A polyclonal antibody was generated using synthetic peptides designed in a specific sequence of the rat D3 receptor (D3R). Using transfected cells expressing recombinant D3R, but not D2 receptor, this antibody labeled 45–80 kDa species in Western blot analysis, immunoprecipitated a soluble fraction of [125I]iodosulpride binding, and generated immunofluorescence, mainly in the cytoplasmic perinuclear region of the cells. In rat brain, the distribution of immunoreactivity matched that of D3R binding, revealed using [125I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino] tetralin ([125I]7-trans-OH-PIPAT), with dense signals in the islands of Calleja and mammillary bodies, and moderate to low signals in the shell of nucleus accumbens (AccSh), frontoparietal cortex, substantia nigra (SN), ventral tegmental area (VTA) and lobules 9 and 10 of the cerebellum. Very low or no signals could be detected in other rat brain regions, including dorsal striatum, or in D3R-deficient mouse brain. Labeling of perikarya of AccSh and SN/VTA appeared with a characteristic punctuate distribution, mostly at the plasma membrane where it was not associated with synaptic boutons, as revealed by synaptophysin immunoreactivity. In SN/VTA, D3R immunoreactivity was found on afferent terminals, arising from AccSh, in which destruction of intrinsic neurons by kainate infusions produced a loss of D3R binding in both AccSh and SN/VTA. D3R-immunoreactivity was also found in all tyrosine hydroxylase (TH)-positive neurons observed in SN, VTA and A8 retrorubral fields, where it could represent D3autoreceptors controlling dopamine neuron activities, in agreement with the elevated dopamine extracellular levels in projection areas of these neurons found in D3R-deficient mice.
- nucleus accumbens shell
- substantia nigra
- ventral tegmental area
- D3 receptor-deficient mice
- tyrosine hydroxylase
Converging pharmacological, genetic and human postmortem studies have implicated the D3 receptor (D3R) in the physiopathology and treatment of schizophrenia, drug addiction and depression (Pilla et al., 1999; Lammers et al., 2000; Schwartz et al., 2000). In rat brain, the largest D3R expression densities occur in granule cells of the islands of Calleja and in medium-sized spiny neurons of the rostral and ventromedial shell of nucleus accumbens (AccSh), which coexpress the D1receptor and neuropeptides (Diaz et al., 1994; Diaz et al., 1995; Le Moine and Bloch, 1996). The neurons from AccSh receive their dopaminergic innervation from the ventral tegmental area (VTA) and other innervations from cerebral cortex, hippocampus and amygdala (Zahm and Brog, 1992; Pennartz et al., 1994), project indirectly to entorhinal and prefrontal cortice and subserve the control of emotion, motivation and reward (Willner and Sheel-Krüger, 1991).
One aspect of the localization and function of the D3R that is still highly debated is its occurrence as an autoreceptor, regulating the activity of dopamine neurons. We originally proposed the existence of D3 autoreceptors on the basis of the expression in substantia nigra (SN) and VTA of D3R mRNA, which strongly decreases after lesion of dopamine neurons (Sokoloff et al., 1990). This lesion, however, also downregulates postsynaptic D3R in AccSh (Lévesque et al., 1995), by deprivation of brain-derived neurotrophic factor (BDNF), an anterograde factor of dopamine neurons (Guillin et al., 1999). Hence the lesion-induced decrease in SN/VTA could reflect a similar process occurring in non-dopaminergic neurons.
Dopamine release (Tang et al., 1994) and synthesis (O'Hara et al., 1996) are inhibited by stimulation of the D3R expressed in a transfected mesencephalic cell line and various agonists, with limited preference for the D3R (Sautel et al., 1995), inhibits dopamine release, synthesis and neuron electrical activity (for review, see Levant, 1997), giving support to the existence of D3 autoreceptors. However, the selectivity of these agonists toward the D3Rin vivo is strongly questioned, because they elicit similar inhibition of dopamine neuron activities in wild-type and D3R-deficient mice (Koeltzow et al., 1998). In addition, dopamine autoreceptor functions are suppressed in D2 receptor-deficient mice (Mercuri et al., 1997;L'hirondel et al., 1998). Nevertheless, dopamine extracellular levels in the nucleus accumbens (Koeltzow et al., 1998) and striatum (R. Gainetdinov and M. G. Caron, personal communication) are twice as high in D3R-deficient as in wild-type mice, suggesting a control of dopamine neurons activity by the D3R.
