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Striatal projection neurons coexpressing dopamine D1 and D2 receptors modulate the motor function of D1- and D2-SPNs

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Abstract

The role of the striatum in motor control is commonly assumed to be mediated by the two striatal efferent pathways characterized by striatal projection neurons (SPNs) expressing dopamine (DA) D1 receptors or D2 receptors (D1-SPNs and D2-SPNs, respectively), without regard to SPNs coexpressing both receptors (D1/D2-SPNs). Here we developed an approach to target these hybrid SPNs in mice and demonstrate that, although these SPNs are less abundant, they have a major role in guiding the motor function of the other two populations. D1/D2-SPNs project exclusively to the external globus pallidus and have specific electrophysiological features with distinctive integration of DA signals. Gain- and loss-of-function experiments indicate that D1/D2-SPNs potentiate the prokinetic and antikinetic functions of D1-SPNs and D2-SPNs, respectively, and restrain the integrated motor response to psychostimulants. Overall, our findings demonstrate the essential role of this population of D1/D2-coexpressing neurons in orchestrating the fine-tuning of DA regulation in thalamo-cortico-striatal loops.

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Fig. 1: Morphologically distinct D1/D2-coexpressing SPNs establish an additional efferent striatal pathway.
Fig. 2: D1/D2-SPNs differ through unique action potential properties.
Fig. 3: D1/D2-SPNs promote immobility and control the effects of D1- and D2-SPNs on locomotion.
Fig. 4: D1/D2-SPN coactivation modulates the temporal dynamics of motor changes elicited by D1- or D2-SPN stimulation.
Fig. 5: Coexpression of D1 and D2 cancels DA-induced changes in excitability.
Fig. 6: D1/D2-SPNs are necessary for proper in vivo integration of DA signals.

Data availability

The data supporting the findings of this study are available within the article and its supplementary materials and are available from the corresponding authors upon request. Source data are provided with this paper.

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Acknowledgements

We thank M. G. Caron, A. Citri, P. Faure, C. Kellendonk, C. Lüsher, M. Picciotto, G. Silberberg and L. Venance for critical reading of the paper. We thank C. Ramakrishnan and K. Deisseroth for providing additional INTRSECT viruses. We thank D. Houtteman, S. Laghmari, P. Hagué and L. Cuvelier for technical assistance and mouse colonies in Brussels, Y. Geng for taking care of the mouse colonies in Montréal and S. De Gois for assistance with imaging techniques. We thank the Imaging Facility of the Faculty of Medicine (LiMiF), which is a ULB Platform supported by the FRS-FNRS. P.B. is a research associate at Fonds de la Recherche Scientifique-FNRS (Belgium). C.V. is a postdoctoral researcher at Fonds de la Recherche Scientifique-FNRS (Belgium), and G.F. is a postdoctoral researcher at FRQS (Canada). P.M.O. and E.P.F. are FRIA grantees. A.D.G. and A.C. are research fellows at Fonds de la Recherche Scientifique-FNRS (Belgium). A.d.K.d’E. is a research director at Fonds de la Recherche Scientifique-FNRS and a WELBIO investigator at the WEL Research Institute. B.G. is a Canada Research Chair and a visiting professor at Université de Paris. This work was supported by grants from the Canadian Institute for Health Research (201309OG-312343-PT, 201803PJT-399980-PT) and the Graham Boeckh Foundation to B.G.; from Research Project of the Fonds de la Recherche Scientifique-FNRS (nos. 23587797, 33659288 and 33659296), WELBIO (no. 30256053), Fondation Simone et Pierre Clerdent (Prize 2018), Fondation ULB and AXA Research Fund (Chaire AXA) to A.d.K.d’E.; and from Research Credit and Incentive Grant for Scientific Research FRS-FNRS (nos. 34793348 and 35285205) to P.B.

Author information

Author notes

  1. These authors contributed equally: Patricia Bonnavion, Christophe Varin.

  2. These authors jointly supervised this work: Alban de Kerchove d’Exaerde, Bruno Giros.

Authors and Affiliations

  1. Neurophy Lab, ULB Neuroscience Institute, Université Libre Bruxelles (ULB), Brussels, Belgium

    Patricia Bonnavion, Christophe Varin, Pilar Martinez Olondo, Aurélie De Groote, Amandine Cornil, Ramiro Lorenzo Lopez, Elisa Pozuelo Fernandez & Alban de Kerchove d’Exaerde

  2. Department of Psychiatry, Douglas Hospital, McGill University, Montreal, Quebec, Canada

    Ghazal Fakhfouri, Elsa Isingrini, Quentin Rainer, Kathleen Xu, Erika Vigneault & Bruno Giros

