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The Journal of Neuroscience, June 15, 2001, 21(12):4390-4399

G_olf Levels Are Regulated by Receptor Usage and Control Dopamine and Adenosine Action in the Striatum

Denis Hervé¹, Catherine Le Moine², Jean-Christophe Corvol¹, Leonardo Belluscio³, Catherine Ledent⁴, Allen A. Fienberg⁵, Mohamed Jaber⁶, Jeanne-Marie Studler¹, and Jean-Antoine Girault¹

¹ Institut National de la Santé et de la Recherche Médicale (INSERM) U536, Institut du Fer à Moulin, 75005 Paris, France, ² Centre National de la Recherche Scientifique Unité Mixte 5541, Laboratoire d'Histologie-Embryologie, Université Victor Segalen Bordeaux 2, 33076 Bordeaux cedex, France, ³ Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, ⁴ Institut de Recherche Interdisciplinaire en Biologie Humaine et Nucléaire, Université Libre de Bruxelles, Campus Erasme, B-1070 Bruxelles, Belgium, ⁵ Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021, and ⁶ INSERM U259, Institut François Magendie, Université Bordeaux II, 33077 Bordeaux cedex, France

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

In the striatum, dopamine D₁ and adenosine A_2A receptors stimulate the production of cAMP, which is involved in neuromodulation and long-lasting changes in gene expression and synaptic function. Positive coupling of receptors to adenylyl cyclase can be mediated through the ubiquitous GTP-binding protein G_S subunit or through the olfactory isoform, G_olf, which predominates in the striatum. In this study, using double in situ hybridization, we show that virtually all striatal efferent neurons, identified by the expression of preproenkephalin A, substance P, or D₁ receptor mRNA, contained high amounts of G_olf mRNA and undetectable levels of G_s mRNA. In contrast, the large cholinergic interneurons contained both G_olf and G_s transcripts. To assess the functional relationship between dopamine or adenosine receptors and G-proteins, we examined G-protein levels in the striatum of D₁ and A_2A receptor knock-out mice. A selective increase in G_olf protein was observed in these animals, without change in mRNA levels. Conversely, G_olf levels were decreased in animals lacking a functional dopamine transporter. These results indicate that G_olf protein levels are regulated through D₁ and A_2A receptor usage. To determine the functional consequences of changes in G_olf levels, we used heterozygous G_olf knock-out mice, which possess half of the normal G_olf levels. In these animals, the locomotor effects of amphetamine and caffeine, two psychostimulant drugs that affect dopamine and adenosine signaling, respectively, were markedly reduced. Together, these results identify G_olf as a critical and regulated component of both dopamine and adenosine signaling.

Key words: Golf; Gs; G-protein; D₁ receptor; A_2A receptor; knock-out mice; striatum; dopamine; adenosine; dopamine transporter; homologous recombination

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The nigrostriatal dopaminergic neurons are essential for proper motor function in both humans and rodents. In the dorsal striatum, dopaminergic influence is predominantly mediated through D₁ (D₁R) and D₂ (D₂R) dopamine receptors that stimulate and inhibit cAMP production, respectively (Sibley and Monsma, 1992; Jaber et al., 1996). cAMP signaling contributes largely to the acute effects of dopamine as well as to long-lasting changes in gene expression and synaptic plasticity (for review, see Greengard et al., 1999; Berke and Hyman, 2000). In the striatum, D₁R and D₂R are enriched in distinct populations of efferent GABAergic spiny neurons (Gerfen et al., 1990; Hersch et al., 1995; Le Moine and Bloch, 1995; Yung et al., 1996), although sensitive measurements reveal some degree of overlap in receptor distribution (Surmeier et al., 1996). The D₁R-enriched population, referred to as striatonigral, projects to the substantia nigra pars reticulata and to the entopeduncular nucleus and contains substance P and dynorphin as cotransmitters, whereas the D₂R-enriched population, referred to as striatopallidal, projects to the external globus pallidus and contains enkephalins (Beckstead and Cruz, 1986; Gerfen and Young, 1988; Gerfen et al., 1990; Le Moine et al., 1990, 1991; Le Moine and Bloch, 1995). In the striatopallidal neurons, which possess little or no D₁R, adenosine A_2A receptors (A_2ARs) are the main receptor type stimulating the cAMP production (Premont et al., 1977; Schiffmann et al., 1991; Svenningsson et al., 1997). Because of this "strategic" location, A_2ARs are potential therapeutic targets for treating psychosis and Parkinson's disease (Ledent et al., 1997; Svenningsson et al., 1999).

In spite of the strong effects of D₁R and A_2AR on cAMP production, the striatum contains small amounts of G_s, the G-protein subunit responsible for adenylyl cyclase stimulation in most cell types, but high concentrations of the olfactory isoform G_olf (Drinnan et al., 1991; Herve et al., 1993). G_olf shares 80% amino acid identity with G_s and mediates olfactory receptor signaling in the olfactory epithelium (Jones and Reed, 1989; Belluscio et al., 1998). Cocaine responses are abolished in G_olf knock-out mice, indicating that G_olf may be necessary for dopamine action (Zhuang et al., 2000), and recent data provide strong evidence that G_olf couples A_2AR to adenylyl cyclase (Kull et al., 2000). Moreover, we have shown that D₁R- and A_2AR-stimulated cAMP production is blocked in the striatum of G_olf knock-out mice (Corvol et al., 2001).