Direct confirmation of the role of the D3R as an autoreceptor requires the demonstration of its occurrence in dopamine neurons, namely by using immunocytochemical methods. In fact, the antibodies directed against the D3R reported so far generated immunolabeling that did not overlap distributions of D3R mRNA and binding sites (Ariano and Sibley, 1994; Larson and Ariano, 1995; Khan et al., 1998), which questions their specificity. In the present study, we have generated a polyclonal antibody against the D3R using sequence-specific peptides and assessed its specificity using recombinant receptors and D3R-deficient mice and by comparison with D3R binding. This antibody has then been used to examine cellular localizations of the D3R by comparison with tyrosine hydroxylase (TH) and synaptophysin immunoreactivities.
MATERIALS AND METHODS
Immunization, antiserum titration and antibody purification. The immunization procedure conformed with local guidelines and has been performed by a person accredited by the French Minister of Agriculture (decree 87848). The peptide H-YGAGMSPVERTRNSL-OH (Y15L) was coupled by copulation with diazotized benzidine to bovine serum albumin (BSA) and subcutaneously injected together with complete Freund's adjuvant once, and then with the incomplete Freund's adjuvant each week for 4 weeks (268 μg of peptide per injection), into female New Zealand rabbits. A booster injection in complete Freund's adjuvant was performed one month latter with the same immunogen. The peptide Y15L was coupled to keyhole Limpet hemocyanin and used for a booster injection (500 μg of the peptide) 3 months later. Three months later, the rabbits were injected with the peptide H-GAGMSPVERTRNSLY-OH (G15Y) coupled by copulation via bisdiazobenzidine to ovalbumin. Five booster injections with the latter peptide (350 μg of peptide/injection) were made during the 3 years that followed. Rabbits were bled every 1–4 weeks and serum titer was assayed using [125I]tyrosyl-labeled G15Y or Y15L and polyethyleneglycol precipitation.
The best titer antiserum collected at the end of the immunization procedure was precipitated with ammonium sulfate, filtered on DEAE-Sephadex and immunopurified on a HiTrap column (Amersham Pharmacia Biotech, Little Chalfont, UK) coupled to peptide G15Y by the amino group. The purified antibody was eluted in glycine-HCl 0.1m, pH 2.3.
Western blot analysis. Wild-type and transfected Chinese hamster ovary (CHO) cells expressing the D2receptor or D3R (Sokoloff et al., 1990) were scrapped, harvested in 50 mm sodium phosphate buffer (PB) containing 150 mm NaCl (PBS), and homogenized in Tris-HCl buffer, 10 mm containing 5 mm EDTA and protease inhibitors (aprotinin, 1 μg/ml; leupeptin, 1 μg/ml; pepstatin, 0.1 μg/ml). Membranes were isolated by centrifugation at 5000 ×g for 10 min and solubilized in PAGE-loading buffer (50 mm Tris-HCl, pH 7.4, 5 mmEDTA, 10% glycerol, 2% SDS, protease inhibitors as above). Proteins (20 μg) were separated by electrophoresis in 12% SDS-polyacrylamide gel and electrophoretically transferred to nitrocellulose filters. Blots were blocked in PBS Blotto (Pierce, New York, NY) with 1% bovine serum albumin at room temperature for 2 hr. The blots were then incubated with purified anti-D3R antibody (1:2000) overnight at 4°C. After three 10 min washes in PBS containing 0.05% Tween 20, blots were incubated with a horseradish peroxydase-conjugated goat anti-rabbit γ-globulins antibody (1:10,000, Pierce) for 1 hr at room temperature and developed using the enhanced chemiluminescence procedure (Amersham Pharmacia Biotech).
Receptor immunoprecipitation. Membranes of CHO cells expressing the D2 receptor or D3R were solubilized in 1% digitonin, 1% sodium cholate, and 1 m NaCl in 50 mm sodium phosphate buffer, pH 7.4, for 30 min at 4°C, diluted twice in sodium phosphate buffer, and centrifuged for 30 min at 50,000 × g. Supernatants (100 μl) diluted 10 times in sodium phosphate buffer were incubated in a final volume of 400 μl with diluted preimmune serum or antiserum and [125I]iodosulpride (0.1 nm, 2200 Ci/mmol) (Amersham) overnight at 4°C. Some incubations were performed with the antibody previously presaturated overnight with the peptide G15Y (1 μg/μl), and some were performed in the presence of 1 μmemonapride to measure nonspecific binding. Fifty microliters of a 50% (v/v) suspension of protein A-Sepharose (Amersham Pharmacia Biotech) were added, and tubes were incubated under gentle agitation for 2 hr at 4°C and then centrifuged for 3 min at 14,000 rpm. Aliquots of supernatants were filtered through GF/B filters coated with 0.3% polyethyleneimine, and filters were rinsed with 3 × 3 ml of cold sodium phosphate buffer. In preliminary experiments, this procedure allowed us to solubilize and recover up to 20% of membrane-bound receptors.
