Cocaine Effects on Dopaminergic Transmission Depend on a Balance between Sigma-1 and Sigma-2 Receptor Expression

Abstract

Sigma σ₁ and σ₂ receptors are targets of cocaine. Despite sharing a similar name, the two receptors are structurally unrelated and their physiological role is unknown. Cocaine increases the level of dopamine, a key neurotransmitter in CNS motor control and reward areas. While the drug also affects dopaminergic signaling by allosteric modulations exerted by σ₁R interacting with dopamine D₁ and D₂ receptors, the potential regulation of dopaminergic transmission by σ₂R is also unknown. We here demonstrate that σ₂R may form heteroreceptor complexes with D₁ but not with D₂ receptors. Remarkably σ₁, σ₂, and D₁ receptors may form heterotrimers with particular signaling properties. Determination of cAMP levels, MAP kinase activation and label-free assays demonstrate allosteric interactions within the trimer. Importantly, the presence of σ₂R induces bias in signal transduction as σ₂R ligands increase cAMP signaling whereas reduce MAP kinase activation. These effects, which are opposite to those exerted via σ₁R, suggest that the D₁ receptor-mediated signaling depends on the degree of trimer formation and the differential balance of sigma receptor and heteroreceptor expression in acute versus chronic cocaine consumption. Although the physiological role is unknown, the heteroreceptor complex formed by σ₁, σ₂, and D₁ receptors arise as relevant to convey the cocaine actions on motor control and reward circuits and as a key factor in acquisition of the addictive habit.

Keywords: ERK1/2 phosphorylation; acute; addiction; cAMP; chronic; dopamine D1 and D2 receptors; label-free; signaling.

FIGURE 1

Expression of σ₂R-containing heteromer complexes in a heterologous expression system. To determine colocalization between σ₂R and dopamine D₁ or D₂ receptors, immunocytochemistry assays were performed in HEK-293T pretreated or not with 30 μM cocaine for 30 min. HEK-293T cells expressing σ₂R-Rluc (1 μg cDNA), D₁R-YFP (1 μg cDNA), D₂R-YFP (1 μg cDNA), σ₂R-Rluc (1 μg cDNA) and D₁R-YFP (1 μg cDNA) or σ₂R-Rluc (1 μg cDNA) and D₂R-YFP (1 μg cDNA) were used. Dopamine receptors were detected by YFP fluorescence (green) and σ₂R was detected by a specific antibody against Rluc (1/100, Millipore, Temecula, CA, United States) followed by a Cy3-conjugated secondary antibody (1/200, Jackson Immunoresearch Laboratories, West Grove, PA, United States) (red). Colocalization is shown in yellow (A). Scale bar 5 μm. In situ proximity ligation assay (PLA) was developed in HEK-293T cells expressing D₁R (1 μg cDNA) or D₂R (1 μg cDNA) and σ₂R (1 μg cDNA) by the use of specific primary antibodies (1/100 dilution) against D₁R, D₂R and/or σ₂R. Nuclei were stained with Hoechst (1/100). Confocal microscopy images (four superimposed sections) were obtained showing D₁R-σ₂R or D₂R-σ₂R complexes as red spots (B). Scale bar 5 μm. Quantification of the PLA provides in the Y-axis the ratio r (number of red spots/cell containing spots) and, above each bar, the percentage of positive cells versus the total number of cells (blue nucleus). Data are the mean ± SEM of four different fields in five independent preparations. One way ANOVA and Dunnett’s post hoc test showed statistically significant differences (^∗∗∗p < 0.001).