The first aim of the present study was to characterize in detail the cellular localization of G_s and G_olf in the striatum using in situ hybridization. We then examined the functional relationship between striatal G_olf levels and dopamine or adenosine transmission, using mutant mice in which the genes for D₁R, A_2AR, or dopamine transporter (DAT) are disrupted. Finally, we examined the functional consequences of decreasing G_olf levels, by measuring the locomotor responses to amphetamine and caffeine in mice, which have reduced levels of G_olf. Our results indicate thus that G_olf is a crucial site for the regulation of D₁R and A_2AR efficacy in the striatum and a potential locus for dysfunction in dopamine and adenosine neurotransmission.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Tissue preparation for in situ hybridization. Brains from adult male Sprague Dawley rats (200-280 gm; Centre d'élevage Janvier, Le Genest. Saint-Isle, France) were dissected out, frozen in liquid nitrogen, and sectioned at the level of the striatum following the atlas of Swanson (1992) into 10-µm-thick sections that were stored at 80°C until use. All experiments were performed in accordance with the guidelines of the French Agriculture and Forestry Ministry for handling animals (decree 87849, license 01499) and with the Centre National de la Recherche Scientifique approval.

Probe synthesis. ³⁵S-labeled or digoxigenin-labeled cRNA probes were prepared by in vitro transcription from rat or mouse (G_s) cDNA clones corresponding to fragments of G_olf, G_s, preproenkephalin A (Yoshikawa et al., 1984), substance P (Bonner et al., 1987), D₁R (Monsma et al., 1990), and choline acetyltransferase (Ibanez et al., 1991) cDNAs. The G_olf cDNA clone corresponded to the clone 8 described by Herve et al. (1995), and the G_s cDNA clone corresponded to the most 3' 550 bp fragment of the murine G_s cDNA sequence (Sullivan et al., 1986). The sequence identity between G_olf and G_s probes was <30%. Transcription was performed from 50 ng of linearized plasmid using either ³⁵S-UTP (>1000 Ci/mmol; NEN Life Science, Paris, France) or digoxigenin-11-UTP (Roche Diagnostic, Meylan, France) and SP6, T3, or T7 RNA polymerases as described (Le Moine and Bloch, 1995). After alkaline hydrolysis to obtain cRNA fragments of ~250 bp, the ³⁵S-labeled probes were purified on Sephadex-G50. The ³⁵S-labeled purified probes and the digoxigenin-labeled probes were precipitated in 3 M sodium acetate, pH 5, and absolute ethanol (0.1/2.5 vol).

In situ hybridization: single detection of G_olf and G_s mRNAs on cryostat sections. Cryostat sections were post-fixed in 4% paraformaldehyde (PFA) for 5 min at room temperature, rinsed twice in 4× SSC, and placed into 0.25% acetic anhydride with 0.1 M triethanolamine in 4× SSC, pH 8, for 10 min at room temperature. After dehydration, the sections were hybridized overnight at 55°C under coverslips with 10⁶ cpm of ³⁵S-labeled G_olf or G_s cRNA probes in 50 µl of hybridization solution (20 mM Tris-HCl, 1 mM EDTA, 300 mM NaCl, 50% formamide, 10% dextran sulfate, 1× Denhardt's reagent, 250 µg/ml yeast tRNA, 100 µg/ml salmon sperm DNA, 100 mM DTT, 0.1% SDS, and 0.1% sodium thiosulfate). Coverslips were removed by rinsing in 4× SSC. After 20 min of RNase A treatment (20 µg/ml) at 37°C, the sections were washed sequentially in 2× SSC (5 min, twice), 1× SSC (5 min), 0.5× SSC (5 min) at room temperature, and rinsed in 0.1× SSC at 65°C (30 min, twice) before dehydration (the latter SSC washes contained 1 mM DTT). Sections were used to expose x-ray films (Kodak Biomax; Eastman Kodak, Rochester, NY) for 3-6 d, and then dipped into Ilford K5 emulsion (diluted 1:3 in 1× SSC), which was developed after an 8 week exposition and stained with toluidine blue.

In situ hybridization: simultaneous detection of two mRNAs on the same sections. Different combinations of cRNA probes were used for the simultaneous detection of two mRNAs on the same sections. Cryostat sections were post-fixed, acetylated, and dehydrated as described above. Combinations of ³⁵S- and digoxigenin-labeled probes (10⁶ cpm of ³⁵S-labeled probe plus 10-20 ng of digoxigenin-labeled probe) were hybridized in the same hybridization solution as described above. After elimination of coverslips, the slides were treated with RNase A and washed in decreasing concentrations of SSC as mentioned above, but without DTT. At the end of the washes, the slides were cooled in 0.1× SSC at room temperature and then processed directly for detection of the digoxigenin signal. The sections were rinsed twice for 5 min in buffer A (1 M NaCl, 0.1 M Tris, and 2 mM MgCl₂, pH 7.5), and then for 30 min in buffer A containing 3% normal goat serum and 0.3% Triton X-100. After a 5 hr incubation at room temperature with alkaline phosphatase-conjugated anti-digoxigenin antiserum (1:1000 in buffer A containing 3% normal goat serum and 0.3% Triton X-100; Roche Diagnostic), the sections were rinsed twice 5 min in buffer A, twice 10 min in 1 M STM buffer (1 M NaCl, 0.1 M Tris, and 5 mM MgCl₂, pH 9.5), and twice for 10 min in 0.1 M STM buffer (0.1 M NaCl, 0.1 M Tris, and 5 mM MgCl₂, pH 9.5). The sections were then incubated overnight in the dark at room temperature in 0.1 M STM buffer containing 0.34 mg/ml nitroblue tetrazolium and 0.18 mg/ml bromo-chloro-indolylphosphate. The sections were rinsed briefly in 0.1 M STM buffer and 2 hr in 1× SSC, dried, and dipped into Ilford K5 emulsion (diluted 1:3 in 1× SSC). After being exposed 10-14 weeks in the dark, the emulsions were developed, and the sections were mounted without counterstaining. Labeled neurons both from single-labeling and double-labeling experiments were counted on sections from three different animals as previously described on similar material (Le Moine and Bloch, 1995).