Animals, tissue, and cell preparation for immunohistochemistry and immunofluorescence. Male Wistar rats (180–250 gm, Iffa-Credo, L'Arbresles, France) or mice (see below) were anesthetized deeply with pentobarbital (30 mg/kg, i.p.) and then perfused transcardially with 50 ml of saline solution (0.9% NaCl warmed at 37°C), followed by 600 ml of an ice-cooled fixative solution containing 2% paraformaldehyde in 0.1 m PB, pH 7.5. The brains were removed, post-fixed in 2% paraformaldehyde for 1–2 hr at 4°C, and rinsed in PB solutions. Some brains were transferred into ascending series of sucrose solutions (10% overnight, 15% for 24 hr, and 20% for 24 hr), frozen and stored at −70°C, and then sectioned in coronal and sagittal planes with a cryostat in 30 μm sections. Other brains were immediately cut in the same planes with a vibratome in 40 μm sections that were collected, cryoprotected in PB containing 30% sucrose, and freeze-thawed (−75°C) to improve penetration of the antibodies. Cryostat or vibratome sections were collected in 0.05m Tris buffer, pH 7.5, containing 150 mm NaCl (TBS) and then treated with blocking serum (5% normal donkey serum, 0.4% BSA, 0.1% gelatin, and 0.1% Tween 20 in TBS) for 1 hr at room temperature. CHO cells were cultured on 20 × 20 mm collagen-coated slides, rinsed with PBS, fixed for 30 min in 2% paraformaldehyde, rinsed again in 0.1 m glycine–PBS, and immersed in the blocking serum as above.
Detection of D3R immunoreactivity by the immunoperoxidase method. The sections were incubated for 24–48 hr at room temperature with the immunopurified anti-D3 receptor antibody diluted 1:2000 in TBS containing 5% normal donkey serum and 0.05% Tween 20 (TBS–NDST20). Some sections were incubated with the antibody previously presaturated overnight with the peptide G15Y (1 μg/μl). The sections were rinsed (four times for 10 min) in TBS containing 0.1% gelatin and 0.05% Tween 20 (TBS–GT20) and immersed for either 1–2 hr at room temperature or overnight at 4°C in biotinylated donkey anti-rabbit γ-globulins (Amersham) diluted 1:200 in TBS–NDST20. The sections were rinsed (three times for 10 min) in TBS–GT20 and then incubated for 1 hr at room temperature in avidin–biotin–HRP complex (ABC reagent, Vectastin-Elite; Vector Laboratories, Burlingame, CA). After rinsing in TBS containing Tween 20 (0.05%) and then in TBS, peroxidase activity was revealed by incubation with 3,3′ diaminobenzidine for 10–30 min at 4°C in the presence of hydrogen peroxide using the Sigma Fast diaminobenzidine tablets (Sigma, St. Louis, MO). The peroxidase reaction was stopped by several rinses in Tris-HCl. The sections were mounted on glass slides, dehydrated in graded ethanols, cleared, and then mounted in Acrytol for observation under a ZeissAxiophot microscope.
Immunofluorescence experiments. Vibratome coronal sections taken at levels of both basal forebrain (nucleus accumbens–ventral pallidum) and midbrain (VTA–SN) regions were incubated for 24–48 hr at room temperature in a mixture of primary immunoreagents diluted in TBS–NDST20. The mixture consisted of purified anti-D3R antibody diluted 1:2000 and mouse monoclonal antibodies directed against either synaptophysin (diluted 1:50) (Boehringer Mannheim Biochemica, Mannheim, Germany) or anti-TH (diluted 1:10,000) (Incstar, Stillwater, MN). Slides with fixated CHO cells were incubated for 48 hr at 4°C with the purified anti-D3R antibody only, diluted 1:2,000. After four washes (10 min each) in TBS–GT20, the sections were incubated in FITC–donkey anti-mouse (Jackson Immunoresearch, West Grove, PA) diluted 1:200 in TBS–NDST20, followed by Cy3–donkey anti-rabbit γ-globulins (Jackson Immunoresearch), diluted 1:1000 in TBS–NDST20, for 1 hr at room temperature. To intensify the D3R fluorescent immunostaining, some double-labeling experiments were performed using a biotinylated donkey anti-rabbit IgG (1:200 in TBS–NDST20) followed by three washes (10 min each) and incubation for 1 hr in Cy3-streptavidin (0.5 μg/ml in TBS–T20). The sections were washed (three times for 10 min), mounted on Super Frost Plus slides, and then coverslipped using Vectashield mounting medium (Vector Laboratories) and nail polish to seal the coverslip. Sections were examined and photographed using ZeissPlan-Neofluar objectives and band-pass filter sets for FITC and rhodamine. Control experiments were performed to ensure that each primary antibody did not react with the non-corresponding secondary antibody-conjugate. In such experiments, sections were incubated as follows: rabbit anti-D3R antibody, followed by FITC–donkey anti-mouse or mouse anti-TH followed by Cy3–donkey anti-rabbit. In these controls, only light autofluorescence and no cross-reactive immunostaining were observed.
