Abstract
The plasticity of glutamatergic transmission in the ventral tegmental area (VTA) represents a fundamental mechanism in the modulation of dopamine neuron burst firing and phasic dopamine release at target regions. These processes encode basic behavioral responses, including locomotor activity, learning and motivated behaviors. Here we describe a hitherto unidentified mechanism of long-term synaptic plasticity in mouse VTA. We found that the burst firing in individual dopamine neurons induces a long-lasting potentiation of excitatory synapses on adjacent dopamine neurons that crucially depends on Ca2+ elevations in astrocytes, mediated by endocannabinoid CB1 and dopamine D2 receptors co-localized at the same astrocytic process, and activation of pre-synaptic metabotropic glutamate receptors. Consistent with these findings, selective in vivo activation of astrocytes increases the burst firing of dopamine neurons in the VTA and induces locomotor hyperactivity. Astrocytes play, therefore, a key role in the modulation of VTA dopamine neuron functional activity.
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Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Change history
25 April 2023
A Correction to this paper has been published: https://doi.org/10.1038/s41593-023-01330-7
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Acknowledgements
The authors dedicate the present study to the memory of Tullio Pozzan, internationally recognized as one of the most eminent cell biologists of our time, great mentor and dear friend of many of us. We are grateful to M. Melis, M. Sessolo, G. Colombo, M. Zordan, M. Santoni and P. Magalhães for helpful discussions and suggestions. We also thank T. Pozzan for valuable comments on the manuscript and discussions; M. Morini, D. Cantatore, B. Chiarenza, A. Monteforte and C. Chiabrera for technical support; and J. Chen for kindly providing IP3R2−/− mice. This research was supported by the European Commission (H2020-MSCA-ITN and 722053 EU-GliaPhD), PRIN 2015-W2N883_001, Premiale CNR-TERABIO, 2017 Premiale MIUR - nano4BRAIN and PRIN 2017 Prot. 20175C22WM to G.C., the Istituto Italiano di Tecnologia and the Ministero della Salute italiano (project GR-2016-02362413) to F.P., grants from UNIVPM (PSA 040046), Fondazione di Medicina Molecolare to F.C., the European Brain Research Institute (EBRI)/National Research Council of Italy (CNR) collaborative agreement to A.L.M. and G.C. and the Euro Bio-Imaging Project Roadmap/ESFRI from the European Commission. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
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L.M.R., M.G.G. and G.C. designed the study. L.M.R. and M.G.G. performed the electrophysiological experiments in brain slices, with the collaboration of M.S. and G.L. L.M.R., M.G.G. and M.S. performed the Ca2+ imaging experiments in brain slices, with the collaboration of A.L. and M.Z. L.M.R. and M.G.G. performed the AAV injections, with the collaboration of A.L. and V.H. A.C. performed the immunohistochemistry experiments. F.M., G.P. and F.P. performed the behavioral experiments. M.C. and A.L.M. performed the in vivo single-unit recordings. M.M., A.P. and F.C. performed the electron microscopy experiments. G.M. provided the Cnr1-floxed mice. All authors discussed the results. M.G.G. and G.C. wrote the paper, with input from all authors.
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Extended data
Extended Data Fig. 1 Electrophysiological properties of DA neurons recorded from VTA slice preparations.
a) Differential interference contrast image of the lateral VTA showing the recording pipettes on a pair of DA neurons and the theta-capillary for extracellular stimulation of rostral glutamatergic afferents (mt, medial terminal nucleus of the accessory optical tract). Scale bar, 100 µm. b) Representative large Ih current elicited by a hyperpolarizing step. Scale bars, 200 pA, 500 ms. c) Representative slow depolarizing potential preceding the action potential during a depolarizing current step injection. Scale bars, 10 mV, 200 ms. d) Representative spontaneous low-frequency tonic firing. Scale bars, 20 mV, 2 s. e) Representative burst and tonic firing evoked by current step injections. Scale bars, 20 mV, 100 ms. f) EPSC amplitude after tonic firing protocol in wt female mice (n = 8 from 5 mice, p = 0.169, two-tailed paired t-test). g) Time course of paired-pulse ratio (PPR) values in female mice (n = 8), before and after tonic firing protocol (arrowhead). Right, mean PPR values before and 30 min after tonic firing (p = 0.827, two-tailed paired t-test). Data are represented as mean ± SEM.
Extended Data Fig. 2 bLTP can be evoked in IP3R2+/+ but not in IP3R2−/− young female littermates.
a) Example of mouse genotyping by PCR amplification of the IP3R2 wt (~200 bp) and mutant (~400 bp) alleles from genomic DNA. b) Top, bLTP can be evoked in IP3R2+/+ (n = 8 from 8 mice, p = 0.029, two-tailed One Sample t-test), but not in IP3R2−/− (n = 5 from 4 mice, p = 0.524, two-tailed One Sample t-test) young female littermates. Bottom, the bLTP in IP3R2+/+ female mice is accompanied by a reduced PPR (p = 0.031, two-tailed paired t-test), similarly to that observed in C57BL/6J young female mice (see Fig. 1). Analysis of the coefficient of variation of EPSCs, 45 min after burst firing for potentiated cells in IP3R2+/+ young mice (black circle, mean value). c) DA neuron bursts evoke an increase of the Ca2+ spike probability/min in astrocytes from IP3R2+/+ (n = 9 from 5 mice, p = 0.017, two-tailed paired t-test), but not in astrocytes from IP3R2−/− (n = 6 from 4 mice, p = 0.533, two-tailed paired t-test) young female littermates. Data are represented as mean ± SEM.