Mutant mice. Pairs of heterozygous mice with a disrupted gene of G_olf (Belluscio et al., 1998) and a hybrid 129 and C57Bl/6 genetic background were crossed. The genomic DNA of the progeny was extracted from tail tissue, digested by HindIII, and hybridized with a G_olf probe corresponding 1.2 kb Pst/Kpn fragment of mouse G_olf gene located 0.5 kb upstream to the ATG codon translational start site. The probe hybridizes with fragments of 2.8 kb when the gene is mutated or 15 kb fragments in wild-type (Belluscio et al., 1998). Pairs of heterozygous mice bearing a null mutation for D₁R or DAT gene and having a hybrid 129 and C57BL/6 genetic background were mated to obtain wild-type and homozygous mutant mice and were typed by Southern blot, as described by Drago et al. (1994) or by Giros et al. (1996). Male wild-type controls and homozygous A_2AR (Ledent et al., 1997) or CB1 cannabinoid receptor (Ledent et al., 1999) knock-out mice used for the experiments were cousins, and their common grandparents were heterozygous mutant mice backcrossed on CD1 background for 10 generations. Mice were kept in stable conditions of temperature (22°C) and humidity (60%) with a constant cycle of 12 hr light and dark and had ad libitum access to food and water.

G protein antibodies. Specific antibodies against G_olf (SL48SP) were obtained by immunizing a rabbit against a recombinant G_olf protein and by purifying the obtained serum with G_olf and G_s columns, as described previously (Corvol et al., 2001). Specific antibodies against G_s (SL22AP) were raised against a G_s-selective peptide and affinity-purified on a peptide column (Penit-Soria et al., 1997). The other antibodies were mouse monoclonal antibodies from commercial sources against G_o (clone L5.6; Neomarkers, Union City, CA), G_i2 (clone 2A.3; Neomarkers), and G (clone 3, Transduction Laboratories, Lexington, KY).

Immunoblot analysis. Wild-type and mutant mice (2-10 month old, age-matched) were killed by decapitation, and their brains were immediately dissected out from the skull and frozen on dry ice. Microdiscs of tissue were punched out from frozen slices (500-µm-thick) within the striatum using a stainless steel cylinder (1.4 mm diameter). Samples were homogenized in 1% SDS, equalized for their content in protein, and analyzed by Western blot as described previously (Herve et al., 1993). Antibody dilutions were 1:1000, 1:500, 1:300, 1:600, and 1:1000 for antibodies against G_olf, G_s, G_i2, G_o, and G, respectively. Antibodies were revealed by the peroxidase-chemiluminescence method (ECL; Amersham, Orsay, France) and autoradiography. The antibodies were stripped one or two times to detect other antigens on the same membranes (Erickson et al., 1982; Herve et al., 1993). Quantification was performed by optical density measurement on autoradiographic films using a computer-assisted densitometer and the NIH Image software. In each membrane, samples from control and mutant mice were alternatively loaded, and the results were normalized as percentage of the mean of controls in each membrane.

cDNA probes and Northern analysis. The G_olf cDNA probe corresponded to a PCR fragment containing the 1156 bp of the coding region of rat cDNA clone (Jones and Reed, 1989; Herve et al., 1995) and the G_s probe was the same as described above. The glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe was a 1.3 kbp full length of rat GAPDH cDNA (Fort et al., 1985). Total RNA was extracted by the guanidinium thiocyanate-phenol-chloroform method (Chomczynski and Sacchi, 1987) from tissue microdiscs taken on frozen slices, and Northern blots were generated as previously described (Herve et al., 1993). The filters were hybridized successively with G_olf, G_s, and GAPDH probes, with a dehybridation step between two hybridations consisting in the incubation of membrane in boiling 0.5% SDS. Radioactivity of positive bands was measured using Instant Imager (Packard, Downers Groves, IL). G_olf and G_s mRNA concentrations were expressed as the counts per minute ratio of G_olf/GAPDH and G_s/GAPDH, respectively.

(³H)SCH23390, (¹²⁵I)iodosulpride, (³H)CGS21680, and (³H)WIN35428 binding. Twenty micrometer coronal brain sections from three adult heterozygous G_olf +/ mice and three wild-type littermates were thaw-mounted onto SuperFrost microscope slides. D₁R, D₂R, A_2AR, and DATs were analyzed by incubating the tissue sections with 2.5 nM (³H)SCH23390 (91 Ci/mmol), 0.2 nM (¹²⁵I)iodosulpride (2000 Ci/mmol), 5 nM (³H)CGS21680 (47 Ci/mmol,) and 4 nM (³H)WIN35428 (86 Ci/mmol), respectively, in conditions as previously described (Bouthenet et al., 1987; Savasta et al., 1988; Jarvis and Williams, 1989; Herbert et al., 1999). The radioligands were obtained from Amersham Pharmacia Biotech (Saclay, France) or from NEN (Boston, MA). Nonspecific binding was determined from adjacent brain sections in the presence of 1 µM SCH23390, 10 µM sulpiride, 10 µM CGS21680, and 30 µM benztropine for blocking D₁R, D₂R, A_2AR, and DATs, respectively. After washing in ice-cold buffer, brain sections were rapidly dried and used to expose tritium-sensitive film (Hyperfilm ³H; Amersham). In autoradiographs, the binding in caudate putamen and nucleus accumbens was evaluated by measuring the optical density with a computer-assisted image analyzer and the NIH image software, and the data were expressed in femtomoles of bound ligand per milligram of tissue using standards (³H-microscales; Amersham Pharmacia Biotech).