D3R-deficient mice. Heterozygotous mice bearing a mutation invalidating the D3R gene, originally obtained from S. Fuchs (Weizmann Institute, Rehovot, Israel) (Accili et al., 1996), were bred and mated. DNA was prepared from a piece of the tail (3–5 mm) using the DNAeasy tissue kit (Qiagen France, Courtaboeuf, France) and amplified with the mixture of primers GCA GTG GTC ATG CCA GTT CAC TAT CAG and CCT GTT GTG TTG AAA CCA AAG AGG AGA GG, amplifying the exon 3 of the wild-type D3R, and TGG ATG TGG AAT GTG TGC GAG and GAA ACC AAA GAG GAG AGG GCA GGA C, amplifying the PGK cassette of the mutated gene. Agarose gel electrophoresis allowed us to detect homozygotous wild-type mice (a single band at 137 bp), homozygotous mutated mice (a single band at 200 bp), and heterozygotous mice (bands at 137 and 200 bp). Homozygotous mutated mice and their wild-type littermates were used in the study.
Lesion studies. Male Sprague Dawley rats (180–200 gm, Iffa Credo, L'Arbresles) were anesthetized with pentobarbital (50 mg/kg). Nucleus accumbens lesions were made by infusion of kainate (1.5 μl of a solution at 2.5 μg/ml in 25 mm Tris-HCl buffer, pH 7.4, at the following coordinates: anterior-posterior +1.7 mm; lateral −1 mm; ventral −7 mm from the dural surface. The infusion cannula was left in position for 3 min after each one-side infusion. Animals were killed 10 d after stereotaxic surgery. Assessment of the placement and extent of the lesion was performed under microscopic observation of Nissl-stained sections.
Receptor autoradiography. Unfixed 10 μm cryostat brain sections were preincubated at room temperature three times for 5 min in 50 mm sodium-HEPES buffer, pH 7.5, containing 1 mm EDTA and 0.1% BSA. They were incubated in the same buffer containing 0.2 nm[125I]R(+)trans-7-hydroxy-2-[N-propyl-N-(3′-iodo-2′-propenyl)amino] tetralin ([125I]trans-7-OH-PIPAT; 2200 Ci/mmol; Amersham) for 45 min at room temperature. Nonspecific binding was determined by incubating adjacent sections in the same medium in the presence of 1 μm dopamine. After incubation, slices were washed four times for 2 min in ice-cold sodium-HEPES buffer containing 100 mm NaCl, dipped in ice-cold distilled water, and then dried under a stream of cold air. Autoradiograms were generated by apposing sections to3H-Hyperfilm (Amersham) for 2–4 d and developed in D-19 developer. Autoradiographic signals were quantified on two to three slices per animal using an image analyzer (IMSTAR, Paris, France). Gray values were converted to microcuries per gram wet weight using 125I standard stripes (Amersham).
In situ hybridization. The procedure has been described previously (Diaz et al., 1995). Briefly, paraformaldehyde-fixed slices (10 μm) were treated with proteinase K (1 μg/ml) and then with 0.25% acetic anhydride in triethanolamine buffer. The sections were hybridized with a 33P-radiolabeled cRNA probe for D3R mRNA (2 × 106 dpm per slide) in 65% deionized formamide, 10% dextran sulfate, 1× Denhardt's solution, 2× SSC, 0.1% sodium pyrophosphate, 100 μg/ml yeast tRNA, and 100 μg/ml denatured salmon sperm DNA. After incubation and rinsing, slices were treated with RNase A (200 μg/ml), washed and dehydrated through a graded series of ethanol, and apposed to films (Hyperfilm β-max, Amersham) for 1 month. Films were developed in D-19 developer (15°C) for 5 min, rinsed rapidly in deionized water, and fixed in 30% sodium thiosulfate for 10–20 min.
