidence for heteromerization among G-protein-coupled receptors (GPCRs) (Marshall et al., 1999; Möhler et al., 2001). Most experiments with cloned GABAB(1) and GABAB(2) subunits expressed in heterologous cells and sympathetic neurons (Filippov et al., 2000) indicate that individual subunits are functionally inert unless they are coexpressed. GABAB receptors therefore appear different from other heterodimeric GPCRs in which individual subunits are functional when expressed alone (Bouvier, 2001). In the GABAB heteromer, the GABAB(1) subunit binds GABA and all competitive GABAB ligands (Kaupmann et al., 1998), whereas the GABAB(2) subunit is responsible for escorting GABAB(1) to the cell surface and for activating the G-protein (Margeta-Mitrovic et al., 2000, 2001b; Calver et al., 2001; Galvez et al., 2001; Pagano et al., 2001; Robbins et al., 2001). Two GABAB(1) isoforms, GABAB(1a) and GABAB(1b), arise by differential promoter usage (Kaupmann et al., 1997; Bettler et al., 2004). Thus far, the data support the existence of two predominant, yet pharmacologically indistinguishable, GABAB receptors in the nervous system, the heteromeric GABAB(1a,2) and GABAB(1b,2) receptors.

GABAB(1)-/- mice do not exhibit detectable GABAB responses in a variety of experimental paradigms, demonstrating that GABAB(1) is not only essential for GABAB signaling in vitro but also in vivo (Prosser et al., 2001; Schuler et al., 2001; Quéva et al., 2003). However, no in vivo experiment addressed whether GABAB(1) can assemble functional GABAB receptors by itself or in association with a protein other than GABAB(2). In support of a separate role, GABAB(1) exhibits a more widespread cellular distribution than does GABAB(2) (Kaupmann et al., 1998; Clark et al., 2000; Ng and Yung, 2001; Burman et al., 2003; Kim et al., 2003; Kulik et al., 2003; Li et al., 2003). Furthermore, at odds with a strict requirement of GABAB(2) for plasma membrane delivery, GABAB(1) was originally cloned by surface expression in mammalian cells (Kaupmann et al., 1997). Additionally, GABAB(1) occasionally yields electrophysiological or biochemical responses when transfected alone into heterologous cells (Kaupmann et al., 1997, 1998). It is therefore conceivable that GABAB(1) is functional either alone or in combination with an unknown protein. However, it remains unclear whether sporadic endogenous expression of GABAB(2) in heterologous cells is responsible for the surface expression and the responses that were seen when GABAB(1) was transfected alone.

To clarify whether GABAB(1) can participate in functional GABAB receptors in the absence of GABAB(2), we generated mice with a loss-of-function mutation in the GABAB(2) gene. Our results show that all well known GABAB responses relate to heteromeric GABAB(1,2) receptors. Surprisingly, the experiments also reveal atypical GABAB responses in hippocampal slices of GABAB(2)-deficient mice, indicating that GABAB(1) assemblies could be operational in neurons that naturally lack GABAB(2).

Materials and Methods

Generation and analysis of GABAB(2)-/- mice. GABAB(2)-/- mice were generated in the BALB/c inbred strain using a newly established BALB/c embryonic stem (ES) cell line. A targeting construct was designed containing a neomycin resistance cassette (pRay-2; GenBank accession number U63120) flanked by 4.5 and 1.8 kb of genomic GABAB(2) DNA that was amplified from a C57BL/6 bacterial artificial chromosome. Homologous recombination was confirmed by Southern blot analysis (Fig. 1A, B). Selected ES cell clones were microinjected into C57BL/6 blastocysts. Chimeric males were crossed with BALB/c females, resulting in an F1 generation of inbred BALB/c GABAB(2)+/- mice. GABAB(2)+/- mice were viable and fertile and allowed the generation of GABAB(2)-/- mice in the F2 generation. The probes used in Northern blot analysis (Fig. 1C) hybridize to exons 4-8 and exons 11-15 upstream (5′ probe) and downstream (3′ probe) of the deletion, respectively (Martin et al., 2001). For in situ hybridization (Fig. 1D), antisense oligonucleotides corresponding to nucleotides 2039-79 and 1810-54 of the rat GABAB(1a) (GenBank accession number Y10369) and GABAB(2) (GenBank accession number AJ011318) cDNAs, respectively, were used. The probes were radiolabeled with [35S]dATP (NEG0345H; NEN, Boston, MA) using terminal deoxynucleotidyl transferase (Promega, Madison, WI). For immunoblot analysis (Fig. 1E, F), polyclonal antibodies directed against the C terminus of GABAB(2) (AB5394; Chemicon, Temecula, CA), the C terminus of GABAB(1) (antibody 174.1) (Malitschek et al., 1998), the N terminus of GABAB(2) (antibody N22) (Kaupmann et al., 1998), and mouse calreticulin (ab4; Abcam, Cambridge, UK) were used. Monoclonal antibodies were used to detect PSD-95 (postsynaptic density protein-95) (MAB1598, Chemicon) and syntaxin (Sigma, St. Louis, MO). Blots were exposed to HRP-conjugated secondary antibodies [NA9340 (Amersham Biosciences, Little Chalfont, UK); A5545 and A0168 (Fluka, Buchs, Switzerland)] and developed using the ECL chemiluminescent detection system (RPN2016; Amersham Biosciences). Brain membrane preparations, ligand binding assays, and receptor autoradiography were performed as described previously (Olpe et al., 1990; Kaupmann et al., 1997). Synaptic plasma membranes were isolated from the P2 pellets of brain lysates by combined flotation-sedimentation density-gradient centrifugation (Jones and Matus, 1974). [3H]CGP62349 (80 Ci/mmol), [3H]CGP54626 (40 Ci/mmol), [125I]CGP71872 (2000 Ci/mmol), and [125I]CGP64213 (2000 Ci/mmol) were purchased from ANAWA (Wangen, Switzerland). [35S]GTPγS (1000 Ci/mmol) was obtained from Amersham Biosciences. Nonradio-active GABAB receptor ligands were from Novartis (Basel, Switzerland). [35S]GTPγS binding was performed with 20 μg of membrane protein, 0.2 nm [35S]GTPγS, and test compounds in 96-well Packard (Meridian, CT) Pico-Plates as described previously (Urwyler et al., 2001).

Figure 1.
Figure 1.

Characterization of GABAB(2)-/- mice. A, Top, GABAB(2) locus encompassing exons 8-11, encoding part of the N-terminal and the transmembrane (TM) domains 1 and 2. Bottom, GABAB(2) allele after homologous recombination with a targeting construct containing a neomycin resistance cassette (neo) flanked by 4.5 and 1.8 kb of genomic DNA (bold lines). Exons 9 and 10 (3.5 kb, black boxes) are deleted. The Southern blot probe used in B is indicated. H, HindIII; E, EcoRI; B, BamHI. B, Southern blot analysis of EcoRI cut genomic DNA from wild-type (+/+), GABAB(2)+/- (+/-), and GABAB(2)-/- (-/-) mice. C, Top, Northern blot analysis of total brain RNA hybridized with GABAB(2) cDNA probes upstream (5′ probe) and downstream of the deletion (3′ probe). The probes hybridize to a band just above the 28 S ribosomal RNA (arrow) in wild-type and GABAB(2)+/-, but not in GABAB(2)-/-, mice. Bottom, Blots from top panels stained with methylene blue, demonstrating equal loading of RNA. Ribosomal RNA bands (18 and 28 S) are labeled. D, In situ hybridization analysis of GABAB(1) (1, top) and GABAB(2) (2, bottom) transcripts of sagittal sections from adult wild-type and GABAB(2)-/- brains. E, Immunoblot analysis of brain extracts from adult mice using antibodies directed against C-terminal and N-terminal epitopes of GABAB(2) and GABAB(1). Antibodies to PSD-95 control for equal loading. GABAB(1a) (1a) and GABAB(1b) (1b) proteins are indicated. F, Immunoblot analysis demonstrating the presence of GABAB(1) protein in synaptic plasma membranes (SPM) purified from the P2 pellet (P2) of brain extracts of wild-type and GABAB(2)-/- mice. Antibodies to calreticulin show that the synaptic plasma membrane fraction is free of ER proteins. Equal loading of samples was controlled with anti-syntaxin antibodies. To detect putative truncated GABAB(2) proteins, we used 15% SDS-PAGE and N-terminal GABAB(2) antibodies. In all other immunoblot experiments, we used 10% SDS-PAGE.