Locomotor activity. Male heterozygous G_olf +/ mice and wild-type littermates were introduced in a circular corridor (4.5 cm width, 17 cm external diameter) crossed by four infrared beams (1.5 cm above the base) placed at every 90° (Imetronic, Pessac, France). The locomotion was counted when the animals interrupted two successive beams and, thus, had traveled one-fourth of the circular corridor. In each session, the spontaneous activity was recorded for 50 min, before the animals were injected with saline or drugs, and their activity was recorded for an additional 60 or 120 min period. In the two first sessions, the animals received saline injections (5 ml/kg, i.p.), and in the third session, they received amphetamine (1, 2, or 3 mg/kg, i.p) or caffeine (25 mg/kg, i.p.) dissolved in saline. For each session an equal number of heterozygous and wild-type mice was studied, and for each drug treatment, groups of 7-14 animals were compared. The tests were performed between 12:00 and 6:00 P.M. in stable conditions of temperature and humidity.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Characterization of striatal neurons expressing G_s and G_olf mRNAs

As previously reported (Drinnan et al., 1991; Herve et al., 1993; Kull et al., 2000), the striatum was the brain region in which the contrast between the levels of G_s and G_olf mRNAs was the most dramatic (Fig. 1). At low magnification, high levels of G_olf mRNAs were observed in all the striatal areas, including caudate putamen (Cp), nucleus accumbens (NA), and olfactory tubercle (OT) (Fig. 1A,C,E,G), which appeared almost devoid of G_s mRNAs (Fig. 1D,F). A strong signal for both G_olf and G_s was detected in a few regions, including piriform cortex (Pir), islands of Calleja, medial habenula (Hb), and dentate gyrus (DG) (Fig. 1A-H), whereas in most other brain areas an intense labeling for G_s mRNAs was associated with low or no labeling for G_olf mRNA (Fig. 1A-H). The use of sense probes showed no labeling for either G_olf (Fig. 1I) or G_s (Fig. 1J) mRNAs.

View larger version (72K): [in this window] [in a new window]   Figure 1.   Distribution of Golf and Gs mRNAs in forebrain. Negative of x-ray films exposed to rat brain adjacent sections hybridized with 35S-labeled probes for Golf and Gs mRNAs. A, C, E, G, Golf mRNA was highly expressed in striatal areas, including caudate putamen, nucleus accumbens, and olfactory tubercle (C, E, G). A signal was also found in the piriform cortex, the islands of Calleja, the medial habenula, and the dentate gyrus (A, C, E, G). All the other areas showed very low or no labeling for Golf mRNA. B, D, F, H, On the contrary, Gs mRNA was highly expressed in many brain areas including all cortical areas, septum, globus pallidus, most of the hypothalamic and thalamic nuclei, hippocampus, and amygdala (B, D, F, H). Caudate putamen, nucleus accumbens, and olfactory tubercles showed almost no labeling, except for some sparse neurons (D, F). A total absence of labeling was observed in control experiments using sense probes for Golf and Gs mRNAs (I, J). Cp, Caudate putamen; NA, nucleus accumbens; OT, olfactory tubercle; GP, globus pallidus; Hb, medial habenula; DG, dentate gyrus; Pir, piriform cortex. Scale bar, 5 mm.

At the cellular level G_olf mRNA was expressed in 96.5 ± 1.3% (n = 1250) of the striatal neurons, mostly medium-sized. Conversely, G_s mRNA-positive neurons were scattered all over the striatal areas, including 13.4 ± 1.2% (n = 1300) of the caudate putamen neurons, from which approximately one-third were large-sized. Double-labeling experiments were performed using various combinations of G_olf and G_s probes labeled with ³⁵S or digoxigenin. Virtually all G_s mRNA-positive neurons expressed G_olf mRNA levels above background, whereas G_olf mRNA was present in numerous neurons with no labeling for G_s mRNA (Fig. 2A,B). In particular, G_olf mRNA was present in almost all striatal medium-sized neurons, very few of which also expressed high levels of G_s mRNA (Fig. 2B, arrow). Simultaneous detection of G_olf mRNA and D₁R mRNA showed that virtually all cells expressing D₁R mRNA were positive for G_olf mRNA (Fig. 2C, arrows). However, approximately half of G_olf-positive neurons did not contain D₁R mRNA (Fig. 2C, white arrowheads). Large-sized neurons were positive for both G_olf and G_s (Fig. 2B, arrowhead). These large neurons, which contained G_olf mRNA, as well as G_s mRNA, were cholinergic interneurons, as demonstrated with a digoxigenin-labeled probe for choline acetyltransferase mRNA (Fig. 2D,E, arrows).

View larger version (129K): [in this window] [in a new window]   Figure 2.   Phenotypical characterization of striatal neurons expressing Golf and Gs mRNAs. Double-labeling in situ hybridization of rat brain sections was performed with various combinations of 35S-labeled probes (indicated by asterisks in the figure) and digoxigenin-labeled probes. In all photomicrographs, silver grains for 35S-labeling are visualized using epi-illumination and appear as bright yellow grains, whereas digoxigenin labeling appears as purple staining. A, Most Golf-positive neurons (silver grains) did not contain Gs mRNA (digoxigenin), which was present in only a few large- or medium-sized neurons (which also contained Golf mRNA; 35S, arrows). B, The reverse combination of probe labeling showed that Golf mRNA (digoxigenin) was present in numerous medium-sized neurons, few of which also expressed significant levels of Gs mRNA (35S, arrow). Most of the large-sized neurons contained both mRNAs (arrowhead). C, Simultaneous detection of Golf mRNA (digoxigenin) and D1R mRNA (silver grains) showed that Golf mRNA was colocalized with D1R mRNA (arrows), but approximately half of Golf mRNA-positive neurons did not contain D1R mRNA (white arrowheads). D, E, Double labeling for choline acetyltransferase mRNA (digoxigenin) and Golf mRNA (35S; D) or Gs mRNA (35S; E). Both Golf and Gs mRNAs were present in choline acetyltransferase-positive large neurons (arrows). Numerous medium-sized neurons expressed only Golf mRNA (D, white arrowhead), whereas very few expressed Gs mRNA (E, white arrowhead). F, G, Double labeling for PPA (digoxigenin) and Golf mRNA (35S; F) or Gs mRNA (35S; G). H, I, Double labeling for substance P mRNA (SP; digoxigenin) and Golf mRNA (35S; H) or Gs mRNA (35S; I). Golf mRNA was present both in preproenkephalin A-positive (F, arrows) and substance P-positive (H, arrows) neurons, as well as in negative cells (F, H, silver grains). Gs mRNA was mainly absent in these digoxigenin-labeled neurons (G, I, arrows). Scale bar, 12 µm.