Generation of anti-D3R antibodies
The immunizing peptide sequence was designed in the putative third intracytoplasmic loop of the rat D3R, which has no homologies with the sequence of other dopamine receptor subtypes (Fig. 1 A). A portion of this sequence has a strong homology with the human D3R. A search in databases did not reveal any other homology with a known protein. A first peptide, Y15L, had an additional tyrosine residue at the N terminus, allowing coupling to the carrier tyrosines with diazotized benzidine, and was used to immunize rabbits. After the initial immunization and a booster injection, rabbits were subsequently boosted with a second peptide, G15Y, having the same sequence, but with the additional tyrosine used for coupling located at the C terminus instead of the N terminus. Immunoreactivity of the antisera was measured with the two peptides [125I]-labeled at either the C or N terminus (Fig. 1 B). Although immunoreactivity against [125I]Y15L raised and reached a similar antiserum titer after each booster injection, immunoreactivity against [125I]G15Y appeared only after G15Y injections, and its titer increased progressively thereafter. At the end of immunization, antisera titers were similar against the two iodinated peptides. Furthermore, antibodies had similar affinities for Y15L and G15Y (EC50 = 0.5 nm; data not shown), as well as a shorter peptide S10L (see sequence on Fig. 1 A), indicating that antibodies indeed recognized internal epitopes.
Characterization of antibodies using recombinant dopamine receptors
In crude membrane preparations of CHO cells expressing the D3R that were subjected to SDS-PAGE, the immunopurified antibody labeled major bands at ∼45 and ∼80 kDa (Fig. 2 A). Minor bands, possibly corresponding to degraded proteins, were also visible at ∼60 kDa and below 35 kDa. All of these bands were absent when the antibody was presaturated with the immunizing peptide G15Y. No signal could be detected with membranes from wild-type or D2receptor-expressing CHO cells.
D3 or D2 receptors expressed by CHO cell lines were solubilized with a digitonin–cholate mixture and labeled with [125I]iodosulpride in the presence of antiserum or preimmune serum. The activity in the supernatant was measured after precipitation of antibody-receptor complexes with immobilized protein A. As shown in Figure 2 B, the antiserum immunoprecipitated >60% of solubilized D3R, but not D2 receptor, and the effect was abolished by presaturation of the antibody with the immunizing peptide. Because the antiserum had no effect on [125I]iodosulpride binding without protein A (<5% inhibition at any dilution starting from 10− 3; data not shown), the depletion of [125I]iodosulpride binding in the supernatant of protein A precipitation represented true immunoprecipitation.
Strong immunofluorescence signals generated with an immunopurified antibody were detected on D3R-expressing CHO cells, but not on wild-type nor D2receptor-expressing CHO cells (Fig. 2 C). Most of the signal appeared in the cytoplasm of D3R-expressing cells, particularly in the perinuclear region, but some cells also displayed immunofluorescence at the plasma membrane (Fig.2 C, arrow). No signal could be detected when the D3R antibody was omitted or presaturated with the immunizing peptide (data not shown).
Distribution of D3 receptor immunoreactivity in rat and wild-type or D3 receptor-deficient mouse brains
In rat brain slices, there was a close overlap between the distributions of D3R immunoreactivity, revealed by immunoperoxidase, and binding sites for [125I]trans-7-OH-PIPAT, a D3R-selective ligand (Burris et al., 1994). Thus, highest immunoreactivity levels were found in the islands of Calleja (Fig. 3 B), which also express the highest density of D3R mRNA (Bouthenet et al., 1991; Diaz et al., 1995). Strong labeling was also observed in AccSh. A much weaker signal was observed in the core of nucleus accumbens (Fig. 3 B), a region where D3R mRNA and binding levels are weak (Fig.3 A) (Bouthenet et al., 1991; Diaz et al., 1995). In normal D3R+/+ mouse brain, the D3R immunolabeling was similar to that found in rat brain and undetectable in D3R-deficient mouse brain (Fig. 3 D,E).
A semiquantitative comparison between D3R immunoreactivity, binding, and mRNA levels in rat brain (Table1) shows an overall agreement between distributions of the three markers. For instance, a high level of D3R immunoreactivity was found in mammillary bodies, a region expressing high levels of D3R binding and mRNA (Bouthenet et al., 1991), whereas a much weaker signal was observed in dorsolateral striatum, where the latter markers are hardly detectable. Moderate signals were observed in the frontoparietal cortex, ventral pallidum, anteroventral thalamic nucleus, and lateral habenula, which also express D3R binding (Table1), whereas other brain regions contained very low or undetectable levels of both D3R immunoreactivity and binding. The only discrepancy was found in the cerebellum, where high levels of D3R binding and mRNA are present in lobules 9 and 10 (Bouthenet et al., 1991; Diaz et al., 1995), whereas weaker D3R immunoreactivity was observed (Table 1). This suggested that the D3R may differ in cerebellum and other brain regions, but cDNAs amplified from mRNAs extracted from cerebellar lobules 9 and 10 and from nucleus accumbens had identical sequences, notably in the region corresponding to the immunizing peptide (data not shown).