  3. Université Paris Cité, INCC UMR 8002, CNRS, Paris, France

    Elsa Isingrini, Eleni Tzavara & Bruno Giros

  4. AP-HM, Hôpital Sainte Marguerite, Pôle Psychiatrie Universitaire Solaris, Marseille, France

    Eleni Tzavara

  5. Oramacell, Paris, France

    Sylvie Dumas

  6. WELBIO, WEL Research Institute, Wavre, Belgium

    Alban de Kerchove d’Exaerde

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  1. Patricia Bonnavion
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Contributions

Conceptualization: B.G., P.B. and A.d.K.d’E. Design and investigation for optogenetic experiments: P.B. with support from P.M.O. Methodology and investigation for motor analysis: P.B., C.V. with support from A.C. Methodology and investigation for electrophysiological experiments: C.V. Investigation for three-dimensional reconstruction: A.D.G. Investigation for behavioral experiments on D1cKO/D2: G.F., Q.R. with support from K.X. and E.I. Analysis: P.B. and C.V. Investigation for FISH experiments: S.D. with support from A.C. for automated analysis. Investigation for viral-based cell quantification and tracing: P.B. in collaboration with A.C. and R.L.L. (automated analysis), with support from E.P.F. (viral injections). Supervision for the animal colonies: E.V. Resources for the D1FlpO strain: E.T. Formal analysis: P.B. and C.V. Validation and supervision: P.B., B.G. and A.d.K.d’E. Writing: P.B., C.V., A.d.K.d’E. and B.G.

Corresponding authors

Correspondence to Alban de Kerchove d’Exaerde or Bruno Giros.

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Nature Neuroscience thanks Nicolas Mallet and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Anatomical characterization of D1/D2-SPNs and generation and validation of a novel D1FlpO/A2aCre transgenic line.

a, Representative coronal section in the dorsal striatum illustrating fluorescent in situ hybridization of Drd1a and Drd2 mRNAs and Dapi staining. Using a CellProfiler pipeline, segmentation was first performed on dapi staining to outline all cells (blue contours). Then D1- and D2-posivive cells are identified using an intensity threshold in red and green channels, respectively (red and green contours). Scale: 20 µm. b, Quantification of D1-, D2-, and D1/D2-positive cells throughout the rostro-caudal extent of the dorsal striatum (n = 3 mice). c, Diagram illustrating the generation of D1FlpO mice. d, Photomicrograph of a coronal brain section at the level of the dorsal striatum after in situ hybridization of FlpO (red) and Drd1a (green) mRNAs illustrating expression of FlpO-recombinase in Drd1a-expressing SPNs. The experiment was repeated on 3 mice. Scales: left: 100 µm; right zoom: 15 µm. e, Coronal brain section after in situ hybridization of Drd2 (green) and Adora2a (red) mRNAs counterstained with Dapi (blue) illustrating specific expression of Adora2 in D2-SPNs34. Some cells expressing Drd2 and not Adora2 (arrows) correspond to cholinergic interneurons. The experiment was repeated on 3 mice. Scales: top left whole section: 500 µm; zoom below: 100 µm. f, INTRSECT ChR2 virus specificity. Representative coronal sections showing Con/Fon- (first line), Con/Foff (second line), Coff/Fon-ChR2eYFP (third line) expression in double D1FlpO x A2aCre mice (left column), in single A2aCre line (middle column) and in single D1FlpO line (right column). We controlled the absence of leaky expression by verifying the absence of recombination of the Con/Fon construct in all single lines, as well as the absence of recombination of the Con/Foff construct in D1FlpO or the Coff/Fon construct in A2aCre. Scale: 500 µm. g, h, Quantification of the density of INTRSECT virus transfected cells using automated detection. g, Representative sections in the dorsal striatum illustrating Con/Fon-, Con/Foff, Coff/Fon-eYFP virus expression, from top to bottom respectively. Red dots: positive cells. Blue dots: false positive cells with lower fluorescence. Scale: 40 µm. h, Density of cells transfected with Con/Fon-eYFP (purple), Con/Foff-eYFP (red) and Coff/Fon-eYFP (blue) virus. We found 228.3 ± 26.9 cells/mm2 for D1/D2-SPNs, 675.1 ± 58.2 cells/mm2 for D2-SPNs, and 561.2 ± 37.1 cells/mm2 for D1-SPNs which correspond to proportions of 16.6 ± 2.4% D1/D2-SPNs, 45.2 ± 5.2% D2-SPNs, and 38.2 ± 0.7% D1-SPNs relative to total SPNs (Dapi normalized, n = 3 mice for Con/Fon-eYFP and Con/Foff-eYFP virus, n = 2 mice for Coff/Fon-eYFP virus). Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