In the dorsal striatum, the striatopallidal and striatonigral medium-sized spiny neurons were identified using digoxigenin-labeled probes for preproenkephalin A mRNA (PPA) (Fig. 2F,G) or for substance P mRNA (SP) (Fig. 2H,I), respectively. G_olf mRNA (³⁵S-labeled probe) was present in both preproenkephalin A- and substance P mRNA-containing neurons (Fig. 2F,H, arrows), whereas the levels of silver grains for G_s mRNA (³⁵S-labeled probe) in these neurons were close to background (Fig. 2G,I, arrows).

Alterations of striatal G-protein levels in mice lacking D₁ receptors

Previous studies had shown that destruction of dopamine neurons resulted in an increase in G_olf protein levels (Herve et al., 1993; Marcotte et al., 1994; Penit-Soria et al., 1997). The simplest explanation of these observations was that the increase in G_olf was the consequence of the chronic absence of dopamine receptor stimulation. We therefore examined whether the complete absence of D₁R was able to alter G_olf, using mice in which the D₁R gene had been disrupted (Drago et al., 1994). As shown in Figure 3, the striatal G_olf concentrations were higher in mutant mice than in wild-type littermates, whereas the levels of other G-protein  subunits G_s, G_o, and G_i2 were not altered (Fig. 3A,B). The levels of G subunit were also significantly increased in D₁R knock-out animals (Fig. 3A,B), probably reflecting the stoichiometric increase in G subunits participating in heterotrimeric complexes with G_olf. The lower increase in G (+36 ± 5%) than in G_olf (+80 ± 10%) levels can be accounted for by the association of G subunits with other  subunits, which remained unaffected. Interestingly, a slight but significant increase in G_olf protein was also detected in the striatum of heterozygous animals having only one null allele for the D₁R gene (116 ± 5% of wild-type; n = 15; p < 0.05), whereas G_s protein levels were unchanged in these animals (103 ± 5% of controls).

View larger version (22K): [in this window] [in a new window]   Figure 3.   Levels of G-proteins in the striatum of D1 receptor knock-out mice. A, Western blot analysis of representative striatal homogenates from D1R knock-out (D1R /) mice or wild-type controls (D1R +/+), with antibodies that recognize specifically Golf, Gs, Gi2, and G. B, The optical density of immunoreactive bands was quantified on films and normalized taking as 100% of the mean optical density of controls on each film (see Materials and Methods). Results correspond to the mean ± SEM of data obtained in 6-16 animals studied in three independent experiments. *p < 0.05 and **p < 0.01 by two-tailed Student's t test. C, Northern analysis of RNA isolated from the striata of D1R knock-out (D1R /) mice and wild-type control (D1R +/+) mice. In each lane, the amounts of 32P-labeled Golf, Gs, and GAPDH probes bound to the membranes were measured with an Instant Imager. The levels of Golf and Gs mRNA were normalized to those of GAPDH mRNA and were expressed as a percentage of mean value measured in wild-type controls (n = 14-16).

We examined G_olf mRNA levels by Northern blot to determine whether the increase in G_olf protein observed in the striatum of mutant mice resulted from an increase in the transcription of the G_olf gene and/or a stabilization of its transcript (Fig. 3C). At least two species of G_olf mRNA were detected in the striatum of wild-type and mutant mice resulting from variations in the length of 5'- and 3'-untranslated regions of G_olf mRNAs, as described previously (Herve et al., 1995). When we measured the total amount of radioactive probe hybridized with all the G_olf mRNA species in Northern blots, the concentration of G_olf transcripts was not significantly different in the striatum of D₁R / mutant mice or wild-type mice (Fig. 3C). When radioactivity was measured independently for the two main bands, no significant variation was observed in the striatum of D₁R-deficient mice, suggesting that the size pattern of G_olf mRNA was unaffected in these mice (data not shown). Thus, the increase in G_olf protein observed in the striatum of D₁R / mutant mice was not the consequence of alterations in G_olf mRNA levels. G_s mRNA concentrations were also not significantly altered in the striatum of D₁R knock-out mice (Fig. 3C).

Alterations of striatal G_olf protein levels in mice lacking dopamine transporter

The results obtained in D₁R-deficient mice, as well as previous experiments using dopamine-depleted animals, indicated that an impairment in dopamine transmission could increase the levels of G_olf in the striatum. To test if constitutive upregulation of dopamine neurotransmission could affect G_olf in the opposite direction, we examined the striatal concentrations of G_olf in mutant mice lacking DAT, responsible for the largest part of dopamine clearance from the extracellular space (Giros et al., 1996). The levels of G_olf, but not G_s, were decreased in the striatum of these mice (Fig. 4). Thus, the chronic increase in the stimulation of D₁R on the one hand, and the absence of D₁R or of dopamine stimulation on the other hand, altered G_olf protein levels in opposite directions.

View larger version (37K): [in this window] [in a new window]   Figure 4.   Levels of Golf and Gs proteins in the striatum of dopamine transporter knock-out mice. A, Western blot analysis of representative striatal homogenates from DAT knock-out mice (DAT/) or wild-type littermates (DAT +/+). Blots were incubated with antibodies that recognize specifically Golf or Gs. B, The optical density of immunoreactive bands was quantified on films as in Figure 3B (n = 8; *p < 0.02; two-tailed Student's t test).