D3 receptor immunoreactivity in the mesencephalon
Moderate D3R immunoreactivity, revealed by the immunoperoxidase method, was found in the rat SN/VTA complex, where individual cell bodies were clearly visualized in all parts of the complex (Fig. 3 H,J). Enlargement shows labeling of individual cell bodies in the SN pars compacta (Fig. 3 J). The immunoreactivity was abolished by presaturation of the antibody with the immunizing peptide G15Y (Fig. 3 I,K). In contrast, [125I]trans-7-OH-PIPAT binding was prominent in a restricted part of the complex, at the junction between SN pars compacta and VTA (Fig. 3 F), and did not overlap the highest D3R mRNA densities (Fig. 3 G). [125I]trans-7-OH-PIPAT binding in this area indeed labeled the D3R, because this binding was completely absent in D3R-deficient mice (Fig.4 A,B). D3R immunoreactivity, revealed by the immunoperoxidase method in SN/VTA, was largely absent in D3R-deficient mice (Fig.4 C,D), and the characteristic punctuate distribution of D3R immunofluorescence in neurons of VTA was completely absent in these mice (Fig.4 E,F). In addition, D3R immunoreactivity, binding, and mRNA were present in the A8 retrorubral dopamine cell group in the rat (Table1).
To investigate the possibility that a fraction of D3R binding in SN/VTA was present on afferent fibers from extrinsic neurons, we performed lesions of neurons intrinsic to AccSh by local infusions of kainate. Ten days later, this lesion elicited disappearance of Nissl-stained cells centered in the shell part of nucleus accumbens, but extended to the core part of nucleus accumbens and medioventral striatum (data not shown). No signs of trans-synaptic degeneration were observed: intrinsic neurons present in SN/VTA were apparently normal in number and shape (data not shown). The lesion elicited large and identical decreases (−62 and −63%, respectively) in D3R binding in both the lesioned area and ipsilateral SN/VTA (Fig.5 A,B).
Comparison of D3 receptor and tyrosine hydroxylase immunofluorescences
To assess the occurrence of D3autoreceptors, double-labeling experiments with antibodies directed against the D3R and TH were performed on sections containing SN/VTA or AccSh, a region of dopamine neurons projection where the D3R is abundant. In SN/VTA, D3R immunofluorescence corresponded to the immunoreactivity revealed with the immunoperoxidase method (Figs.3 H, 6 A). At lower magnification (Fig.6 A,B), distributions of D3R and TH immunofluorescences were overlapping in the SN pars compacta and VTA but not in SN pars reticulata and SN pars lateralis, where most D3R-positive cells were TH negative. Microscopic examination of every section from 10 animals (15–20 sections per animal) at high magnifications (see representative examples in Fig.6 C-F) indicated that all TH-positive cells also displayed D3R immunofluorescence; we could not find any TH-positive cell not expressing D3R immunofluorescence in SN and VTA. In contrast, some D3R-positive cells (a few of them are designated by arrows in Fig. 6 D) did not display TH immunofluorescence. At the highest magnification used (Fig.6 E,F), there were clearly distinct cellular localizations of the two immunofluorescences: D3R immunofluorescence appeared with a characteristic punctuate distribution at the plasma cell membrane and within the cytoplasm, whereas TH immunofluorescence was homogeneously distributed in the cytoplasm. Both immunofluorescences were absent from the cell nucleus. In addition, on a limited number of sections (n = 3) taken at the level of the retrorubral A8 dopamine cell group, we also observed that all TH-positive cells also displayed D3R immunofluorescence (data not shown).
In AccSh, D3R immunofluorescence also appeared with a punctuate distribution at the plasma membrane of cell bodies and within the neuropil (Fig. 6 G). TH immunofluorescence was mainly distinct from D3 receptor immunofluorescence, with very rare apparent coincidences (Fig.6 H).
Comparison of D3 receptor and synaptophysin immunofluorescences
To assess whether the D3R immunofluorescence was localized at the vicinity of synaptic components, we compared distributions of D3R and synaptophysin immunofluorescences in AccSh (Fig. 6 I), islands of Calleja (Fig. 6 J), and VTA (Fig.6 K). The punctuate distribution of D3R immunofluorescence both in the cell bodies, mostly near the plasma membrane, and in the neuropil in the three regions did not correspond to the distribution of synaptophysin immunofluorescence, and only few apparent coincidences could be found.