Immunohistochemistry. Immunoperoxidase staining was performed in brain sections of adult mice using guinea pig antisera against GABAB(2) (1:5000; AB5394; Chemicon) and GABAB(1) (1:3000; AB1531; Chemicon). Mice were deeply anesthetized with Nembutal (50 mg/kg) and perfused through the ascending aorta with 4% paraformaldehyde in 0.15 m phosphate buffer. Brains were postfixed for 3 hr, processed for antigen retrieval using microwave irradiation (Fritschy et al., 1998), cryoprotected in sucrose, and cut at 40 μm with a sliding microtome. The immunoperoxidase staining was performed using diaminobenzidine as chromogen (Fritschy et al., 1999). Tissue from different genotypes was processed together to minimize variability attributable to the staining procedure. Sections were analyzed by light microscopy (Axioskop; Zeiss, Jena, Germany) and photographed with a high-resolution digital camera.

Electrophysiology. Transverse hippocampal slices (350-μm-thick) from 3- to 4-week-old mice were prepared. Slices were maintained for 45 min at 35°C in an interface chamber containing saline equilibrated with 95% O2 and 5% CO2 and containing the following (in mm): 124 NaCl, 2.7 KCl, 2 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.24 NaH2PO4, 18 glucose, and 2.25 ascorbate. Slices were then kept for at least 45 min at room temperature before being transferred to a superfusing recording chamber. Whole-cell recordings from CA1 pyramidal cells were performed at 30-32°C using infrared videomicroscopy to visualize cells. Patch electrodes (3-5 MΩ) were filled with a solution containing the following (in mm): 140 Cs-gluconate, 10 HEPES, pH 7.25, 10 phosphocreatine, 5 QX-314-Cl, 4 Mg-ATP, and 0.3 Na-GTP (295 mOsm). For measurements of postsynaptic holding currents (at -50 mV, in 0.5 μm TTX), Csgluconate was replaced by equimolar K-gluconate, and QX-314 was omitted. Synaptic currents were elicited every 15 sec using a bipolar platinum-iridium electrode (diameter, 25 μm). EPSCs were measured at -70 mV in the presence of picrotoxin (100 μm). IPSCs were measured at 0 mV in the presence of kynurenic acid (2 mm). All experiments assessing presynaptic GABAB receptor function were performed in the presence of BaCl2 (200 μm) to prevent the activation of postsynaptic Kir3 channels. BaCl2 did not affect the EPSC or IPSC amplitudes. Current-voltage (I-V) relationships were assessed by ramp-command protocols (from -40 to -140 mV, 250 msec duration) before and after the application of agonists, and the agonist-induced I-V relationship was derived by subtraction. Data were recorded with an Axopatch 200B (Axon Instruments, Union City, CA), filtered at 2 kHz, and digitized at 10 kHz. Data were acquired and analyzed with the LTP Program (W. Anderson, University of Bristol, Bristol, UK) (Anderson and Collingridge, 2001) or with pClamp8.0 (Axon Instruments). All membrane potentials were corrected for the experimentally measured liquid junction potential of 11 mV for the internal K-gluconate solution. Slope conductance was determined between -140 mV and the reversal potential. Series resistance was monitored throughout the experiments by applying a hyperpolarizing pulse, and, if it changed >15%, the data were not included in the analysis. All values are expressed as means ± SEM. Statistical comparisons were done with paired or unpaired Student's t test as appropriate, at a significance level of 0.05. GABAB receptor ligands were from Novartis. Non-GABAergic drugs were from Fluka.

Electroencephalogram measurements. Electroencephalogram (EEG) measurements were performed as described previously (Schuler et al., 2001; Kaupmann et al., 2003). The behavior of the mice, which were individually housed in wooden observation cages, was monitored with a video system. EEGs were amplified using an isolated four-channel bipolar EEG amplifier (EEG-2104; Spectralab, Maharashtra, India), recorded on a thermo recorder (MTK95; Astro-Med, West Warwick, RI), and stored on disk.

Measurement of core body temperature, locomotor activity, rotarod, and memory performance. Rectal temperature was determined to the nearest 0.1°C as described previously (Schuler et al., 2001; Kaupmann et al., 2003). Statistical analysis was performed using repeated-measures ANOVA, followed by Fisher's least significant difference test when appropriate. Locomotor activity was recorded using a color video camera, surveying the open field, and analyzed using EthoVision 1.90 software (Noldus Information Technology, Wageningen, The Netherlands). To assess rotarod performance, mice were trained to stay on the rotarod (12 rpm) for 300 sec over two separate sessions the day before the experiment. During the test day, the length of time each mouse remained on the cylinder (“endurance time”; maximal score of 300 sec) was measured immediately before (time 0) and 1, 2, and 4 hr after the application of l-baclofen (12.5 mg/kg) or vehicle (0.5% methylcellulose). The dose of baclofen that shows maximal effects on rotarod performance was determined in previous studies (Schuler et al., 2001). Memory performance in the passive avoidance test was performed as described previously (Venable and Kelly, 1990; Schuler et al., 2001).

Nociceptive tests. Heat or mechanical nociceptive stimuli were used in the antinociceptive tests as described previously (Schuler et al., 2001). The hotplate (Eddy and Leimbach, 1953), tail-flick (D'Amour and Smith, 1941), and the paw-pressure tests (Randall and Selitto, 1957) are well established techniques to assess acute pain. The tail flick is a reflex response to a noxious thermal stimulus applied to the tail and is generally held to represent a spinal reflex response, whereas the hotplate response to a noxious thermal stimulus to the plantar surface of the paws is thought to involve supraspinal sites.