Alterations of striatal G_olf protein levels in mice lacking A_2A receptors

Because G_olf was expressed in neurons containing A_2AR and because the lack of G_olf abolished the cAMP production induced by an A2 agonist (Corvol et al., 2001), we examined whether the absence of A_2AR could alter G_olf levels in a manner similar to D₁R. To evaluate this possibility, we measured G_olf protein and other G-protein subunits in the striatum of mutant mice lacking A_2AR (Ledent et al., 1997). By comparison to wild-type controls, an increase in G_olf protein levels was observed in A_2AR knock-out mice, whereas G_s, G_o, and G_i2 subunits were not significantly altered in the same striatal samples (Fig. 5A,B). In contrast to what was seen in D₁R knock-out mice, no change in striatal concentrations of G was detected in A_2AR knock-out mice (Fig. 5B). This may be attributable to the fact that the increase in the concentration of G_olf was less pronounced than in D₁R / mice, and not sufficient to enhance significantly the total levels of G. As in the case of D₁R, the total amounts of G_olf and G_s mRNA (Fig. 5C) and the expression pattern of the various forms of G_olf mRNA (data not shown) were not altered in A_2AR knock-out mice.

View larger version (23K): [in this window] [in a new window]   Figure 5.   Levels of G-proteins in striatum of A2A receptor knock-out mice. A, Western blot analysis of representative striatal homogenates from A2AR knock-out mice (A2AR /) or wild-type controls (A2AR +/+), with antibodies that recognize specifically Golf, Gs, and Go. B, The optical density of immunoreactive bands was quantified on films as in Figure 3B (n = 4-7; *p < 0.01; two-tailed Student's t test). C, Northern analysis of RNA isolated from the striata of A2AR knock-out (A2AR /) mice and wild-type control (A2AR +/+) mice. Results were obtained as described in the legend to C (n = 10-11).

The observed increases in G_olf protein levels seen in D₁R and A_2AR knock-out mice could be an unspecific consequence of the absence of any G-protein-coupled receptor. To rule out this possibility, we measured the G-protein subunits levels in knock-out mice lacking CB1 cannabinoid receptors, which are expressed at very high levels in medium-sized spiny neurons, but are negatively coupled to adenylyl cyclase unlike the D₁R and A_2AR (Ledent et al., 1999). The lack of expression of CB1 receptors in the striatum induced no significant modification in the striatal concentrations of G_olf (93 ± 5% of wild-type; n = 4). The striatal levels of G_s, G_o, G_i1, G_i2, and G subunits were also unchanged in these mice (data not shown). It is noteworthy that the deficiency in functional D₂R was reported to have no influence on G_olf mRNA expression in the striatum (Zahniser et al., 2000).

Locomotor responses to amphetamine and caffeine in mice heterozygous for a null mutation of G_olf

Because alterations of dopamine or adenosine transmission affected G_olf levels, we investigated whether changes in G-protein levels could have functional consequences on behavioral responses mediated through D₁R and A_2AR. In the striatum of mice heterozygous for a null mutation of G_olf gene (G_olf +/), the levels of G_olf were approximately half of those in wild-type mice, and the D₁R- and A_2AR-stimulated adenylyl cyclase responses were significantly reduced (Corvol et al., 2001). Thus, these mice provided an excellent model to test the effects of reduced amounts of G_olf, such as those observed in DAT-deficient mice, without the interference of other alterations of dopamine neurotransmission.

We examined the locomotor activity of G_olf +/ mice and wild-type littermates in response to D-amphetamine and caffeine, two drugs whose actions depend on D₁R and A_2AR, respectively (Crawford et al., 1997; Ledent et al., 1997). The locomotor activity of mice was tested in three sessions, in which their basal activity was first monitored (Fig. 6A). During the second and third sessions, G_olf +/ mice had a slightly lower spontaneous activity than wild-type littermates (Fig. 6A). The response to drugs was studied during the third session. When G_olf +/ mice were challenged with 1, 2, or 3 mg/kg of D-amphetamine (Fig. 6B) or with 25 mg/kg of caffeine (Fig. 6C), their locomotor activity was stimulated but remained significantly lower than that of wild-type mice receiving the same treatments (Fig. 6B,C). Interestingly, the slight increase in locomotor activity induced by saline injection was also decreased in G_olf +/ mice (Fig. 6B).

View larger version (20K): [in this window] [in a new window]   Figure 6.   Effects of amphetamine and caffeine on the locomotor activity of Golf +/ mice. The horizontal activity of male heterozygous (Golf +/) and wild-type (Golf +/+) mice was measured in three sessions. In each session, animals were allowed to habituate to the testing apparatus for 50 min and received saline injections in the two first sessions and amphetamine (1, 2, or 3 mg/kg) or caffeine (25 mg/kg) in the third session. A, Spontaneous activity of Golf +/ and Golf +/+ mice during the 50 min habituation period in the three sessions. Activities were significantly lower in mutant mice during sessions 2 and 3 (two-way ANOVA; F(1,260) = 22.3 and F(1,260) = 33.1, respectively; p < 0.001). B, Effects of amphetamine or saline injections on the locomotor activity of Golf +/ mice. Data for saline correspond to the results obtained in the second session. C, Effects of caffeine on the locomotor activity of Golf +/ mice. The results are the mean ± SEM of data obtained with 7-14 animals. *p < 0.05 and **p < 0.01, significantly different from wild-type controls with the same treatment.