Extended Data Fig. 3 Expression of CB1, D2, D3, D4, D1, mGluR1α and mGluR1β receptors in neuronal and astrocytic compartments in the VTA of P16 female and male mice.
a) EM image showing CB1, D2, D3 and D4R immunoreactivity at neuronal compartments (Den, dendrites; Ax, axons; AxT, axon terminals) and astrocytic processes (AsP), in the lateral VTA of P16 female and male mice. Quantitative analysis of the distribution of immunoreactive profiles in female and male mice is reported in Supplementary Table 1. Scale bar, 250 nm. b) Representative pre-embedding EM images showing the expression of D3 and D4Rs at astrocytic processes (AsP) from the lateral VTA of a P16 female mouse. Green arrows indicate the presence of immunopositive products in AsP (AsP+). Scale bar, 300 nm. c) EM images of mGlu1α and mGlu1βR-immunoreactivity in the lateral VTA of P16 female and male mice. mGluR1α is largely detectable in dendrites (Den), in some astrocytic processes (AsP) and axons (Ax; see Supplementary Table 3 for quantitative distribution of mGluR1α immunoreactivity in both female and male). The mGluR1β is detectable in AsP, AxT (including those making an asymmetric synaptic contact) and Den (see Supplementary Table 3 for quantitative distribution of mGluR1β immunoreactivity in both female and male). Scale bar, 250 nm. d) Upper panel, the same as in (b), but in the lateral VTA of a P16 male mouse. Lower panel, quantification and comparison (two sided contingency Fisher’s test) of D3 (p < 0.0001) and D4R (p > 0.999) expression in female and male young mice. e) Representative ultrastructural fields of D1 immunoreactivity in the neuropil of lateral VTA in P16 female mice. Examples of neuronal (Den, dendrites) and astroglial (AsP, astrocytic processes) D1 immunoreactivity are illustrated. Quantitative analysis of the distribution of immunoreactive profiles is reported in Supplementary Table 4. Scale bar: 250 nm. f) Upper panel, representative fluorescence images showing two SR-101-positive astrocytes and the Ca2+ increase evoked in one of them (arrowhead, detected with Fluo-4), after locally applying the D1-type R agonist SKF 38393 (1 mM in glass pipette). Scale bar, 5 μm. Lower panel, time course of the Ca2+ transient shown on the left. Scale bars, 10 s, 10 %. g) Left, time course of the mean Ca2+ spike probability, in 10 sec bins, at basal conditions and after SKF 38393 challenge, both in the absence and presence of the D1-type R antagonist SCH-23390 (10 μM). Right, bar chart of the mean Ca2+ spike probability/min before and immediately after SKF 38393 challenge to show the Ca2+ response of VTA astrocytes to SKF 38393 (without SCH-23390, n = 6 from 4 mice, p = 0.022; with SCH-23390, n = 6 from 4 mice, p = 0.325; two-tailed paired t-test). h) Same as in g), but after ATP (4 mM in glass pipette) in five of the six slices previously challenged with SKF 38393 (n = 5 from 3 mice, p = 0.007, two-tailed paired t-test). Note that, compared to the strong astrocyte response to ATP, VTA astrocytes show a small, but significant Ca2+ response to D1-type receptor activation that is abolished in the presence of the D1-type receptor antagonist SCH-23390. Data are represented as mean ± SEM.
Extended Data Fig. 4 Targeted expression of mCherry-hM3D in VTA astrocytes from young male mice.
a) High magnification fluorescence images of the VTA from a mouse injected with AAV-9/2-hGFAP-hM3D(Gq)_mCherry-WPRE-hGHp(A), showing colocalization in astrocyte processes of mCherry-hM3D and the astrocyte marker S100β. Scale bar, upper panel 20 μm, lower panel 10 μm. b) Bar chart showing the percentage of mCherry positive cells that are astrocytes (S100β positive) or neurons (NeuN positive). ɑS100β; n = 1106 mCherry-hM3D+ cells from 4 mice, 8 slices; ɑNeuN, n = 1039 mCherry-hM3D+ cells from 4 mice, 8 slices. Data are represented as mean ± SEM.
