Article Figures & Data
Figures
- Figure 1.
Cocaine alters the functional characteristics of D1R-H3R heteromers in transfected cells. a–c, f, Neuroblastoma SK-N-MC cells stable expressing D1R and H3R were not treated or treated overnight with 30 μm cocaine. a, b, cAMP production was determined after 10 min stimulation with D1R agonist SKF 38393 (1 μm) or the H3R agonist imetit (1 μm) (a) or with D1R agonist SKF 81297 (100 nm) or the H3R agonist RAMH (100 nm) (b) in the absence or in the presence of 0.5 μm forskolin. Values (cAMP produced in each condition minus basal stimulation in the absence of forskolin or agonists) represent mean ± SEM, n = 4–8. One-way ANOVA followed by Bonferroni post hoc test showed a significant (*p < 0.05, **p < 0.01, ***p < 0.001) effect over basal (cells not stimulated) or (#p < 0.05, ###p < 0.001) over forskolin effect (cells stimulated with forskolin). c, f, Cells infected with the empty virus vector (c) or infected with lentiviral particles expressing shRNA (shσ1R) to silence the receptor (f) were treated for 30 min with the D1R antagonist SCH 23390 (1 μm) or the H3R antagonist thioperamide (1 μm) before the addition of SKF 38393 (100 nm) or imetit (100 nm) for an additional incubation period of 10 min and ERK 1/2 phosphorylation was determined by Western blot. Values represent mean ± SEM (n = 3–5) of percentage of phosphorylation relative to basal levels found in untreated cells. One-way ANOVA followed by Bonferroni post hoc tests showed a significant (*p < 0.05, **p < 0.01) effect over basal or of the antagonist plus agonist treatment over the agonist treatment (#p < 0.05). d, β-Arrestin 1 recruitment was measured by BRET experiments in HEK293T cells 48 h post-transfection with 2 μg of cDNA corresponding to D1R, 1 μg of cDNA corresponding to β-arrestin 1-Rluc, and 3 μg of cDNA corresponding to H3R-YFP. Cells were not treated (BRET < 10) or treated for 30 min with 30 μm cocaine, 1 μm SCH 23390, or 1 μm thioperamide alone or in combination before 10 min stimulation with SKF 38393 (100 nm) or imetit (100 nm). Values represent mean ± SEM, n = 4. One-way ANOVA followed by Bonferroni post hoc tests showed a significant effect over basal (**p < 0.01, ***p < 0.001) or of the antagonist plus agonist treatment over the agonist treatment (##p < 0.01, ###p < 0.001). e, σ1R expression was determined after silencing of σ1R by shRNA. Neuroblastoma SK-N-MC cells stable expressing D1R and H3R were infected with lentiviral particles containing either control (LV control) or shRNA specific for σ1R (LV sh σ1R) and the relative expression levels were tested using quantitative RT-PCR. Results are expressed as an average of three replicates.
- Figure 2.
σ1R-D1R-H3R complexes detected by energy transfer experiments. a, c, d, BRET saturation experiments were performed using HEK293T cells 48 h post-transfection with 0.2 μg of cDNA corresponding to H3R-Rluc (a) or 0.15 μg of cDNA corresponding to D1R-Rluc (c, d) and increasing amounts of cDNA corresponding to σ1R-YFP (a; 0.1–0.5 μg cDNA) or H3R-YFP (c, d; 0.2–1.5 μg cDNA). Cells were treated for 30 min (a, c) or 24 h (d) with medium (red lines) or with 30 μm cocaine (black lines) prior BRET determination. c, d, Cells were also transfected with siRNA for σ1R (see Materials and Methods; green line). Both fluorescence and luminescence of each sample were measured before every experiment to confirm similar donor expressions (∼110,000 bioluminescence units) while monitoring the increase in acceptor expression (10,000–40,000 fluorescence units). b, SRET saturation experiments were performed using HEK293T cells 48 h post-transfection with 0.3 μg of cDNA corresponding to H3R-Rluc, 0.23 μg of cDNA corresponding to σ1R-GFP2, and increasing amounts of cDNA corresponding to D1R-YFP (0.05–0.6 μg). Cells were treated for 30 min with medium (red lines) or with 30 μm cocaine (black lines) prior to SRET determination. The relative amount of BRET or SRET is given as a function of 100 × the ratio between the fluorescence of the acceptor (YFP) and the luciferase activity of the donor (Rluc). BRET and SRET are expressed as mBU or mSU, respectively, and values are means ± SEM; three to four different experiments grouped as a function of the amount of acceptor. a, b, Top, A schematic representation of the BRET (a) and SRET (b) techniques is shown.
- Figure 3.