Source data

Extended Data Fig. 2 Electrophysiological characterization of the three SPNs subpopulations.

a-c, Representative example of voltage response to current steps in one D1/D2-SPN (a), one D1-SPN (b) and one D2-SPN (c), with their corresponding photomicrograph under infrared illumination (IR, top left) and under fluorescent illumination (eYFP, top right). d, Two-dimensional projection of the set of neurons recorded following PCA into the first- and second-order components. Individual cells are represented in violet (D1/D2), blue (D1) and red (D2) circles; crosses represent centroids of D1/D2-SPNs (violet), D1-SPNs (blue) and D2-SPNs (red). e, Percent of variance explained as a function of the number of principal components. f, Two-dimensional projection of orthonormal principal component coefficients for each parameter. (Abbreviations: RMP: resting membrane potential; Rm: input membrane resistance; τm: membrane time constant; Cm: membrane capacitance; Sag: Sag index; Ith: step intensity to first spike; lat: first spike latency; Vth: action potential; Ath: adaptation around threshold; Fmin: minimal steady state frequency around threshold; I–f slope: early slope of the current-frequency relationship; Isat: maximum intensity before depolarization block; Asat: amplitude of early adaptation at high firing; τsat: time constant of early adaptation at high firing; msat: slope of late adaptation at high firing; Fmax: maximal steady state frequency at high firing; SFA: spike frequency adaptation at high firing; A1: amplitude of the first action potential; A2: amplitude of the second action potential; D1: duration of the first action potential; D2: duration of the second action potential; W1: spike half-widths of the first action potential; W2: spike half-widths of the second action potential; AHP1: AHP maximum of the first action potential; AHP2: AHP potential maximum of the second action potential; tAHP1: AHP latency after of the first action potential; tAHP2: AHP latency of the second action potential; A.Red: amplitude reduction; D.Inc: duration increase; W.Inc: half-width duration increase). g, Clusters obtained after k-means partitioning (left, k = 2; middle, k = 3) based on all the electrophysiological parameters recorded, and contingency table (right) comparing clusters assignments (lines) and cell identity defined by INTRSECT eYFP labelling. Source data are provided as a Source Data file.

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Extended Data Fig. 3 Comparison between optogenetic stimulation protocols to elicit behavioral changes.

a, Effect of four different optogenetic stimulation protocols (constant light ON during 30 s, black; 5 Hz stimulation during 30 s, orange; one pulse at 20 Hz lasting 0.5 s, violet; 20 Hz stimulation during 30 s, green; D1/D2, n = 21, 8, 8, 13, respectively; D2, n = 15, 8, 8, 8, respectively; D2 + D1/D2, n = 11, 6, 6, 5, respectively; D1, n = 16, 6, 6, 11, respectively; D1 + D1/D2, n = 10, 5, 5, 5, respectively; D1/D2eYFP, n = 11, 5, 5, 6, respectively) on the proportion of time spent in locomotion, small movements, and immobility (three-ways ANOVA followed by two-sided Tukey’s tests pre vs. light ON: for locomotion, ††p < 0.01, †††p < 0.001; for small movements, #p < 0.05, ###p < 0.001; for immobility, *p < 0.05, ***p < 0.001). Data are presented as mean values ± SEM. b, Temporal evolution of changes in locomotion (top), small movements (middle), and immobility (bottom) occurrences induced by the four different optogenetic stimulation protocols constant light ON during 30 s, black; 5 Hz stimulation during 30 s, orange; one pulse at 20 Hz lasting 0.5 s, violet; 20 Hz stimulation during 30 s, green) (solid line, mean; shading, bootstrap 95% CI). Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

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Extended Data Fig. 4 Architecture of motor states and locomotion speed during optogenetic stimulation.

a-c, Effect of optogenetic stimulation motor architecture during locomotion (a), small movement (b), and immobility (c) quantified by the proportion of time spent in each state (left panels), bouts frequency (middle panels), and the average episode duration (right panels) in D1/D2, control D1/D2eYFP, D1, D1 + D1/D2, D2, and D2 + D1/D2 mice (two-ways RM ANOVA followed by two-sided Tukey’s tests pre vs. light ON: *p < 0.05, **p < 0.01, ***p < 0.001). d, Effect of laser illumination on average speed (black) and top speed (9th decile, green) during locomotion (two-ways RM ANOVA followed by two-sided Tukey’s tests pre vs. light ON: ***p < 0.001). e, f, Averaged temporal evolution of mice speed (e) and acceleration (f) around locomotion onset during laser ‘OFF’ periods between stimulation trials. For all graphs: D1/D2, n = 21; D2, n = 15; D2 + D1/D2, n = 11; D1, n = 16; D1 + D1/D2, n = 10 and control D1/D2eYFP, n = 11. Data are presented as mean values ± SEM. Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