Results

Previous experiments showed that only GABAB(1)-/- mice generated in the inbred BALB/c genetic background are viable (Prosser et al., 2001; Schuler et al., 2001; Quéva et al., 2003). We therefore ablated the GABAB(2) gene in BALB/c ES cells (Fig. 1A). Southern blot analysis confirms deletion of exons 9 (81 bp) and 10 (151 bp), encoding part of the N-terminal extracellular and the first transmembrane domain of GABAB(2) (Fig. 1B). BALB/c GABAB(2)-/- mice are viable, occur at a Mendelian ratio, and do not express detectable GABAB(2) mRNA, as shown by Northern blot analysis using hybridization probes flanking the GABAB(2) gene deletion (Fig. 1C). This demonstrates that any truncated mRNA produced from the 5′ part of the GABAB(2) gene is highly unstable. The complete lack of GABAB(2) mRNA is confirmed by in situ hybridization (Fig. 1D). Hence, GABAB(2)-/- mice do not express any full-length or truncated GABAB(2) protein, as shown by immunoblotting using antibodies directed against extreme C- or N-terminal epitopes (Fig. 1E). Immunoblot analysis further reveals that GABAB(2)+/- mice express less GABAB(2) protein than wild-type mice. A densitometric analysis of in situ hybridizations from several brain sections reveals that GABAB(1) mRNA expression in GABAB(2)-/- mice is not significantly changed when compared with wild-type littermates (Fig. 1D). However, immunoblot analysis indicates an ∼50 and 90% reduction of GABAB(1) protein in GABAB(2)+/- and GABAB(2)-/- mice, respectively (Fig. 1E). This is reminiscent of the almost complete absence of GABAB(2) protein previously seen in GABAB(1)-/- mice and yet again demonstrates that the two subunits cross-stabilize each other (Prosser et al., 2001; Schuler et al., 2001; Quéva et al., 2003). Despite this considerable downregulation, we clearly detect GABAB(1) protein in synaptic plasma membrane preparations of GABAB(2)-/- mice (Fig. 1F). This indicates that in vivo some GABAB(1) protein exits the endoplasmatic reticulum (ER) in the absence of the GABAB(2) subunit.

Redistribution of GABAB(1) in GABAB(2)-/- neurons

The regional and cellular distribution of GABAB subunits was investigated using antibodies recognizing GABAB(2) or the common C terminus of GABAB(1a) and GABAB(1b) (Fig. 2). A comparison of GABAB(2)-immunoreactivity (IR) and GABAB(1)-IR in adjacent sections of wild-type mice reveals a mostly overlapping distribution throughout the brain, with strong staining in cerebellum, thalamus, and hippocampal formation (Fig. 2A). In GABAB(2)+/- mice, GABAB(2)-IR is reduced in all brain regions, whereas GABAB(1)-IR remains similar to wild-type mice. In GABAB(2)-/- mice, a partial expression of GABAB(1)-IR is still seen in most brain regions, contrasting with the complete loss of GABAB(2) expression. At higher magnification, GABAB(1)-IR in GABAB(2)-/- mice exhibits a strikingly different cellular distribution than in wild-type mice, as illustrated for the hippocampal formation (Fig. 2B). The homogeneous, diffuse staining of the neuropil is almost reduced to background level, whereas the cell body layers, which normally are weakly labeled in wild-type mice, now appear very prominent. In addition, some scattered hippocampal interneurons are more evident in GABAB(2)-/- than in wild-type mice. The GABAB(1)-IR prominently outlines the soma and the proximal dendrites of these isolated interneurons, shown at higher magnification in Figure 2C. Similar results were observed throughout the brain, with an apparent accumulation of GABAB(1)-IR in the soma and proximal dendrites and a corresponding reduction of neuropil staining (data not shown). This staining was specific because no GABAB(1)-IR was detected in brain sections from GABAB(1)-/- mice, which were used as a control (data not shown). It is impossible to conclusively determine whether the GABAB(1)-IR seen in GABAB(2)-/- mice is partly associated with the plasma membrane or not. However, the strong GABAB(1)-IR in proximal dendrites of scattered interneurons, as shown in the hippocampal formation (Fig. 2B,C) and the biochemical (Fig. 1F) and electrophysiological data (see below) (see Fig. 6) all suggest that this is the case. Altogether, our immunohistochemical analysis suggests that GABAB(1) fails to efficiently localize at distal neuronal sites in the absence of GABAB(2).

Figure 2.
Figure 2.

Alteration of GABAB receptor-IR in GABAB(2)-/- brains. A, Effect of GABAB(2) gene deletion on the distribution of GABAB(2)-IR (2) and GABAB(1)-IR (1), as visualized in color-coded parasagittal sections from adult wild-type (+/+), GABAB(2)+/- (+/-), and GABAB(2)-/- (-/-) mice. The color scale is indicated. The reduced expression of GABAB(2) in GABAB(2)+/- mice and the complete loss of expression in GABAB(2)-/- mice are evident throughout the brain (top). GABAB(1)-IR is retained in GABAB(2)+/- mice and partly reduced in GABAB(2)-/- mice, in which it exhibits an altered cellular distribution, as seen in the hippocampus (bottom). The residual GABAB(1)-IR in GABAB(2)-/- mice is not caused by nonspecific binding of the secondary antibodies, which are the same for GABAB(1) and GABAB(2). The specificity of the GABAB(1) antiserum was also tested in GABAB(1)-/- mice, in which no specific staining was observed (data not shown). B, Color photomicrographs of the hippocampal formation stained for GABAB(1) in adult wild-type and GABAB(2)-/- mice. The pronounced increase of IR in the CA1-CA3 pyramidal cell layer and in the dentate gyrus granule cell layer (DG) contrasts with the strong reduction in the dendritic layers [stratum oriens (so), stratum radiatum (sr), stratum lucidum (sl), and molecular layer (ml)]. C, Enlargement of the framed areas in B. Numerous interneurons, which are primarily hidden in sections from wild-type mice because of the homogeneous staining, appear more strongly labeled in GABAB(2)-/- mice but with a normal distribution and morphology. Scale bars: A, 2 mm; B, 200 μm.

Figure 6.
Figure 6.

Baclofen inhibits a postsynaptic K+ conductance in CA1 pyramidal cells of GABAB(2)-/- mice. A, Holding current (at -50 mV) plotted versus time for wild-type (top, filled circles) and GABAB(2)-/- (bottom, open circles) mice. Whereas both baclofen (50 μm) and adenosine (100 μm) induce an outward current in wild-type mice, baclofen induces an inward current in GABAB(2)-/- mice. Baclofen-induced effects were blocked by application of the GABAB receptor antagonist CGP55845A (2 μm) in wild-type as well as in GABAB(2)-/- mice. B, Summary graph illustrating the baclofen-induced inward current at -50 mV in GABAB(2)-/- mice. Baclofen-induced currents: wild-type, n = 5; GABAB(2)-/-, n = 9. Adenosine-induced currents: wild-type, n = 5; GABAB(2)-/-, n = 4. C, Current-voltage relationship of the baclofen-induced conductance in wild-type (black trace) and GABAB(2)-/- (gray trace) mice. Currents were obtained by calculating the difference between the I-V curves before and after addition of baclofen. Whereas a current with a positive slope conductance is induced by baclofen in wild-type mice, a current with negative slope conductance is induced in GABAB(2)-/- mice. D, Current-voltage relationship of the adenosine-induced conductance in wild-type mice (black trace) is not different from GABAB(2)-/- mice (gray trace). E, Baclofen induces the closure of K+ channels in GABAB(2)-/- mice. Raising extracellular [K+] concentration shifts the reversal potential of the baclofen-induced current. F, The baclofen-induced conductance change is mediated by G-protein activation. In the presence of intracellular GDPβS (1 mm for 25 min), both the baclofen-induced (control, n = 5; GDPβS, n = 5) and adenosine-induced (control, n = 4; GDPβS, n = 5) currents are inhibited in GABAB(2)-/- mice. G, Changes in the holding current (at -50 mV) in response to baclofen (Bacl.) after preincubation with adenosine. H, Summary graph illustrating that the effects of adenosine and baclofen are not fully additive. In wild-type neurons (+/+), the effect of a combined application of adenosine and baclofen is lower than the sum of the individual effects [Adenosine + Bacl. (calculated)]. In GABAB(2)-/- (-/-) neurons, the effects of adenosine and baclofen are not fully additive. Application of baclofen does not obliterate the adenosine response. I, Left, Summary graph illustrating postsynaptic conductance changes induced by baclofen in wild-type (n = 5), GABAB(2)+/- (n = 10), and GABAB(1)-/- (n = 4) mice. The conductance changes were blocked by application of the GABAB(1) receptor antagonist CGP55845A (2 μm; wild-type, n = 4; GABAB(2)-/-, n = 8). Adenosine-induced conductance changes are not different between genotypes (wild-type, n = 4; GABAB(2)-/-, n = 4; GABAB(1)-/-, n = 3). *p < 0.05; **p < 0.01.