Expression of σ1R-D1R-H3R complexes in the mouse striatum detected by coimmunoprecipitation and PLA experiments. a–d, Coimmunoprecipitation experiments are shown. Striatal slices from WT and σ1R KO mice were untreated or treated with 150 μm cocaine for 30 min (cocaine). Solubilized striatal membranes (a) and immunoprecipitates with anti-D1R antibody or anti-calnexin antibody as negative control (NC; b–d) were analyzed by SDS-PAGE and immunoblotted using anti-D1R antibody (b), anti-H3R antibody (c), or anti-σ1R antibody (c). IP, immunoprecipitation; WB, Western blotting; MW, molecular mass. e–m, PLAs were performed as indicated in Materials and Methods, using WT (e–g) or σ1R KO mice (h–j) striatal slices. D1R-H3R (e, h), σ1R-D1R (f, i), and σ1R-H3R (g, j) heteromers were visualized as red spots around blue-colored DAPI-stained nucleus. k–m, Negative controls were performed doing the PLA experiments in WT mouse striatal slices incubated with only anti D1R (k), anti-σ1R (l), or anti-H3R (m) antibody as primary antibodies. Cell nuclei were stained with DAPI (blue). For each part a magnification is shown. Scale bar, 20 μm.
- Figure 4.
Expression of σ1R-D1R-H3R complexes in the rat striatum detected by coimmunoprecipitation and PLA experiments. a–d, Coimmunoprecipitation experiments are shown. Rat striatal slices were untreated or treated with 150 μm cocaine for 30 min (cocaine). Solubilized striatal membranes (a) and immunoprecipitates with anti-D1R antibody or anti-calnexin antibody as negative control (NC; b–d) were analyzed by SDS-PAGE and immunoblotted using anti-D1R antibody (b), anti-H3R antibody (c), or anti-σ1R antibody (c). IP, immunoprecipitation; WB, Western blotting; MW, molecular mass. e–g, PLAs were performed as indicated in Materials and Methods, using rat striatal slices. D1R-H3R (e), σ1R-D1R (f), and σ1R-H3R (g) heteromers were visualized as red spots around blue–colored DAPI-stained nucleus. Scale bar, 20 μm.
- Figure 5.
Cocaine inhibits the H3R signaling and the D1R-H3R cross-antagonism in ERK 1/2 phosphorylation in mouse striatum. WT (a) and σ1R KO (b) mouse striatal slices were treated or not with 150 μm cocaine for 2 h and were preincubated for 20 min with medium, the D1R antagonist SCH 23390 (10 μm), or the H3R antagonist thioperamide (10 μm) before the addition of the D1R agonist SKF 38393 (1 μm) or the H3R agonist imetit (1 μm) for an additional incubation period of 10 min. ERK 1/2 phosphorylation was determined by Western blot. Immunoreactive bands from three to eight slices obtained from eight WT or KO animals were quantified for each condition. Values represent mean ± SEM percentage of phosphorylation relative to basal levels found in untreated slices. No significant differences were obtained between the basal levels of the WT and the σ1R KO mice. One-way ANOVA followed by Bonferroni post hoc tests showed a significant (*p < 0.05) effect over basal or of the antagonist plus agonist treatment over the agonist treatment (#p < 0.05, ##p < 0.01).
- Figure 6.
Cocaine inhibits the H3R-mediated modulation of D1R-promoted ERK 1/2 phosphorylation in rat striatum. Striatal slices from naive rats treated or not with 150 μm cocaine for 2 h (a, b) and striatal slices from sham rats or rats acutely treated (24 h) with cocaine (c, d), or striatal slices from sham rats or 9 weeks cocaine self-administered rats (e, f) were preincubated for 20 min with medium, the D1R antagonist SCH 23390 (10 μm), or the H3R antagonist thioperamide (10 μm) before the addition of the D1R agonist SKF 38393 (1 μm) or the H3R agonist imetit (1 μm) for an additional incubation period of 10 min and ERK 1/2 phosphorylation was determined by Western blot. Immunoreactive bands from 3 to 10 slices obtained from four animals were quantified for each condition. Values represent mean ± SEM percentage of phosphorylation relative to basal levels found in untreated slices. One-way ANOVA followed by Bonferroni post hoc tests showed a significant (*p < 0.05, **p < 0.01, ***p < 0.001) effect over basal or of the antagonist plus agonist treatment over the agonist treatment (#p < 0.05, ##p < 0.01, ###p < 0.001).
- Figure 7.
Receptor expression in the striatum of cocaine acutely treated rats compared with sham rats. The expression of H3R (white bars), D1R (gray bars), and σ1R (black bars) in striatal membranes from a pool (n = 5) of sham rats or rats acutely treated (24 h) with cocaine (cocaine) was determined by competition experiments of [3H]RAMH (3 nm) versus RAMH, [3H]SCH 23390 (2 nm) versus SCH 23390, or [3H] YM-09151-02 (2 nm) versus PRE-084, respectively. Competition curves were monophasic and binding data were fitted to Equations 4 or 5 (see Materials and Methods) to calculate the maximum binding.
- Figure 8.
