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. 2019 Feb;56(2):797-811.
doi: 10.1007/s12035-018-1120-y. Epub 2018 May 24.

Adenosine A 1-Dopamine D 1 Receptor Heteromers Control the Excitability of the Spinal Motoneuron

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Free PMC article

Adenosine A 1-Dopamine D 1 Receptor Heteromers Control the Excitability of the Spinal Motoneuron

Marla Rivera-Oliver et al. Mol Neurobiol. .
Free PMC article

Abstract

While the role of the ascending dopaminergic system in brain function and dysfunction has been a subject of extensive research, the role of the descending dopaminergic system in spinal cord function and dysfunction is just beginning to be understood. Adenosine plays a key role in the inhibitory control of the ascending dopaminergic system, largely dependent on functional complexes of specific subtypes of adenosine and dopamine receptors. Combining a selective destabilizing peptide strategy with a proximity ligation assay and patch-clamp electrophysiology in slices from male mouse lumbar spinal cord, the present study demonstrates the existence of adenosine A1-dopamine D1 receptor heteromers in the spinal motoneuron by which adenosine tonically inhibits D1 receptor-mediated signaling. A1-D1 receptor heteromers play a significant control of the motoneuron excitability, represent main targets for the excitatory effects of caffeine in the spinal cord and can constitute new targets for the pharmacological therapy after spinal cord injury, motor aging-associated disorders and restless legs syndrome.

Keywords: Adenosine A1 receptor; Dopamine D1 receptor; Motoneuron; Receptor heteromers; Spinal cord.