Many studies have used antibodies raised against carrier-coupled synthetic peptides, the sequence of which is chosen in unique and predicted antigenic regions of the target protein. The antibodies raised in this manner are likely to recognize mainly the free, not the coupled, extremity of the peptide, which is a valuable strategy if the chosen sequence is at the N or C termini of the target protein, but a possible drawback if the sequence is internal. In the case of the D3R, the N-terminal sequence has several putative sites for N-glycosylation, which may hinder recognition of the native protein, and the C terminus displays significant homologies with D2 and D4 receptor corresponding sequences. Hence, we decided to generate antibodies by using a synthetic peptide taken at the level of the third intracytoplasmic loop, coupled to the carrier by its N terminus, and then using a peptide having the same sequence but coupled by its C terminus. As expected, this strategy indeed favored generation of antibodies recognizing internal epitopes, because these antibodies display similar titer and affinity for the immunizing peptides, whether they are [125I]tyrosyl-labeled at the C or N terminus. The immunopurified antibody also binds to denatured as well as native recombinant D3R expressed by transfected CHO cells. The major immunoreactive species are ∼40 and 80 kDa in size, and the smallest species may correspond to a degraded, incompletely synthesized, or nonglycosylated form (Mr 45,500 Da deduced from the predicted amino acid sequence), whereas the largest species may correspond to either SDS and reduction-resistant dimers or glycosylated forms. The species at ∼80 kDa have the same apparent size as D2/D3 receptor binding sites labeled with photoaffinity radioligands using brain tissue homogenates (Amlaiky and Caron, 1985; Redouane et al., 1985), suggesting the largest as the active form. This hypothesis needs confirmation, however, because we could not reliably measure the apparent size of the native D3R from rat brain, which is not surprising given its very low expression level: 10–100 times lower than that of the D2 receptor (Lévesque et al., 1992).
Thus, in transfected CHO cells, the overexpressed D3R recognized by the antibody is largely present as immature or degraded protein, which is in agreement with its main occurrence in the cytoplasm, particularly in the perinuclear region of the cell. This cellular localization markedly contrasts with that found in neurons, where the D3R is apparently mainly present at the plasma membrane, with only few occurrences as cytoplasmic patches, probably associated with recycling or synthesizing vesicles. In addition, the antibody recognizes recombinant solubilized D3R, in an active form able to bind a radioligand and precipitable by immobilized protein A. This antibody would therefore be a valuable tool for studying the D3R regulation, e.g., phosphorylation.
In rat brain sections, the pattern of immunolabeling matched that of D3R binding and transcripts, with highest levels present in the islands of Calleja and mammillary bodies, moderate to low levels in AccSh, frontoparietal cortex, lobules 9 and 10 in cerebellum, and SN/VTA, and very low levels in other brain structures including striatum. The observation that immunolabeling and D3R binding patterns overlap is a strong criterion for assessing the specificity of this antibody, which was apparently not fulfilled in previous studies. In the latter, the specificity of the antibodies used was questionable because highest levels of alleged D3R-like immunoreactivity were not found in AccSh or islands of Calleja but in striatum and core of nucleus accumbens (Ariano and Sibley, 1994) or in hippocampus (Khan et al., 1998), which contains low or undetectable levels of D3R binding or mRNA. The selectivity of the antibody developed herein is also based on the solid evidence, lacking in previous studies, that the immunolabeling it generates is absent in D3R-deficient mice. This antibody therefore appears to have the specificity required for reliable immunocytochemistry.
The only minor discrepancy identified so far is the lowest level of immunolabeling in lobules 9 and 10 of the cerebellum, as compared with D3R binding and mRNA. Because we have presently confirmed that the D3R in the cerebellum has a sequence identical to that found in the nucleus accumbens, this apparent mismatch could be because of an inappropriate tissue preparation, leading to a loss of accessibility of the antibody or antigenicity in this particular brain area. D3R binding is present in dendritic trees of Purkinje cells of the cerebellar molecular layer (Diaz et al., 1995), which may not be totally preserved by the fixatives. Alternatively, the D3R in the cerebellum could partly be in a form not recognized by the antibody. The sequence of the immunizing peptide contains a putative site for casein kinase 2 (S/T-X-X-D/E), a protein kinase very abundant in brain (Blanquet, 2000). Phosphorylation by this or a similar enzyme activity at this site may hinder recognition by the antibody.