FIGURE 2

Identification of D₁R-σ₁R-σ₂R heteromers in a heterologous expression system. Bioluminescence energy transfer (BRET) was developed in HEK-293T cells expressing (i) a constant amount of D₁R-Rluc (0.3 μg cDNA) (A) or D₂R-Rluc (0.25 μg cDNA) (B) and increasing amounts of σ₂R-YFP (0.5–4 μg cDNA), or (ii) a constant amount of D₁R-Rluc (0.3 μg cDNA) and increasing amounts (0.5–4.5 μg cDNA) of σ₁R-YFP (C) or (iii) a constant amount of σ₁R-Rluc (0.1 μg cDNA) and increasing amounts of σ₂R-YFP (1–6 μg cDNA) (D). Cells were treated (red line) or not (black line) with 30 μM cocaine for 30 min. BRET is expressed as milli BRET units (mBU) and is given as the mean ± SEM of seven different experiments. Competition experiments were developed in HEK-293T cells expressing a constant amount of σ₁R-Rluc (0.1 μg cDNA) and D₁-YFP (1.5 μg cDNA) and increasing amounts of unfused σ₂R (0–5 μg cDNA) (E) or a constant amount of D₁-Rluc (0.05 μg cDNA) and σ₂R-YFP (0.3 μg cDNA) and increasing amounts of unfused σ₁R (0–5 μg cDNA) (F). Transfer of energy was expressed as milli BRET units (mBU) and results are given as the mean ± SEM of 10 different experiments. One way ANOVA and Dunnett’s post hoc test showed statistically significant differences (^∗p < 0.05, ^∗∗p < 0.01). (G) Sequential resonance energy transfer (SRET) assay developed in HEK-293T cells transfected with constant amounts of σ₁R-Rluc (0.2 μg cDNA) and D₁R-YFP (1.5 μg cDNA) and increasing amounts of σ₂R-RFP (0.5–4 μg cDNA). A negative control was performed using cDNA for the cannabinoid CB₁ receptor (fused to RFP) instead of cDNA for σ₂R-RFP. SRET is expressed as milli SRET units (mSU) and are given as the mean ± SEM of 6 different experiments. (H) Schematic representation of SRET.

FIGURE 3

σ₂R modulation of D₁ receptor-mediated signaling in a heterologous expression system. cAMP determination experiments were developed in HEK-293T cells expressing D₁R (A–D) or D₂R (E–H) in the absence (C,G) or presence of 3 μg siRNA for σ₁R (A,E), 3 μg siRNA for σ₂R (B,F) or both (D,H). Cells were pretreated with 30 μM cocaine for 30 min, 300 nM PB-28 or vehicle 15 min prior to receptor activation using 200 nM SKF-81297 or 500 nM sumanirole. In cells expressing D₂R 0.5 μM forskolin was used to induce increases in cAMP levels. Basal [cAMP] is considered 100% in cells expressing D₁R, whereas forskolin-induced [cAMP] is considered 100% in cells expressing D₂R. Values are the mean ± SEM of 12–15 different experiments. One way ANOVA followed by a Dunnett’s multiple comparison post hoc test showed a significant effect of treatments versus control (^∗∗∗p < 0.001), a significant effect of treatments versus SKF-81297 (^#p < 0.05, ^##p < 0.01, and ^###p < 0.001) and a significant effect of treatments versus sumanirole (^&&p < 0.01 and ^&&&p < 0.001).

FIGURE 4

Cocaine effects on D₁R-mediated signaling. MAP kinase activation was determined in HEK-293T cells transfected with 0.75 μg cDNA for D₁R in the absence (C) or presence of 3 μg siRNA for σ₁R (A), 3 μg siRNA for σ₂R (B) or both (D). The culture medium was replaced by non-supplemented DMEM and 2 h later cells were treated for 30 min with 30 μM cocaine, 300 nM PB-28 or vehicle followed by a 200 nM SKF-81297 stimulation (7 min). The basal level of pERK1/2 is considered 100%. Values are the mean ± SEM of 10–12 different experiments. One way ANOVA followed by a Dunnett’s multiple comparison post hoc test showed a significant effect of treatments versus control (^∗p < 0.05, ^∗∗∗p < 0.01) and a significant effect of treatments versus SKF-81297 (^#p < 0.05, ^##p < 0.01, and ^###p < 0.001). Real-time DMR signal 60 min recordings in HEK-293T cells transfected with 0.75 μg cDNA for D₁R in the absence (G) or presence of 3 μg siRNA for σ₁R (E), 3 μg siRNA for σ₂R (F) or both (H) that were treated with 30 μM cocaine (red), 300 nM PB-28 (dark blue) or vehicle (green) for 30 min previous to 200 nM SKF-81297 stimulation.