Radioligand binding studies in GABAB(2)-/- mice

All known competitive GABAB ligands bind exclusively to the GABAB(1) subunit (Kaupmann et al., 1998; Kniazeff et al., 2002). We therefore used antagonist radioligand binding to analyze GABAB(1) binding sites in GABAB(2)-/- mice. Saturation binding experiments at brain membrane preparations with [125I]CGP64213 failed to detect significant numbers of GABAB(1) binding sites in GABAB(2)-/- mice (Fig. 3A). The failure to detect antagonist radioligand binding at neuronal membranes from GABAB(2)-/- brains precludes agonist displacement studies. We were therefore unable to determine whether GABAB agonist affinity is lower in GABAB(2)-/- mice, as one would expect from previous recombinant work showing that GABAB(2) increases agonist affinity at GABAB(1) by ∼100-fold (Marshall et al., 1999). More sensitive detection systems, such as [125I]CGP71872 photoaffinity labeling (Fig. 3B) and [3H]CGP62349 autoradiography (Fig. 3C,D), reveal low but significant numbers of GABAB(1) binding sites in GABAB(2)-/- mice. Photoaffinity labeling detects both GABAB(1a) and GABAB(1b) in GABAB(2)-/- tissue (Fig. 3B), in agreement with the immunoblot analysis (Fig. 1E).

Figure 3.
Figure 3.

GABAB(1) binding sites in GABAB(2)-/- brains. A, Saturation isotherms for [125I]CGP64213 antagonist binding to cortex membranes. No significant binding is detected in membranes from GABAB(2)-/- mice. The number of binding sites is reduced in GABAB(2)+/- versus wild-type mice. The maximal number of binding sites (Bmax) for wild-type and GABAB(2)+/- mice are 1.4 ± 0.12 and 0.7 ± 0.05 pmol/mg protein, respectively; Kd values were 1.1 ± 0.06 and 0.9 ± 0.05 nm, respectively (mean ± SEM; n = 3). B, Autoradiograms of brain extracts from wild-type (+/+), GABAB(2)+/- (+/-), and GABAB(2)-/- (-/-) mice, labeled with the photoaffinity antagonist [125I]CGP71872 (0.5 nm) and analyzed by SDS-PAGE. Exposure for 8 d (8d exp.) reveals low amounts of labeled GABAB(1a) (1a) and GABAB(1b) (1b) proteins in GABAB(2)-/- brains. C, GABAB(1) subunit autoradiography. Sagittal cryostat sections were incubated with the GABAB antagonist [3H]CGP62349. Nonspecific binding was determined in the presence of an excess of 100 μm unlabeled l-baclofen. Tritium-sensitive x-ray films were exposed for 24 hr and developed using a Cyclone Storage Phosphor screen (PerkinElmer Life Sciences, Boston, MA). D, Quantitative analysis of [3H]CGP62349 receptor autoradiography. Individual brain regions (n = 3) were counted using the MCID software package (Imaging Research, St. Catharines, Ontario, Canada). The differences in radioligand binding between the three genotypes are significant (two-sided Dunnett test; p < 0.001 for combined analysis of all brain regions). A-C, Representative experiments, which were repeated three times.

We next used [35S]GTPγS binding to investigate whether the residual GABAB(1) protein in GABAB(2)-/- mice participates in functional receptors (Fig. 4). The [35S]GTPγS binding assay preferentially detects receptors that are coupled to Gαi/o-type G-proteins, the main effectors of native GABAB receptors. We did not detect any significant GABA- or baclofen-induced [35S]GTPγS binding in GABAB(2)-/- cortical (Fig. 4) or hippocampal (data not shown) membrane preparations. This indicates that the GABAB(1) protein expressed in GABAB(2)-/- mice is either not coupled to Gαi/o or not present in sufficient amounts to generate detectable [35S]GTPγS binding. In GABAB(2)+/- cortical membranes, baclofen and GABA elicit <50% of the [35S]GTPγS binding seen with wild-type membrane preparations (Fig. 4), consistent with the reduced expression levels of GABAB(1) and GABAB(2) proteins (Figs. 1E, 3).

Figure 4.
Figure 4.

[35S]GTPγS binding to cortex membranes. No significant GABA-stimulated (filled symbols, filled lines) or baclofen-stimulated (Bac; open symbols, dotted lines) [35S] GTPγS binding is detected in GABAB(2)-/- membranes. [35S]GTPγS binding to membranes from GABAB(2)+/- mice is significantly reduced compared with wild-type mice. Values are normalized to the maximal response obtained with wild-type mice.

Loss of presynaptic GABAB functions in GABAB(2)-/- mice

Electrophysiology provides a more sensitive means than [35S]GTPγS binding for detecting functional GABAB receptors expressed by individual neurons. We therefore used whole-cell patch-clamp recording to examine GABAB(2)-/- mice for the presence of GABAB heteroreceptors and autoreceptors on excitatory and inhibitory terminals, respectively. We first studied excitatory synaptic transmission in the hippocampus (Fig. 5A,B). Stimulation in the Schaffer collateral-commissural fibers induces EPSCs in CA1 pyramidal neurons. The amplitude of these EPSCs is reduced by the activation of GABAB heteroreceptors or A1 adenosine receptors that inhibit glutamate release (Schuler et al., 2001). Accordingly, in slices from wild-type mice, both baclofen and adenosine evoke the expected depression of the EPSCs (baclofen, 74.0 ± 3.2% inhibition, n = 4, p < 0.01; adenosine, 85.5 ± 5.3% inhibition, n = 4, p < 0.01). However, only adenosine has an effect in slices from GABAB(2)-/- mice (baclofen, 0.9 ± 12.6% inhibition, n = 8; adenosine, 82.1 ± 7.3% inhibition, n = 6, p < 0.001). This demonstrates that GABAB(2)-/- mice lack functional GABAB heteroreceptors on Schaffer collateral terminals, whereas adenosine receptors are still operational and inhibit glutamate release. We next analyzed inhibitory synaptic transmission in the presence of ionotropic glutamate receptor antagonists (Fig. 5C,D). Activation of GABAB autoreceptors on interneurons attenuates IPSCs recorded from CA1 pyramidal neurons of wild-type mice (55.3 ± 5.8% inhibition; n = 7; p < 0.001). In contrast, baclofen is unable to inhibit IPSCs in GABAB(2)-/- mice (-0.5 ± 3.9% inhibition; n = 6), although the μ-opioid receptor agonist [d-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin (DAMGO) is still effective in both genotypes (wild-type, 46.5 ± 4.5% inhibition, n = 7, p < 0.001; GABAB(2)-/- mice, 58.1 ± 3.7% inhibition, n = 5, p < 0.001). These latter experiments show that hippocampal interneurons lack GABAB autoreceptors in GABAB(2)-/- mice.

Figure 5.
Figure 5.