Cocaine inhibits the H3R-mediated modulation of D1R-promoted cell death in organotypic striatal slice cultures. Organotypic cultures of rat striatal slices were prepared as indicated (see Materials and Methods) and after 24 h, culture medium was replaced by fresh medium containing no ligands (a) or 150 μm cocaine (c). After 1 h, vehicle or 10 μm SCH 23390, imetit, or thioperamide were added and incubated for an additional 1 h before the addition of the D1R agonist SKF 38393 (50 μm) and slices were maintained 48 h more in culture. As a control, cell death induced by 1 mm glutamate in the presence or in the absence of 10 μm imetit was analyzed (b). Cell death was determined by DAPI and PI staining as indicated (see Materials and Methods). Values represent mean ± SEM percentage of PI stained cells versus total DAPI-stained cells determined in six to eight fields from two to three independent organotypic cultures. Bifactorial ANOVA showed a significant (***p < 0.001) effect over basal corresponding to nontreated organotypic cultures. One-way ANOVA followed by Bonferroni post hoc tests showed a significant (***p < 0.001) effect over basal corresponding to nontreated organotypic cultures or of organotypic cultures treated with two ligands respect to the cultures treated with SKF 38393 (###p < 0.001).
- Figure 9.
Effect of σ1R agonist and antagonists on the H3R-mediated modulation of the D1R-promoted cell death and signaling in the striatum. Rat striatal organotypic cultures (a–c) or striatal slices (d–f) were prepared as indicated (see Materials and Methods) and were treated for 1 h with medium (a, d) or PD 144418 (10 μm; b, c, e, f) before adding 1 μm PRE-084 (a, d), vehicle (b, e), or 150 μm cocaine (c, f). At 24 h of organotypic culture (a–c) or after 2 h of slice incubation (d–f) the D1R antagonist SCH 23390 (50 μm, a–c or 10 μm, d–f) or the H3R antagonist thioperamide (10 μm) were added 20 min before the addition of the D1R agonist SKF 38393 (50 μm, a–c or 1 μm, d–f) or the H3R agonist imetit (10 μm, a–c or 1 μm, d–f) alone or in combination. Striatal organotypic cultures were cultured for an additional 48 h and slices were incubated for an additional 10 min and were processed to determine cell death or ERK 1/2 phosphorylation, respectively, as indicated in Materials and Methods. a–c, Values represent mean ± SEM percentage of PI stained cells versus total DAPI-stained cells determined in four to six fields from two to three independent organotypic cultures. d–f, Values represent mean ± SEM phosphorylation relative to basal levels found in untreated slices, determined in 3–10 slices obtained from four animals. One-way ANOVA followed by Bonferroni post hoc tests showed a significant (*p < 0.05, ***p < 0.001) effect over basal (nontreated organotypic cultures or slices) or of the two ligands treatment over the agonist treatment (#p < 0.05, ###p < 0.001).
- Figure 10.
Scheme representing the D1R signaling in the D1R-H3R heteromer in the absence or presence of cocaine. In GABAergic neurons (gray), in the absence of cocaine, H3R (blue) and D1R (red) form heteromers to which σ1R (yellow) can bind. Activation of only the D1R protomer (a) promotes a decrease of cAMP levels in accordance with Gi (orange) coupling of both receptors in the heteromer. Moreover, D1R activation induces β-arrestin (purple) recruitment and promotes ERK 1/2 phosphorylation. Upon dual stimulation of the heteromer with both H3R and D1R agonists in the absence of cocaine (b), a negative cross-talk (the ability of one receptor agonist to negatively modulate the partner receptor ligand binding or signaling) between receptors in the heteromer occurs, inhibiting β-arrestin recruitment and subsequently reducing ERK 1/2 phosphorylation from the heteromer. When cocaine (brown) binds to σ1R (c) this induces structural changes in the heteromer disrupting the negative cross-talk. Thus, the H3R-mediated brake on D1R is lost, allowing D1R (green) to couple to Gs (pale green) and increasing cAMP. Importantly, β-arrestin recruitment and ERK 1/2 phosphorylation are also detected in the presence of cocaine.
Tables
Competition experiment ([3H]SCH vs SKF38393) Parameters KD1 (nm) KD2 (nm) Not treated rats in the absence of cocaine Control 31 ± 4 1100 ± 200 +RAMH 14 ± 1* 320 ± 10* Not treated rats in the presence of cocaine Control 28 ± 2 1000 ± 100 +RAMH 30 ± 4 1100 ± 200 Cocaine acutely treated rats Control 28 ± 4 440 ± 60 +RAMH 19 ± 4 390 ± 40 Sham rats Control 26 ± 4 700 ± 100 +RAMH 3 ± 2** 130 ± 40** Cocaine self-administered rats Control 33 ± 6 800 ± 200 +RAMH 37 ± 9 1900 ± 600 ↵Competition curves of the D1R antagonist [3H]SCH 23390 (2 nm) binding versus increasing concentrations of the D1R agonist SKF 38393 were performed in the absence or the presence of 2 nm of the H3R agonist RAMH using striatal membranes from control rats treated or not with 30 μm cocaine for 30 min, rats acutely treated with cocaine, or cocaine self-administered rats. KD1 and KD2 are, respectively, the equilibrium dissociation constants of SKF 38393 binding to D1R and were determined by fitting binding data to Equation 3. Parameters are mean ± SEM (n = 3). For each group, significant differences with respect to control were calculated by an unpaired Student's t test (*p < 0.05, **p < 0.01).