In G_olf +/ mice, the alterations in responses to amphetamine and caffeine were not caused by changes in D₁R, D₂R, or A_2AR levels in the caudate putamen or nucleus accumbens because the binding of (³H)SCH23390, (¹²⁵I)iodosulpride, and (³H)CGS21680 was unchanged in these brain regions (Fig. 7). The measurements of the (³H)WIN35428 binding in the caudate putamen and nucleus accumbens of G_olf +/ mice (Fig. 7) indicated also a normal density of DAT, which is known to be crucial for the psychostimulant effect of D-amphetamine in mouse (Giros et al., 1996).

View larger version (65K): [in this window] [in a new window]   Figure 7.   Lack of change in D1, D2, and A2A receptors and in dopamine transporter in the caudate putamen and nucleus accumbens of heterozygous Golf knock-out mice. Coronal brain sections at the level of the striatum were incubated with (3H)SCH23390, (125I)iodosulpride, (3H)CGS21680, or (3H)WIN35428 to analyze the D1R, D2R, A2AR, and DAT, respectively. The optical densities in the caudate putamen and the nucleus accumbens were measured in at least six different sections in each mouse and compared with standards. The results correspond to the mean ± SEM of data obtained in three mutant mice and three wild-type littermates. Scale bar, 5 mm.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Neuronal expression of G_olf and G_s in the striatum

Receptor-regulated production of cAMP in the striatum plays a central role in the physiology of the basal ganglia as well as in the long-term modifications that take place after repeated administration of drugs of abuse (for review, see Greengard et al., 1999; Berke and Hyman, 2000). The striatum contains high levels of G_olf and very low levels of G_s (Drinnan et al., 1991; Herve et al., 1993). Recent studies have demonstrated the role of G_olf in receptor-regulated cAMP production of the striatum and its functional consequences (Zhuang et al., 2000; Corvol et al., 2001). We confirm here that most medium-sized neurons, which correspond to GABAergic output spiny neurons and represent the vast majority of striatal neurons (Kemp and Powell, 1971), express G_olf and very little or no G_s transcripts (Kull et al., 2000). In the rat dorsal striatum these output neurons are divided in two populations identified on the basis of the neuropeptides they express (Gerfen and Young, 1988). We found that G_olf mRNA was expressed in both types of neurons, which did not contain significant amounts of G_s mRNA. Moreover, D₁R-positive neurons expressed G_olf transcripts, but no detectable G_s. The rare medium-sized neurons containing G_s mRNA also expressed G_olf in double-labeling experiments. Because substance P- or preproenkephalin A-positive neurons did not express G_s, the medium-sized neurons containing both G_s and G_olf mRNA are likely to be aspiny interneurons (Kawaguchi et al., 1995). Interestingly, we show here that large-sized cholinergic interneurons, identified by the presence of choline acetyltransferase mRNAs, expressed both G_olf and G_s mRNAs. Striatal cholinergic neurons contain no A_2AR (Svenningsson et al., 1997), but a few express low amounts of D₁R (Le Moine et al., 1991; Surmeier et al., 1996). Recent experiments using in situ hybridization have also confirmed the presence of D₅ receptor mRNA in these neurons (C. Le Moine, unpublished data), as previously indicated by single-cell RT-PCR experiments (Yan and Surmeier, 1997) and an immunohistochemical study in primate (Bergson et al., 1995). Our results indicate that both G_olf and G_s may couple D₁R and D₅ receptors to adenylyl cyclase in striatal cholinergic neurons. Thus, it appears that virtually all the striatal output neurons express only G_olf, whereas interneurons express both G_olf and G_s. The presence of G_olf transcripts, but not of G_s, in D₁R- and A_2AR-rich output neurons, accounts for the fact that cAMP formation by stimulation of these receptors was virtually abolished in G_olf mutant mice (Corvol et al., 2001).

Regulation of G_olf levels in mice with genetically altered D₁ or A_2A signaling

In a previous study we have shown that the disappearance of striatal dopamine increased G_olf levels by 40-50% (Herve et al., 1993; Penit-Soria et al., 1997). In mutant mice lacking either D₁R or A_2AR we found a selective increase in G_olf protein. Together these observations reveal that a lack of stimulation of D₁R, as well as the absence of either D₁R or A_2AR, result in an upregulation of G_olf levels, suggesting that receptor usage may control G_olf levels. This hypothesis is corroborated by the study of DAT / mice, which provide a model of chronic hyperstimulation of dopamine receptors in vivo (Giros et al., 1996). In these mice the levels of G_olf protein were decreased in the striatum. These variations are reminiscent, at the level of a G-protein, of the classical "denervation hypersensitivity" and "agonist-induced desensitization," well characterized at the level of receptors (Freedman and Lefkowitz, 1996; Bloch et al., 1999).

Our results show that in A_2AR and D₁R mutant mice, the changes in G_olf occur at a post-transcriptional level and do not result from a change in gene expression. Preliminary evidence suggests that regulation of G_olf levels may also be independent of cAMP-regulated protein phosphorylation pathways, because these levels were unchanged in mice lacking functional DARPP-32 (D. Herve, A. Fienberg, and J. A. Girault, unpublished observations), a key mediator in striatal cAMP-dependent protein phosphorylation (Fienberg et al., 1998). A more attractive possibility is that alterations in G_olf protein levels result directly from changes in its rate of activation. This hypothesis is supported by several cell culture studies on G_s. In NG108-15 cells, stimulation of receptors activating adenylyl cyclase induced a downregulation of G_s at a post-translational level, which was independent of cAMP production (McKenzie and Milligan, 1990; Adie and Milligan, 1994). Similarly, strong activation of G_s by cholera toxin or by an activating mutation dramatically decreased its cellular concentration, possibly by an increased degradation (Levis and Bourne, 1992; Milligan, 1993). We suggest that analogous changes in G_olf degradation rate may account for the changes in its levels observed here in vivo. Normal stimulation of D₁R and A_2AR by endogenous dopamine and adenosine would result in a basal rate of G_olf degradation, which is reduced in mice lacking dopamine or functional D₁R or A_2AR. Conversely, in mice lacking DAT, overstimulation of D₁R would increase G_olf rate of degradation. According to this hypothesis, the changes in G_olf concentration are expected to occur only in the striatal cells affected by the mutation, i.e., striatonigral cells in mice lacking functional D₁R or DAT or striatopallidal cells in mice lacking functional A_2AR.