Lack of baclofen-induced presynaptic inhibition in CA1 pyramidal cells of GABAB(2)-/- mice. A, Excitatory synaptic transmission. Monosynaptic EPSC peak amplitudes plotted versus time and representative traces from wild-type (top, filled circles) and GABAB(2)-/- (bottom, open circles) mice. Both baclofen (50 μm) and adenosine (100 μm) significantly depress the EPSC amplitude in wild-type mice, whereas baclofen and CGP55845A (2 μm) have no effect on the EPSC amplitude in GABAB(2)-/- mice. The effect of adenosine is similar in both genotypes. Traces are averages of 10 consecutive sweeps. Calibration: 40 msec, 100 pA. B, Summary graph showing the lack of baclofen-induced presynaptic inhibition of excitatory synaptic transmission in GABAB(2)-/- mice (wild-type, n = 4; GABAB(2)-/-, n = 8). Adenosine-induced inhibition is similar in both genotypes (wild-type, n = 4; GABAB(2)-/-, n = 6). C, Inhibitory synaptic transmission. Monosynaptic IPSC peak amplitudes plotted versus time and representative traces from wild-type (top, filled circles) and GABAB(2)-/- (bottom, open circles) mice. Both baclofen (50 μm) and the μ-opioid agonist DAMGO (1 μm) significantly depress the IPSC amplitude in wild-type mice, whereas baclofen and CGP55845A (2 μm) have no effect on the IPSC amplitude in GABAB(2)-/- mice. The effect of DAMGO was similar in both genotypes. Traces are averages of 10 consecutive sweeps. Calibration: 100 msec, 200 pA. D, Summary graph showing the lack of baclofen-induced presynaptic inhibition of inhibitory synaptic transmission in GABAB(2)-/- mice (wild-type, n = 7; GABAB(2)-/-, n = 6). DAMGO-induced inhibition was similar in both genotypes (wild-type, n = 7; GABAB(2)-/-, n = 5). **p < 0.01; ***p < 0.001.

GABAB receptors inhibit instead of activate K+ channels in GABAB(2)-/- mice

Postsynaptic GABAB and adenosine receptors activate a Kir3-mediated K+ conductance in CA1 pyramidal neurons (Lüscher et al., 1997; Schuler et al., 2001). The GABAB receptor-activated K+ conductance underlies the late IPSP (Lüscher et al., 1997). Accordingly, at a holding potential of -50 mV and in physiological [K+]ext, baclofen elicits an outward current in CA1 pyramidal cells of wild-type mice (116.2 ± 26.7 pA; n = 5; p < 0.05) (Fig. 6A,B) that is blocked by the GABAB(1) antagonist CGP55845A (2 μm; 93.4 ± 12.5% inhibition; n = 4). Surprisingly, but consistent with the strong GABAB(1)-IR observed on the soma and proximal dendrites (Fig. 2B,C), a baclofen-induced current is also seen in CA1 pyramidal neurons of GABAB(2)-/- mice. However, baclofen elicits an inward instead of the typical outward current (-19.2 ± 4.5 pA; n = 9; p < 0.01) (Fig. 6A,B). This inward current can be blocked by the GABAB(1) antagonists CGP55845A (2 μm; 99.4 ± 2.7% inhibition; n = 8) (Fig. 6A) and CGP62349 (4 μm; 87.5 ± 8.3% inhibition; n = 5), the ligand that was used for autoradiographic detection of GABAB(1) (Fig. 3C). Whereas the baclofen-induced outward current in wild-type mice is associated with a decrease in the input resistance (-93.3 ± 25.8 MΩ; n = 5; p < 0.05), the inward current in GABAB(2)-/- mice is associated with an increase in input resistance (35.2 ± 12.1 MΩ; n = 9; p < 0.05). Adenosine-induced currents are similar in wild-type and GABAB(2)-/- mice (wild-type, 67.0 ± 11.1 pA, n = 5, p < 0.01; GABAB(2)-/-, 51.2 ± 3.8 pA, n = 4, p < 0.01) (Fig. 6A,B). The current-voltage relationship of baclofen-induced currents reveals a positive slope conductance in wild-type mice (3.0 ± 0.7 nS; n = 5; p < 0.05) (Fig. 6C,I), whereas a negative slope conductance is induced in GABAB(2)-/- mice (-2.3 ± 0.5 nS; n = 10; p < 0.01) (Fig. 6C,I). Consistent with the baclofen-induced increase in input resistance, a negative slope conductance indicates that baclofen application leads to the closure of ion channels in GABAB(2)-/- mice. The baclofen-induced conductance changes in wild-type and in GABAB(2)-/- mice are completely blocked by the GABAB(1) antagonist CGP55845A (2 μm; wild-type, -0.2 ± 0.4 nS, n = 4, p < 0.05; GABAB(2)-/-, -0.01 ± 0.06 nS, n = 8, p < 0.001) (Fig. 6I). Adenosine-induced conductance changes are similar in wild-type (conductance, 4.83 ± 0.91 nS; n = 4; p < 0.01; Vrev, -94.5 ± 1.2 mV; n = 4) and GABAB(2)-/- (4.63 ± 0.83 nS; n = 4; p < 0.01; Vrev, -93.5 ± 2.5 mV; n = 4) mice (Fig. 6D,I). The reversal potential of the baclofen-induced current in GABAB(2)-/- cells is shifted by raising the extracellular [K+] from 2.7 mm (Vrev, -96.7 ± 3.6 mV; n = 10; calculated Vrev for K+, -99.5 mV) to 20 mm (Vrev, -47.6 ± 7.4 mV; n = 6; calculated Vrev for K+, -45.8 mV) (Fig. 6E), indicating that a closure of K+ channels underlies the baclofen-induced conductance change in GABAB(2)-/- neurons. Barium at a concentration of 300 μm completely occludes the baclofen-induced channel closure in wild-type and GABAB(2)-/- CA1 pyramidal cells (data not shown). It is therefore conceivable that the GABAB receptors in GABAB(2)-/- CA1 neurons and the GABAB(1,2) receptors in wild-type CA1 neurons both couple to Kir3 channels but with opposite effects on channel activity. A large body of in vitro data supports that, within the heteromeric GABAB(1,2) receptor, the now-missing GABAB(2) subunit is absolutely necessary for G-protein coupling (Galvez et al., 2001; Margeta-Mitrovic et al., 2001b; Robbins et al., 2001; Duthey et al., 2002; Havlickova et al., 2002). We therefore investigated whether the baclofen-induced closure of K+ channels in GABAB(2)-/- cells is mediated by G-proteins or not. We recorded postsynaptic responses in the presence of GDPβS, which prevents G-protein activation. Intracellular dialysis of CA1 pyramidal cells from GABAB(2)-/- mice with GDPβS (1 mm for at least 25 min) specifically blocks the induction of postsynaptic currents by baclofen (control, -19.2 ± 4.5 pA, n = 5; GDPβS, 0.1 ± 3.9 pA, n = 5, p < 0.01) (Fig. 6F) or adenosine (control, 51.2 ± 3.8 pA, n = 4; GDPβS, 2.1 ± 4.9 pA, n = 5, p < 0.05) (Fig. 6F), demonstrating that the baclofen-induced conductance change in GABAB(2)-/- CA1 pyramidal cells is G-protein mediated. It is conceivable that the baclofen-induced inhibition of a K+ current is the consequence of a dominant-negative effect. For example, GABAB(1) activation in GABAB(2)-/- neurons may sequester G-proteins that are normally associated with other GPCR-activating K+ channels. Such a baclofen-dependent sequestering of G-proteins would reduce K+ currents and could underlie the inward current observed in GABAB(2)-/- neurons. We investigated whether baclofen can cross-inhibit the adenosine response by first applying adenosine to CA1 pyramidal cells, followed by a combined application of adenosine and baclofen (Fig. 6G,H). In both wild-type and GABAB(2)-/- neurons, the effects of adenosine and baclofen are not fully additive, indicating that adenosine and GABAB receptors share G-proteins and/or effector K+ channels. However, the cross-inhibitory effect was not larger in GABAB(2)-/- than in wild-type neurons. Although the outcome of these experiments does not completely exclude sequestering, it clearly does not support it. The fact that the baclofen-induced current is blocked by GDPβS also argues against a passive sequestering of G-proteins and shows that activation of G-proteins is necessary to trigger the inward current (Fig. 6F). Others and we reported previously a complete loss of postsynaptic baclofen responses in GABAB(1)-/- mice (Prosser et al., 2001; Schuler et al., 2001). We therefore reinvestigated GABAB(1)-/- mice for baclofen-induced responses under identical experimental conditions as used for the analysis of GABAB(2)-/- mice (Fig. 6I). Consistent with our previous findings, we do not observe any postsynaptic conductance changes induced by baclofen in GABAB(1)-/- mice. Therefore, exclusively GABAB(2)-/- mice express residual functional GABAB receptors.