Figures

Figure 1
Figure 1. A1R-D1R heteromer expression in transiently transfected HEK-293T cells
In (A) BRET saturation experiments were performed in HEK-293T cells transfected with 0.5 μg of A1R-Rluc cDNA and increasing amounts of D1R-YFP cDNA (1 μg to 5 μg, black curve) or, as negative control, with 0.5 μg of A1R-Rluc cDNA and increasing amounts of 5HT2BR-YFP cDNA (0.5 μg to 5 μg, red line). The relative amount of BRET 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 is expressed as milli BRET units (mBU) and is given as the mean ± SD of 5–6 experiments grouped as a function of the amount of BRET acceptor. At the top a schematic representation of BRET is given. In (B), BiFC experiments were performed in HEK-293T cells transfected with cDNA (4 μg each one) of A1R-nYFP and D1R-cYFP or, as negative controls, 5HT2A-nYFP and D1R-cYFP or D1R-cYFP and CB1R-nYFP. Cells were treated for 4 h with vehicle or 4 μM of D1R TM5 or TM7 peptides and fluorescence at 530 nm was read. Values are mean ± S.E.M. of 5-6 experiments (100% represents 30000 fluorescence units); ***: p < 0.001, as compared with A1R-nYFP and D1R-cYFP expressing cells not treated with peptides (one-way ANOVA followed by Dunnett's comparisons). In (C and D), HEK-293T cells transfected with 0.3 μg of A1R and 0.5 μg of D1R cDNA (A1R-D1R HEK) or with 0.4 μg of CB1R and 0.5 μg of D1R cDNA as negative control (CB1R-D1R HEK) were treated for 4 h with vehicle or with 4 μM of D1R TM5 or TM7 peptides before performing proximity ligation assays. In (C), confocal microscopy images (superimposed sections) are shown in which A1R-D1R heteromers appear as red spots in vehicle and TM7 treated cells and not in cells treated with TM5 peptide or in the negative control. In all cases, cell nuclei were stained with DAPI (blue). Scale bars = 20 μM. In (D), the percentage of cells showing red spots related to the total cell number determined as stained blue nuclei is given in each case as well as the ratio (r) between the number of red spots and cells showing spots (top columns). Values are mean ± S.E.M. of n = 5-6; **: p < 0.01 as compared with cells not treated with peptides (one-way ANOVA followed by Dunnett's comparisons).
Figure 2
Figure 2. A1R-D1R heteromer expression and adenylyl cyclase signaling in fibroblast Ltk- cells
In (A and B), Ltk- cells expressing A1R and D1R (A1R-D1R Ltk) or only D1R as negative control (D1R Ltk-) were treated for 4 h with vehicle or with 4 μM of D1R TM5or TM7 peptides before performing proximity ligation assays. In (A), confocal microscopy images (superimposed sections) are shown in which heteromers appear as red spots in vehicle and TM7 treated cells and not in cells treated with TM5 peptide or in the negative control. In all cases, cell nuclei were stained with DAPI (blue). Scale bars = 20 μM. In (B) the percentage of cells showing spots related to the total cells determined as stained blue nuclei is given in each case as well as the ratio (r) between the number of red spots and cells showing spots (top columns). Values are mean ± S.E.M. of n = 8-16; **: p < 0.01 as compared with D1R Ltk-cells (one-way ANOVA followed by Dunnett's comparisons). In (C-E), A1R-D1R Ltk- were pre-treated for 4 h with vehicle (C) or with 4 μM of D1R TM 5 (D) or TM 7 (E) peptides. Cells were then treated for 10 min with vehicle or with A1R antagonist DPCPX (1 μM) or the D1R antagonist SCH23390 (1 μM) prior being stimulated with medium, the A1R agonist R-PIA (100 nM) or the D1R agonist SKF38393 (200 nM) in the absence or in the presence of 20 μM forskolin (FK). Values are mean ± S.E.M. of n = 4–5 and are expressed as percentage of FK treated cells in each condition (100% represents 80-100 pmols cAMP/106 cells) ***: p < 0.001 versus basal; ###: p < 0.001 versus FK; &&&, && and &: p < 0.001, p < 0.01 and p < 0.05 versus SKF38393, respectively (one-way ANOVA followed by Bonferroni's comparisons).
Figure 3
Figure 3. Immunohistochemical co-localization of A1R and D1R in the ventral lumbar spinal cord
In (A), low-magnification confocal images of a ventrolateral L2 section (P3 mouse), showing the distributions of A1R (green fluorescence from Alexa Fluor 488-conjugated secondary antibodies), D1R (red fluorescence from rhodamine-conjugated secondary antibodies) and the overlay of both fluorescent labels. Hoechst staining allows identification of cell nuclei (blue channel). In (B), higher-magnification of the framed area in (A), showing the co-localization of A1R and D1R in lamina IX of the ventral spinal cord, where motoneurons can be identified by size and position (asterisks); vr: ventral root; scale bar: 50 μM.
Figure 4
Figure 4. A1R-D1R heteromer expression in mouse spinal cord motoneurons
In situ proximity ligation assays were performed using slices from mouse lumbar spinal cord from P4, P6 and P11 animals. Slices from lamina IX (motoneurons) and laminae VIII and X (interneurons) were analyzed using specific primary antibodies directed against A1R and D1R (A1R-D1R) or only against D1R as negative control (D1). Slices were treated for 4 h with vehicle (A, B and control in C and D) or with 4 μM of D1R TM 5 or TM7 peptides (C, D). In (A and C), confocal microscopy images (superimposed sections) from (A) laminae IX and X from P11 animals in which heteromers appear as red spots in lamina IX, but not in lamina X or in negative controls, and from (C) lamina IX from P5 animals in which heteromers appear as red spots in non peptide treated slices (control) and treated with D1R TM 7 but not in slices treated with TM5. In all cases, cell nuclei were stained with DAPI (blue). Scale bars = 20 μM. In (B and D) the percentage of cells showing red spots related to the total cell number determined as stained blue nuclei is given in each case as well as the ratio (r) between the number of red spots and cells showing spots (top columns). Values are mean ± S.E.M. of n =7-15. **: p < 0.01 as compared with the negative control D1 in (B); ** p < 0.01 as compared with the control non-treated with peptides in (D) (one-way ANOVA followed by Dunnett's comparisons).
Figure 5
Figure 5. A1R-D1R heteromer-dependent excitatory effects of caffeine in the spinal motoneuron
WCR and PPR recordings of spinal motoneurons with application of dopamine (3 μM) or the D1R agonist SKF81297 (1 μM) and caffeine (50 μM) or the selective A1R antagonist DPCPX (1 μM). In (A), the effect of caffeine + dopamine is compared with the effect of dopamine alone and with the non-drug control conditions. In (B), the effect of DPCPX plus dopamine is compared with the non-drug control conditions. In (C and D), the effect of caffeine + SKF81297 is compared with the effect of SKF81297 alone and with non-drug conditions and with the application of synthetic peptides with the sequence of TM5 (C) and TM7 (D) of the D1R. (A1, B1, C1 and D1) show representative responses of a motoneuron to increasing pA depolarizing steps; (A2, B2, C2 and D2) show FI plots of the average spike frequency over a range of current step amplitudes; (A3, B3, C3 and D3) show the quantified averaged response from several motoneurons in mean ± S.E.M. of n = 5–10. Repeated-measures ANOVA followed by Dunnett's comparisons or paired t test showed significant increases in spike frequency of the motoneurons treated with caffeine + dopamine (*: p < 0.05), DPCPX + dopamine (***: p < 0.001), and caffeine + SKF81297 in the presence of TM7 of D1R (**: p < 0.01), but not TM5, as compared with control. Specific parameters (threshold, rheobase and AP amplitude; see Materials and Methods) for each group of experiments are shown as Supplementary Information.

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