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Extended Data Fig. 5 Effect of optogenetic stimulation on real-time place preference.

a, Proportion of time spent in the laser-paired area before (Pre) and during (ON) the optical activation (continuous blue light) of D1/D2-SPNs (n = 17), D2-SPNs (n = 11), D2 + D1/D2-SPNs (n = 13), D1-SPNs (n = 13), D1 + D1/D2-SPNs (n = 8) or in control (D1/D2eYFP; n = 9) mice in the laser-paired zone (two-ways RM ANOVA followed by two-sided Tukey’s tests Pre vs. LaserON: ns p > 0.05, ***p < 0.001). b, Average temporal evolution of mice speed aligned to locomotion episode onsets when the animal is exploring the laser-paired zone (green curves) or the unpaired zone (black curves) during the baseline period (Pre, top panels) and during the optogenetic stimulation in the laser paired zone (Laser ON, bottom panels). Note during the Laser ON condition, the large increase in locomotion speed elicited by the stimulation of D1/D2, D2, and D2 + D1/D2-SPNs suggesting the aversive effect of the stimulation, and the decrease in speed elicited by the stimulation of D1, and D1 + D1/D2-SPNs suggesting the appetitive effect of the stimulation. Data are presented as mean values ± SEM. Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

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Extended Data Fig. 6 Dynamic changes in small movements and immobility occurrence during optogenetic stimulation.

Temporal evolution of changes in small movements (left panels), and immobility (right panels) occurrences induced by optogenetic stimulation (constant light ON during 30 s; D1/D2, n = 21; D1/D2eYFP, n = 11; D2, n = 15; D2 + D1/D2, n = 11; D1, n = 16; D1 + D1/D2) (solid line, mean; shading, bootstrap 95% CI). X-axes correspond to time (s) and optogenetic stimulation is applied for 30s from time 0. Source data are provided as a Source Data file.

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Extended Data Fig. 7 Temporal organization of motor states.

a, Representation of transition probabilities between the three motor states during baseline (top) and during three periods occurring early (0–5 s), in the middle (10–15 s) or at the end (20–25 s) of the laser illumination (bottom) of D1/D2-SPNs (n = 21 mice), D2-SPNs (n = 15 mice), and D2 + D1/D2-SPNs (n = 11 mice), D1-SPNs (n = 16 mice), D1 + D1/D2-SPNs (n = 10 mice), and control D1/D2eYFP-SPNs (n = 11 mice). For Laser ON condition, colored arrows indicate significant increases (solid lines) or decreases (dashed lines) in transition probabilities in comparison with baseline condition. b, Survival curves of ongoing locomotion (top panels), small movements (middle panels), and immobility (bottom panels) episodes when light stimulation starts (D1/D2, n = 21; D2, n = 15; D2 + D1/D2, n = 11; D1, n = 16; D1 + D1/D2, n = 10; D1/D2eYFP, n = 11) (two-sided Kolmogorov-Smirnov test vs D1/D2eYFP: *p < 0.05, **p < 0.01, ***p < 0.001). Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

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Extended Data Fig. 8 Effects of DA application on SPNs subpopulations.