Lack of behavioral responses to baclofen in GABAB(2)-/- mice

In addition to inducing electrophysiological responses, baclofen may also cause detectable behavioral responses in GABAB(2)-/- mice. We therefore studied well known physiological responses to baclofen in GABAB(2)-/- mice. First, we investigated whether baclofen still induces delta waves in the EEG, as shown previously for wild-type mice (Schuler et al., 2001; Kaupmann et al., 2003). Twenty minutes after baclofen application (10 mg/kg, i.p.), delta waves appeared in the EEG of wild-type mice but not in the EEG of GABAB(2)-/- mice (Fig. 7). Ten hours after baclofen administration, the EEG of wild-type mice reverted to normal. No significant EEG changes were observed in GABAB(2)-/- mice during the entire duration of the experiment. This indicates that the baclofen-induced electrophysiological responses in GABAB(2)-/- mice (Fig. 6) do not result in detectable changes of electrical activity at the network level.

Figure 7.
Figure 7.

Lack of baclofen-induced delta waves in GABAB(2)-/- mice. A, Effect of l-baclofen (10 mg/kg, i.p.) on the EEG of freely moving wild-type (+/+) and GABAB(2)-/- (-/-) mice. The EEG of wild-type and GABAB(2)-/- mice were similar 10 min before baclofen application (-10 min). Twenty minutes after baclofen application, delta waves were observed in the EEG of wild-type, but not of GABAB(2)-/-, mice (+20 min). Single spikes appeared sporadically in the EEG of wild-type mice (+40 min), followed by delta waves that lasted for several hours (+7 hr). Ten hours after baclofen application, the EEG traces of wild-type and GABAB(2)-/- mice were again similar (+10 hr). B, Quantification of baclofen-induced delta waves in the EEG of wild-type and GABAB(2)-/- mice. The percentage of delta waves of the total power amplitude was calculated over periods of 10 min. Three to four mice per genotype were analyzed.

We next investigated whether GABAB receptors in GABAB(2)-/- mice are able to mediate the well known muscle-relaxant effect of baclofen. Baclofen induces muscle relaxation in wild-type but not in GABAB(2)-/- mice, as shown by the inability or ability, respectively, of the mice to stay on the rotarod during a 5 min period (Fig. 8A). Similarly, GABAB(2)-/- mice demonstrate a lack of baclofen-induced hypothermia (Fig. 8B). Together, these data indicate that residual GABAB receptors in GABAB(2)-/- mice are unable to influence muscle relaxation or body temperature.

Figure 8.
Figure 8.

Lack of baclofen-induced motor impairment and hypothermia in GABAB(2)-/- mice. A, No baclofen-induced impairment of rotarod endurance is observed in GABAB(2)-/- (-/-) mice (n = 7-10). In contrast, wild-type mice (+/+) show a marked reduction in rotarod performance after baclofen application (p < 0.05; Fisher's post hoc tests). The vehicle-treated control groups stayed on the rotarod during the entire experiment (300 sec) at all time points examined. Thus, in the graph, the data points for the wild-type vehicle control (black dots) are hidden behind the data points for the GABAB(2)-/- vehicle control (white dots). At all time points after baclofen application (1, 2, and 4 hr), the GABAB(2)-/- group (white triangles) differed significantly from the wild-type control group (black triangles) (p < 0.05; Fisher's post hoc tests). All data points represent mean ± SEM values. B, Baclofen induces a potent reduction in body temperature in wild-type mice (black triangles) compared with the vehicle control group (black dots) (p < 0.05; Fisher's post hoc tests), whereas it is without effect on basal temperature in GABAB(2)-/- mice (n = 7-10). However, GABAB(2)-/- mice (white dots) exhibit a slight but significantly reduced basal temperature compared with wild-type littermates (black dots) (p < 0.05; Fisher's post hoc tests). All data points represent mean ± SEM values.

GABAB(2)-/- mice exhibit spontaneous epileptiform activity, hyperlocomotor activity, hyperalgesia, and impaired passive avoidance learning

We reported previously that adult GABAB(1)-/- mice exhibit pronounced spontaneous epileptiform activity (Schuler et al., 2001). We therefore investigated whether adult GABAB(2)-/- mice are epileptic and recorded continuous EEG in freely moving animals using implanted electrodes. GABAB(2)-/- mice displayed several episodes of spontaneous seizures per day. The analysis of three GABAB(2)-/- mice over a 96 hr period revealed an average of 3.75 (0, 11, 3, 1), 0.5 (1, 0, 0, 1), and 2.0 (3, 2, 2, 1) seizures per day. The recorded seizures were exclusively of the clonic type. This is in contrast to GABAB(1)-/- mice, in which additionally absence-type and spontaneous tonic-clonic seizures occurred with low frequency (Schuler et al., 2001). Epileptiform activity was never observed in wild-type littermates (n = 3).

GABAB(1)-/- mice exhibit a sporadic hyperlocomotor phenotype when exposed to a new environment (Schuler et al., 2001). We similarly studied the locomotor activity of GABAB(2)-/- mice using the Ethovision recording system. During a 1 hr observation period, GABAB(2)-/- mice moved over a significantly larger distance with significantly increased speed compared with wild-type and heterozygous littermates (Fig. 9A). These experiments demonstrate that functional GABAB receptors in GABAB(2)-/- mice do not rescue the hyperlocomotor phenotype seen with GABAB(1)-/- mice, which completely lack functional GABAB receptors.

Figure 9.
Figure 9.