The antibody developed in the present study allowed us to not only confirm the localization of the D3R in rat brain but also to address the important issue of its occurrence as an autoreceptor. Three distinct kinds of immunolabeling localization were found in SN/VTA that most likely correspond to the D3R, because the immunolabeling herein is absent in D3R-deficient mice. The first is expressed by non-dopaminergic neurons, which are the most abundant in the SN pars lateralis and also contain the highest levels of D3R mRNA. This suggests that a large fraction of D3R gene transcripts are expressed by non-dopaminergic neurons, which may be liable to downregulation of D3R expression after dopamine neuron denervation by 6-hydroxydopamine (Sokoloff et al., 1990), similar to that occurring in the nucleus accumbens (Lévesque et al., 1995) as a result of BDNF deprivation (Guillin et al., 1999). It remains to be determined whether the putative downregulation of D3R expression in non-dopaminergic mesencephalic neurons after dopamine neuron ablation also results from the loss of BDNF, as is the case of striatal neurons (Guillin et al., 1999), or from the loss of another factor. BDNF and its receptor trkB are expressed in mesencephalon (Altar et al., 1994; Seroogy et al., 1994), together with other neurotrophic factors (Fallon and Loughlin, 1995).
The second localization of D3R immunolabeling in SN/VTA most likely corresponds to presynaptic heteroreceptors present on terminals of neurons originating from AccSh. Such a pathway has been identified by tract-tracing experiments (Berendse et al., 1992). In agreement, we found that a lesion by kainate of accumbal neurons decreased D3R binding to a similar extent in AccSh and SN/VTA. This indicates that the D3R in this latter area is mainly present on afferent terminals.
In SN, VTA, and the A8 retrorubral field, we found a third localization of D3R immunoreactivity in all dopamine neurons observed, which were identified as TH-positive neurons. This observation is at variance with our previous double in situhybridization studies (Diaz et al., 1995), which allowed us to detect only a few neurons expressing both tyrosine hydroxylase and D3R mRNAs. In fact, long exposure of photographic emulsion after in situ hybridization with a D3R mRNA probe produced weak but distinct signals evenly distributed within cells of SN/VTA (J. Diaz, unpublished results), in agreement with the occurrence of D3autoreceptors in all dopamine neurons and the higher sensitivity of double immunofluorescence compared with double in situhybridization.
In dopamine neurons, as in postsynaptic neurons in AccSh, D3R immunoreactivity appears distinct from cytoplasmic TH immunoreactivity, with a striking punctuate distribution along the plasma membrane, not overlapping synaptophysin immunoreactivity. The D3R therefore appears not to be present in the vicinity of synaptic boutons, and very rare apparent co-occurrences of D3R and TH immunoreactivities were noticed in fibers of both SN/VTA and AccSh. This result suggests that the D3R is mainly extrasynaptic, like the D1 and D2 receptors (Yung et al., 1995), and that dopamine acts through the D3R at some distance of its releasing sites, i.e., in a paracrine manner suggested previously (Diaz et al., 1995). This situation is not exceptional for neuromodulators, because other examples exist, e.g., for serotonin (Bunin and Wightman, 1999) or neurotensin (Boudin et al., 1998). Insofar as the sensitivities of immunofluorescence detection at nerve terminals and perikarya are identical, the results also imply that D3 autoreceptors are rather somatodendritic, but confirmation of this localization requires the use of higher resolution methods.
In agreement with the selective somatodendritic localization of D3 autoreceptors, the inhibitory control of dopamine release by nerve terminals seems to be exerted exclusively by D2 autoreceptors (L'hirondel et al., 1998). D3 autoreceptors, together with D2 autoreceptors (Mercuri et al., 1997), may thus rather control the electrical activity of dopamine neurons, which would explain the elevated extracellular dopamine in projection areas of these neurons in D3R-deficient mice. This control could have been masked in experiments using compounds inadequately selective of the D3R (Koeltzow et al., 1998), because the compounds used also activate the D2receptor. Alternatively, D3 autoreceptors could not be operant in anesthetized animals or in vitro in brain slices used in electrophysiological studies (Mercuri et al., 1997;Koeltzow et al., 1998), whereas elevated dopamine extracellular levels were measured in freely moving D3R-deficient mice. Finally, D3 autoreceptors may mediate yet unrecognized control of other dopamine neuron activities, such as synthesis or release of neuropeptides coexpressed with dopamine in these neurons, e.g., neurotensin, cholecystokinin, or neurotrophins.
This work was supported by a grant from the European Commission (FP5 Programme QLG4-CT-1999-00075).
Correspondence should be addressed to Dr. Pierre Sokoloff, Unitéde Neurobiologie et Pharmacologie Moléculaire (Institut National de la Santé et de la Recherche Médicale U 109), Centre Paul Broca, 2ter, rue d'Alésia, 75014 Paris, France. E-mail:.