a, Representative example of the effect of bath applied DA (40 µM, 4 min) on membrane potential and evoked firing activity in one D1/D2-SPN. b, Voltage traces recorded in one representative D1/D2-SPN (top), one D1-SPN (middle), and one D2-SPN (bottom) in response to depolarizing and hyperpolarizing current steps during baseline (first column), at peak of DA effect from the same holding current than in the beginning of the recording (second column), at peak of DA from holding current to restore voltage at the start of the recording (third column), and after DA washout (last column). For the D1/D2-SPN example i-iv represent times indicated in a. c, d, Quantification of changes in membrane resistance (around –60 mV) induced by DA application (c), and change in holding necessary to maintain cells at the same potential than at the begging of the recording before and after DA application (d) in D1/D2 (n = 10 cells from 6 mice, violet), D1- (n = 9 cells from 4 mice, blue), and D2-SPNs (n = 7 cells from 4 mice, red) and (two-sided paired t-tests between baseline and after DA application: *p < 0.05, **p < 0.01, ***p < 0.001; two-sided t-tests comparing the magnitude of DA effect between SPN subpopulations: #p < 0.05, ##p < 0.01, ###p < 0.001). e, Changes in average current-frequency relationship evaluated from ramp current injection during baseline and at the peak of DA effect in D1/D2- (n = 11 cells from 6 mice), D1- (n = 6 cells from 4 mice), and D2-SPNs (n = 8 cells from 4 mice) and (two-sided paired t-tests between baseline and after DA application: *p < 0.05). f, g, Effect of D1R antagonist (SCH23390, 10 µM) and D2R antagonist (sulpiride, 10 µM) application on membrane potential (f) and number of spikes elicited by a depolarizing current step (g) in D1/D2-SPNs (SCH23390, n = 5 cells from 3 mice; sulpiride, n = 5 cells from 3 mice) (two-sided paired t-tests between baseline and after antagonist application: *p < 0.05, **p < 0.01; two-sided t-tests comparing the magnitude of effect between treatments: #p < 0.05). h-j, Effect of DA application on D1/D2-SPNs without any pre-treatment, in the presence of SCH23390, or in the presence of sulpiride on the membrane potential (h), the number of elicited spiked by a depolarizing pulse from current membrane potential (i), and the number of spikes elicited from initial membrane potential (j) (no treatment, n = 11 cells from 6 mice; SCH23390, n = 5 cells from 3 mice; sulpiride, n = 5 cells from 3 mice) (two-sided paired t-tests between baseline and after DA application: *p < 0.05, **p < 0.01, ***p < 0.001; two-sided t-tests comparing the magnitude of DA effect between treatments: #p < 0.05, ##p < 0.01, ###p < 0.001). k, Voltage response and APs of representative D1/D2-SPNs to ramp current injection before (black) and after (green) DA application without pre-treatment (left), in the presence of SCH23390 (middle), or in the presence of sulpiride (right). l-m, Effect of DA application on AP threshold (l) and minimal ramp current to elicit discharge (m) in D1/D2-SPNs without antagonists (n = 10 cells from 6 mice), or in the presence of SCH23390 (n = 5 cells from 3 mice), or sulpiride (n = 5 cells from 3 mice) (two-sided paired t-tests between baseline and after DA application: *p < 0.05, **p < 0.01; two-sided t-tests comparing the magnitude of DA effect between conditions: #p < 0.05). Data are presented as mean values ± SEM. Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

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Extended Data Fig. 9 Supplements on the phenotypic characterization of D1cKO/D2 mice.

a, Comparison of spontaneous locomotor behavior between D1wt/D2 (n = 13) and D1cKO/D2 (n = 15) mice in term of average and top speed during locomotion episodes (left panel) and motor states architecture (two-sided t-tests, D1wt/D2 vs. D1cKO/D2 mice: ns p > 0.05, *p < 0.05). b, Temporal distribution of the locomotor effects of acute amphetamine or saline injections in D1wt/D2 (NaCl, n = 8; 1 mg/kg, n = 6; 3 mg/kg, n = 7) and D1cKO/D2 mice (NaCl, n = 6; 1 mg/kg, n = 6; 3 mg/kg, n = 7) and (inserts) total distance traveled over the first 60 min after injection (three-ways ANOVA followed by post-hoc two-ways ANOVA for interaction genotype x time: NaCl, F(23,276) = 1.9; 1 mg/kg, F(23,230) = 5.2; 3 mg/kg, F(23,276) = 0.57; ns p > 0.05, ##p < 0.01, ###p < 0.001; and post-hoc two-sided Mann Whitney tests D1wt/D2 vs. D1cKO/D2 mice on distance traveled over 60 min: ns p > 0.05, *p < 0.05, ***p < 0.001). c, Temporal evolution of traveled distance across repeated 10 mg/kg cocaine injection in D1wt/D2 (black) and D1cKO/D2 mice (orange) (three-ways ANOVA followed by post-hoc two-ways ANOVA for interaction genotype x time: Day1, F(26,806) = 1.9; Day2, F(26,806) = 2.29; Day3, F(26,806) = 6.55; Day4, F(26,806) = 4.96; Day5, F(26,806) = 6.08; Day6, F(26,806) = 4.27; Day14, F(26,806) = 2.72; ###p < 0.001). Data are presented as mean values ± SEM. Detailed statistics are displayed in Supplementary Table 1. Source data are provided as a Source Data file.

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Extended Data Table 1 Complete list of electrophysiological properties recorded in D1/D2-, D1- and D2-SPNs
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Bonnavion, P., Varin, C., Fakhfouri, G. et al. Striatal projection neurons coexpressing dopamine D1 and D2 receptors modulate the motor function of D1- and D2-SPNs. Nat Neurosci (2024). https://doi.org/10.1038/s41593-024-01694-4

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