Behavioral analysis of GABAB(2)-/- mice. A, Hypolocomotor activity in GABAB(2)-/- mice. During a 1 hr observation period, GABAB(2) knock-out mice (-/-) moved over significantly longer distances (left histogram) with significantly greater speed (right histogram) than heterozygous (+/-) and wild-type (+/+) control mice. n = 7-8 per genotype; mean ± SEM; *p < 0.05. B, Response latencies of wild-type (+/+), heterozygous (+/-), and GABAB(2) knock-out (-/-) mice in the hotplate test assessed at 55°C. GABAB(2)-/- mice show significantly reduced paw-lick latencies compared with wild-type and heterozygous control groups. n = 19-20 per genotype; mean ± SEM; ***p < 0.001. C, Response latencies of wild-type (+/+), heterozygous (+/-), and GABAB(2) knock-out (-/-) mice in the tail-flick test assessed at infrared intensity 14. GABAB(2)-/- mice show significantly reduced tail-flick latencies compared with wild-type and heterozygous control groups. n = 19-21 per group; mean ± SEM; *p < 0.05. D, Paw-withdrawal thresholds for wild-type (+/+), heterozygous (+/-), and GABAB(2) knock-out (-/-) mice in response to a mechanical stimulus. Withdrawal thresholds of the left hindpaw were assessed for each genotype. GABAB(2)-/- mice show a significantly reduced withdrawal threshold compared with wild-type and heterozygous control groups. n = 19-21 per group; ***p < 0.001. Nociception tests were analyzed with Tukey's honestly significant difference test. In all tests, there were no significant differences in the behavior of wild-type or heterozygous mice. E, Impaired passive avoidance learning in GABAB(2)-/- mice. Step-through latencies of wild-type (+/+) and GABAB(2) knock-out (-/-) mice into the dark (shock) compartment on the training day (white bars) and in the retention test (black bars). GABAB(2)-/- mice were slower to enter on training day but faster in the retention test compared with the wild-type control mice. Wild-type, but not GABAB(2)-/-, mice show significantly longer latencies to enter the dark compartment in the retention test compared with the training trial, which is taken as an index of memory of the initial experience. n = 6-11 per group; mean ± SEM; ***p < 0.001 versus training; #p < 0.05 versus genotype; ##p < 0.01 versus genotype.

GABAB agonists exhibit antinociceptive properties in models of acute and chronic pain (Patel et al., 2001). These properties are likely mediated by supraspinal and spinal GABAB receptors (Malcangio et al., 1991). Consistent with these pharmacological findings, GABAB(1)-/- mice exhibit pronounced hyperalgesia, suggesting that GABAB receptors exert a tonic control over nociceptive processes (Schuler et al., 2001). We used the hotplate (Fig. 9B), tail-flick (Fig. 9C), and paw-pressure (Fig. 9D) tests to measure acute pain behaviors in GABAB(2)-/- mice. Similar to the GABAB(1)-/- mice, GABAB(2)-/- mice exhibit hyperalgesia in all three tests, showing significantly reduced response latencies or withdrawal thresholds when compared with wild-type or heterozygous littermate mice. In all three tests, we did not observe significant differences in the behavior of wild-type or heterozygous mice.

GABAB antagonists are reported to have profound effects on memory processing. They can either enhance (Getova and Bowery, 1998; Nakagawa et al., 1999; Stäubli et al., 1999; Getova and Bowery, 2001) or attenuate (Brucato et al., 1996) cognitive performance in a variety of learning paradigms in mice and rats. We reported previously that GABAB(1)-/- mice exhibit a severe impairment of passive avoidance learning (Schuler et al., 2001). We therefore investigated the memory performance of GABAB(2)-/- mice (Fig. 9E). GABAB(2)-/- mice, in contrast to wild-type mice, show no increased latency in entering the darkened shock compartment in the retention trial that followed the training trial. This indicates that GABAB(2)-/- mice exhibit an impairment of passive avoidance learning, similar to GABAB(1)-/- mice. We further observed that GABAB(2)-/- mice show increased latencies to enter the darkened shock compartment on the training trial compared with wild-type littermate mice (p < 0.01). This excludes the possibility that GABAB(2)-/- mice have a tendency to enter the dark compartment more quickly, independent of the training experience.

Discussion

Pharmacological and behavioral analyses of GABAB(2)-/- mice indicate that deletion of the GABAB(2) subunit is sufficient to abolish all well known responses to GABAB agonists, such as [35S]GTPγS binding (Fig. 4), muscle relaxation (Fig. 8A), hypothermia (Fig. 8B), and EEG delta wave induction (Fig. 7). These findings are paralleled by a loss of typical electrophysiological GABAB responses in the GABAB(2)-/- hippocampus (Figs. 5, 6). These results are analogous to the results obtained with GABAB(1)-/- mice and suggest that all classical GABAB responses relate to heteromeric GABAB(1,2) receptors. The heteromeric nature of predominant native GABAB receptors is further emphasized by the substantial downregulation of GABAB(1) protein in GABAB(2)-/- mice (Fig. 1E). An analogous requirement of GABAB(1) for stable expression of GABAB(2) was observed in GABAB(1)-/- mice (Prosser et al., 2001; Schuler et al., 2001; Quéva et al., 2003).

Strikingly, the remaining GABAB(1) protein in GABAB(2)-/- neurons accumulates in distinct cellular compartments than in wild-type neurons. Throughout the GABAB(2)-/- brain, we observed a redistribution of the GABAB(1)-IR from the neuropil to the soma (Fig. 2 and data not shown). We also noticed some scattered hippocampal interneurons that are more evident in GABAB(2)-/- than wild-type brains (Fig. 2B,C). The GABAB(1)-IR prominently outlines the soma and proximal dendrites of these cells. This is reminiscent of the strong somatic GABAB(1)-IR observed in a subset of GABAergic hippocampal interneurons lacking GABAB(2)-IR (Fritschy et al., 1999; Sloviter et al., 1999; Kulik et al., 2003). Presumably, both a genetically induced and a natural lack of GABAB(2) expression leads to a relocation of GABAB(1) protein to the soma and proximal dendrites. Because GABAB(2) is important for exit of GABAB(1) from the ER, most of the somatic GABAB(1)-IR likely reflects protein that fails to exit the ER. However, some of the GABAB(1)-IR on the soma and proximal dendrites may also represent GABAB(1) protein at the cell surface. This is supported by biochemical (Fig. 1F) and electrophysiological (Fig. 6) data that reveal GABAB(1) expression in synaptic membranes and functional receptors in the somatodendritic compartment of CA1 pyramidal neurons, respectively. Besides being important for G-protein coupling and export from the ER (Margeta-Mitrovic et al., 2000, 2001b; Calver et al., 2001; Galvez et al., 2001; Pagano et al., 2001; Robbins et al., 2001), GABAB(2) may therefore also be necessary for the targeting of GABAB(1,2) receptors to the distal zones of neuronal processes.

Whether a physiologically relevant signaling underlies the electrophysiological GABAB responses that we observe in CA1 neurons of GABAB(2)-/- mice is unclear. It is possible that these GABAB responses are a consequence of the knock-out situation, in which GABAB(1) is expressed in the absence of its usual dimerization partner. An abnormal intracellular accumulation of GABAB(1) protein in GABAB(2)-/- pyramidal cells may overload the ER-retention machinery, thereby allowing some GABAB(1) to escape to the cell surface and to couple to G-proteins. Consistent with this possibility, GABAB(1) was originally expression cloned using [125I]CGP64213 binding on the surface of live COS-1 cells (Kaupmann et al., 1997), showing that some GABAB(1) protein can overcome ER retention in the absence of GABAB(2). In further support of this possibility, no GABAB responses were detected in CA1 pyramidal neurons of mice expressing a C-terminally truncated version of the GABAB(2) protein (A. Calver, personal communication). Apparently, the truncated GABAB(2) protein dimerizes with GABAB(1) in the ER, generating a dominant-negative situation that impedes transit of GABAB(1) protein through intracellular compartments.