Role of G_olf in locomotor activity

We have recently shown that reduced levels of G_olf in heterozygous mutant mice (G_olf +/) resulted in a reduced stimulation of adenylyl cyclase by D₁R and A_2AR in the striatum (Corvol et al., 2001). The present study demonstrates that reduced availability of G_olf in these mice has also important functional consequences on spontaneous and drug-stimulated locomotor activity, revealing a novel phenotype of haplo-insufficiency.

The ventral striatum, including the nucleus accumbens, is involved in spontaneous and drug-stimulated motor activity (Pennartz et al., 1994). A role for D₁R in the control of locomotor activity is supported by several studies. Microinjection of D₁R antagonist in the ventral striatum of rats reduces their locomotor activity, whereas D₁R agonists have the opposite effect (Meyer, 1993). Accordingly, the decreased coupling of D₁R to adenylyl cyclase in the ventral striatum of G_olf +/ mice could account for their slightly lower locomotor activity. By contrast it should be noted that an increase in basal activity has been reported in G_olf / mice (Belluscio et al., 1998; Zhuang et al., 2000) and D₁R / mice (Xu et al., 1994). These surprising observations contrasted with the pharmacological evidence for a stimulatory role on motor activity of D₁R in the nucleus accumbens (Meyer, 1993; Vezina et al., 1994). These paradoxical behavioral effects in G_olf / and D₁R / mice could be caused by differences in the methods used for measuring locomotor activity or alternatively may be the consequences of developmental changes and/or compensatory actions of other neuromodulatory systems such as serotoninergic or noradrenergic systems (Tassin et al., 1992; Trovero et al., 1992; Gainetdinov et al., 1999). Nevertheless, in both G_olf / and D₁R / mice, the locomotor responses to cocaine or amphetamine are lost, unambiguously showing that both G_olf and D₁R are necessary for the locomotor responses induced by these psychostimulants (Xu et al., 1994; Crawford et al., 1997; Zhuang et al., 2000). Interestingly, the acute effects of D-amphetamine on motor activity diminished in G_olf +/ mice, further demonstrating that G_olf levels determine the amplitude of D₁R-mediated responses.

In G_olf +/ mice, the effects of caffeine on motor activity were reduced, indicating that G_olf levels are an important factor for caffeine-induced behavioral response. The psychostimulant effects of caffeine are mediated through A_2AR and are absent in mutant mice lacking these receptors (El Yacoubi et al., 2000). Caffeine is a nonselective antagonist of A_2AR, and caffeine could have a reduced effect in G_olf +/ mice because the ongoing A_2AR signaling, which is produced by endogenous adenosine, is blunted in these mice. This explanation is likely because endogenous levels of adenosine are sufficient to cause the activation of adenosine receptors in brain (for review, see Svenningsson et al., 1999). However, D₁R activity is required for caffeine-induced motor effect (Garrett and Holtzman, 1994), and the lower D₁R signaling in G_olf +/ mice could also contribute to the attenuation of caffeine effects. Further experiments are needed to explore the respective contribution of these two mechanisms, both leading to a diminished effect of caffeine.

Functional implications

The present study demonstrates that experimental alterations in G_olf concentrations affect two important types of neurotransmission in basal ganglia and provides strong evidence that G_olf levels in striatal neurons are regulated in vivo by its use. Whereas much effort has been devoted to understand desensitization and downregulation of G-protein-coupled receptors in vitro and in vivo (Freedman and Lefkowitz, 1996; Bloch et al., 1999), little is known about the regulations that take place at the level of G-proteins. Our previous results in 6-hydroxydopamine-lesioned rats, as well as the present results in mice with genetically altered neurotransmission, show that a loss of D₁R activation or the absence of D₁R or A_2AR result in an upregulation of G_olf. Thus, G_olf constitutes an attractive locus for the regulation of D₁R and A_2AR signaling in the striatum, either by modulation of its levels, or, possibly, by functionally relevant post-translational modifications. Alterations in G_olf levels and/or activity are parameters that will have to be carefully considered in the function of the basal ganglia in physiological and pathological conditions. In this respect, our results introduce G_olf heterozygous mutant mice as a potential animal model for studying striatum-dependent behavioral dysfunctions.

	FOOTNOTES

Received Jan. 29, 2001; revised March 20, 2001; accepted March 27, 2001.

This work has been supported by Institut National de la Santé et de la Recherche Médicale, by a grant from Mission Interministérielle de lutte contre la Drogue et la Toxicomanie addiction program, and by Grant 97H0003 from Ministère de Education Nationale de l'Enseignement Supérieur et de la Recherche. We thank F. Laujay for his help with in situ hybridization experiments, R. Axel, J. Drago, M. Parmentier, B. Giros, and M. Caron for kindly allowing the use of G_olf, D₁R, A_2AR, and DAT mutant mice, respectively, F. Gonon and E. Borrelli for providing us with some mutant mice, P. Ingrassia, R. Urbe, and H. Ibrahim for their help with animal breeding, and C. Drouin and J. P. Tassin for their advice about the measure of locomotor activity. We thank J. Glowinski and his colleagues of INSERM U114 for their support.

Correspondence should be addressed to D. Hervé, INSERM U536, Collège de France, 11, place Marcelin Berthelot, 75231 Paris, France. E-mail: denis.herve{at}infobiogen.fr.