FIGURE 5

Expression and function of σ₂R-D₁R complexes in primary cultures of striatal neurons. In (A,B) PLA assay was developed in striatal primary cultures of neurons pretreated or not with cocaine 30 μM for 30 min. σ₂R-D₁R or σ₂R-D₂R heteromer complexes were detected by the use of specific antibodies (1/100 dilution) against σ₂R and D₁R or σ₂R and D₂R. Confocal microscopy images (four superimposed sections) were obtained where nuclei were stained with Hoechst (1/100). Scale bar 5 μm (A). Quantification of the PLA provides in the Y-axis the ratio r (number of red spots/cell containing spots) and, above each bar, the percentage of positive cells versus the total number of cells (blue nucleus) (B). Data are the mean ± SEM of four different fields in five independent preparations. One way ANOVA and Dunnett’s multiple comparison post hoc test showed statistically significant differences versus control (^∗∗p < 0.01). Primary cultures of striatal neurons, control (E,I) or transfected with siRNA for σ₁R (C,G), σ₂R (D,H) or both (F,J) were treated with 30 μM cocaine for 30 min or 300 nM PB-28 prior to 200 nM SKF-81297 stimulation. cAMP levels (C–F) or MAP kinase activation l (G–J) were determined. Basal [cAMP] is considered 100%. The basal level of pERK1/2 is considered 100%. Values are the mean ± SEM of 10–15 different experiments. One way ANOVA followed by a Dunnett’s multiple comparison post hoc test showed a significant effect of treatments versus not treated cells (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001) and a significant effect of treatments versus SKF-81297 (^#p < 0.05, ^##p < 0.01, and ^###p < 0.001).

FIGURE 6

D₁R-mediated signaling is modulated by σ₁R in cocaine acute and by σ₂R in cocaine chronic exposure. In (A,B) PLA assay was developed in brain sections from male Sprague–Dawley rats i.p. injected with vehicle or 15 mg/kg cocaine under acute or chronic regimes (see section “Materials and Methods”). D₁R-σ₁R or D₁R-σ₂R heteromer complexes were detected by PLA using of specific antibodies against D₁R (1/100), σ₁R (1/100, Santa Cruz Biotechnology, Dallas, TX, United States) or σ₂R (1/100). Confocal microscopy images (four superimposed sections) were obtained where nuclei were stained with Hoechst (1/100). Scale bar 5 μm (A). Quantification of the PLA provides in the Y-axis the ratio r (number of red spots/cell containing spots) and, above each bar, the percentage of positive cells versus the total number of cells (blue nucleus) (B). Data are the mean ± SEM of six different fields in five independent preparations. One way ANOVA and Dunnett’s multiple comparison post hoc test showed statistically significant differences versus control (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001), significant differences in D₁R-σ₁R complex amount between acute and chronic treatments (^&&&p < 0.001) and significant differences in D₁R-σ₂R heteromer complex amount between acute and chronic treatments (^$$p < 0.01). cAMP determination experiments were developed in primary cultures of striatal neurons, control (C), transfected with 3 μg siRNA for σ₁R (D) or 3 μg siRNA for σ₂R (E). Cultures were divided into 9 groups and pretreated with vehicle or 30 μM cocaine for different time periods (from 0.5 h to 7 days) prior to receptor activation using 200 nM SKF-81297. Basal [cAMP] is considered 100%. Values are the mean ± SEM of five different experiments. One way ANOVA followed by a Dunnett’s multiple comparison post hoc test showed a significant effect of treatments versus basal (^∗p < 0.05, ^∗∗∗p < 0.001) and a significant effect of cocaine treatments (white bars) versus cocaine non-exposed neurons (black bar) (^#p < 0.05, ^##p < 0.01, and ^###p < 0.001).