Normally, postsynaptic GABAB receptors activate a K+ conductance underlying the late IPSP (Lüscher et al., 1997). However, in GABAB(2)-/- mice, baclofen induces a G-protein-dependent inward current instead of the expected outward current, most likely reflecting the closure of K+ channels (Fig. 6). Barium at 300 μm occludes the baclofen-induced current inhibition seen in GABAB(2)-/- mice (data not shown). Kir3 channels could therefore not only be the cause of the typical outward current seen in wild-type neurons (Lüscher et al., 1997) but could also be responsible for the atypical inward current seen in GABAB(2)-/- neurons. The GABAB(1) antagonists CGP55845A (Fig. 6A,I) and CGP62349 (see Results) block the baclofen-induced inward current seen in GABAB(2)-/- CA1 pyramidal cells. A radioactive version of the antagonist used in electrophysiology, [3H]CGP62349, specifically recognizes residual GABAB(1) protein in the GABAB(2)-/- brain (Fig. 3C). Moreover, the inward current is not observed in GABAB(1)-/- CA1 pyramidal cells (Fig. 6I). Together, this suggests that the baclofen-sensitive current is triggered by receptors incorporating GABAB(1). Baclofen-sensitive currents were seen in the majority of GABAB(2)-/- CA1 neurons analyzed, indicating that the neuronal environment reliably assists functioning of GABAB(1) in the absence of GABAB(2). It remains unclear why the GABAB(1)-mediated electrophysiological responses in GABAB(2)-/- CA1 neurons are opposite to those recorded in wild-type CA1 neurons. We addressed whether activation of GABAB(1) in GABAB(2)-/- neurons takes on a dominant-negative effect by sequestering G-proteins that normally activate Kir channels. We did not observe increased cross-inhibition of the adenosine response by baclofen in GABAB(2)-/- as opposed to wild-type neurons, rendering sequestering unlikely (Fig. 6G,H). Further arguing against a passive sequestering of G-proteins, the baclofen-induced inward current in GABAB(2)-/- neurons is blocked by GDPβS (Fig. 6F). Some G-proteins are reported to inhibit rather than to activate Kir3 channels (e.g., by phospholipase C-mediated phosphatidylinositol-4, 5-biphosphate hydrolysis or PKC activation) (Schreibmayer et al., 1996; Sharon et al., 1997; Lei et al., 2000, 2001; Blanchet and Lüscher, 2002; Mao et al., 2004). Similar to what is now observed, metabotropic glutamate receptors not only activate but also inhibit K+ channels, presumably by coupling to distinct G-proteins (Sharon et al., 1997). For example, they were shown to be able to suppress a barium-sensitive K+ current in CA3 pyramidal cells (Lüthi et al., 1997) and to downregulate Kir3 channels in Xenopus oocytes (Sharon et al., 1997). It is therefore conceivable that the somatic redistribution of GABAB receptors (Fig. 2) in GABAB(2)-/- neurons leads to a promiscuous coupling to G-proteins that are not normally associated with heteromeric GABAB(1,2) receptors. This would explain why no significant [35S]GTPγS binding is detectable in neuronal membranes from GABAB(2)-/- mice (Fig. 4) because this assay preferentially detects Gi/o-proteins that are typically associated with native GABAB(1,2) receptors. A promiscuous coupling to G-proteins in neurons may also explain why we never observed GABAB responses opposite to those of heteromeric GABAB(1,2) receptors when GABAB(1) was functional by itself in transfected cells (Kaupmann et al., 1997, 1998). There is compelling in vitro evidence to show that, in the heteromer, the GABAB(2) subunit is necessary to engage and activate G-proteins (Galvez et al., 2001; Margeta-Mitrovic et al., 2001b; Robbins et al., 2001; Duthey et al., 2002; Havlickova et al., 2002). GABAB(1) may therefore also function in association with another, yet unknown GPCR subunit, which couples to G-proteins other than GABAB(2). In that respect, a GABAB receptor-related protein has been identified (Calver et al., 2003). However, in heterologous cells, this protein does not appear to participate in typical GABAB signaling. Furthermore, because “Family C” GPCRs preferentially assemble homodimers, the existence of homodimeric GABAB(1) receptors cannot be excluded (Bouvier, 2001). It is possible that homodimeric GABAB(1) receptors couple to G-proteins other than heterodimeric GABAB(1,2) receptors. Moreover, they may exhibit a constitutive activity that can be inhibited by agonists. Of note, it was reported that chimeric GABAB receptors with two GABAB(1) extracellular domains exhibit an increased basal activity and, for unknown reasons, respond to GABA agonists with inhibition rather than activation of Kir3 channels (Margeta-Mitrovic et al., 2001a). Similar observations were made in a related study (Galvez et al., 2001).

It is not ruled out that the baclofen-induced inward current is also present in wild-type CA1 pyramidal cells, in which it would be masked by simultaneous larger outward currents activated by heteromeric GABAB(1,2) receptors. Unfortunately, because we lack ligands that distinguish molecular subtypes of GABAB receptors, genetic manipulation is currently the only means to functionally dissociate native GABAB assemblies with and without a GABAB(2) subunit. Regardless of whether the baclofen-induced current seen in GABAB(2)-/- CA1 neurons is a consequence of the knock-out situation or not, the observation of a functional GABAB receptor in the absence of GABAB(2)-/- may be important. An increasing number of studies suggest that various cellular populations in the nervous system express GABAB(1) without GABAB(2) (Billinton et al., 2000; Calver et al., 2000; Clark et al., 2000; Ng and Yung, 2001; Burman et al., 2003; Kim et al., 2003; Kulik et al., 2003; Li et al., 2003; Straessle et al., 2003). Our results imply that neurons that naturally lack a GABAB(2) subunit nevertheless have the potential to express functional GABAB receptors. Unfortunately, it is currently impossible to identify such cells for electrophysiological recordings. This, together with the finding that the GABAB receptors seen in GABAB(2)-/- mice do not appear to be involved in classical GABAB functions, makes it currently difficult to address the possible physiological role of such receptors.

In conclusion, it clearly emerges that heteromeric GABAB(1,2) receptors are the prevalent GABAB receptors in the nervous system and that virtually all GABAB(2) protein is normally associated with GABAB(1). However, our genetic experiments also suggest that GABAB(1) could be functional in neurons that naturally lack GABAB(2) expression.

Footnotes

  • This work was supported by Swiss Science Foundation Grant 3100-067100.01 (B.B.). We thank Rita Meyerhofer, Hugo Buerki, C. Sidler, and F. Parpan for technical support, K. Sauter for genotyping, and D. Benke, C. Lüscher, and all members of the Bettler laboratory for helpful discussions.

  • Correspondence should be addressed to either of the following: Bernhard Bettler, Biozentrum/Pharmazentrum, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland, E-mail: bernhard.bettler{at}unibas.ch; or Klemens Kaupmann, Novartis Institutes for Biomedical Research, Novartis Pharma AG, CH-4002 Basel, Switzerland. E-mail: klemens.kaupmann{at}pharma.novartis.com.

  • Copyright © 2004 Society for Neuroscience 0270-6474/04/246086-12$15.00/0

  • ↵* M.G., H.S., and R.V. contributed equally to this work.

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Redistribution of GABAB(1) Protein and Atypical GABAB Responses in GABAB(2)-Deficient Mice
Martin Gassmann, Hamdy Shaban, Réjan Vigot, Gilles Sansig, Corinne Haller, Samuel Barbieri, Yann Humeau, Valérie Schuler, Matthias Müller, Bernd Kinzel, Klaus Klebs, Markus Schmutz, Wolfgang Froestl, Jakob Heid, Peter H. Kelly, Clive Gentry, Anne-Lise Jaton, Herman Van der Putten, Cédric Mombereau, Lucas Lecourtier, Johannes Mosbacher, John F. Cryan, Jean-Marc Fritschy, Andreas Lüthi, Klemens Kaupmann, Bernhard Bettler
Journal of Neuroscience 7 July 2004, 24 (27) 6086-6097; DOI: 10.1523/JNEUROSCI.5635-03.2004
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