Extended Data Fig. 5 Effects of the NO synthase inhibitor L-NAME on bLTP and astrocyte Ca2+ response to DA neuron burst firing.
a) Time course and bar chart of EPSC amplitude in the presence of the NO synthase inhibitor L-NAME (100 μM in the patch pipette of the burst firing DA neuron, n = 12 from 9 mice, p = 0.277; two-tailed One sample t-test). b) Mean amplitude of normalized EPSCs in female mice, 6 min after bursts, in the presence of different antagonists (AM251, n = 7 from 4 mice, p = 0.105; eticlopride, n = 10 from 8 mice, p = 0.291; LY-367385, n = 12 from 9 mice, p = 0.215; L-NAME, n = 12, from 9 mice p = 0.044; two-tailed One sample t-test). c) Time course and bar chart of astrocytic Ca2+ spike probability/min in the presence of L-NAME before and after burst firing (100 μM, n = 7 from 4 mice, p = 0.075; two-tailed paired t-test). d) Mean astrocytic Ca2+ spike probability/min in female mice, at basal conditions and 4.5 min after burst, in the presence of different antagonists (AM251, n = 6 from 3 mice, p = 0.671; eticlopride, n = 6 from 3 mice, p = 0.673; LY-367385, n = 6 from 4 mice, p = 0.048; L-NAME, n = 7 from 4 mice, p = 0.009; two-tailed paired t-test). e) A 5 min bath perfusion of CNO (10 μM), in the absence and presence of DEA NONOate (10 μM), transiently (in the first 9 min) increases EPSC amplitude of DA neurons in male mice expressing hM3D in astrocytes (CNO, n = 7 from 6 mice, p = 0.016, two-tailed One sample Wilcoxon Signed Rank test; CNO + DEA NONOate, n = 13 from 9 mice, p = 0.013, two-tailed One sample t-test). These experiments were performed in the presence of AM251 and eticlopride. Data are represented as mean ± SEM.
Extended Data Fig. 6 The mechanism of bLTP generation in young female mice is preserved in adult mice.
a) Representative EM images of mGluR1β expression at axon terminals (AxT+) forming asymmetric synaptic contacts (arrowheads) with dendrites (Den) and CB1 and D2R localization at astrocytic processes (AsP+) from adult female and male mice. Green and blue arrows indicate the presence of immunopositive products in female and male, respectively. Scale bar, 300 nm. b) Time course and bar chart of the mean amplitude of normalized EPSCs in adult male mice in the presence of different antagonists (L-741,626 (D2R) 10 µM, n = 9 from 7 mice, p = 0.34, two-tailed One Sample t-test; AM251 (CB1R), n = 11 from 8 mice, p = 0.24, two-tailed One Sample Wilcoxon signed Rank test; LY-367385 (mGluR1), n = 8 from 7 mice, p = 0.096, two-tailed One Sample t-test; L-NAME (NO synthase), n = 7 from 5 mice, p = 0.604, two-tailed One Sample t-test). As in young mice, bLTP generation in adult mice requires eCB-DA signaling and mGluR activation. Data are represented as mean ± SEM.
Extended Data Fig. 7 Targeted expression of mCherry-Cre, GCaMP6f and mCherry-hPMCA2w/b in VTA astrocytes from adult mice.
a) High magnification fluorescence images of the VTA from an adult male mouse injected with AAV9-hGFAP-mCherry_iCre-WPRE-hGHp(A), illustrating the nuclear localization of mCherry-Cre in GFAP-positive astrocytes. Scale bar, 10 μm. b) Bar chart showing the percentage of nuclear mCherry-Cre positive cells that are astrocytes (GFAP positive) or neurons (NeuN positive). αGFAP; n = 1265 mCherry-Cre+ cells from three mice, 8 slices; αNeuN, n = 747 mCherry-Cre+ cells from three mice, 5 slices. c) Confocal microscope fluorescence images of the VTA from an adult male mouse injected with AAV5.GfaABC1D.cytoGCaMP6f.SV40, showing the green fluorescence of GCaMP6f (α-GFP), nuclear Top-Ro3 (blue) and the specific red staining for either neurons (α-NeuN) or astrocytes (α-S100β). Merged images, localization of GCaMP6f in astrocytes (S100β-positive cells) and not in neurons (NeuN-positive cells). Scale bar, 25 μm. d) Bar chart showing the percentage of GCaMP6f positive cells that are astrocytes (S100β positive) or neurons (NeuN positive). αS100β; n = 1383 GCaMP6f+ cells from four mice, 10 slices; αNeuN, n = 1586 GCaMP6f+ cells from four mice, 12 slices. e) High magnification fluorescence images of the VTA from an IP3R2−/− adult mouse injected with AAV5-GfaABC1D-mCherry-hPMCA2w/b.SV40, showing the expression of the Ca2+ pump hPMCA2w/b (α-RFP red staining) in GLT1-positive astrocytic processes. Scale bar, 10 μm. f) Bar chart showing the percentage of cells expressing the Ca2+ pump hPMCA2w/b that are astrocytes (GLT-1 positive) or neurons (NeuN positive). αGLT1; n = 2164 mCherry-hPMCA2w/b(αRFP)+ cells from four mice, 13 slices; αNeuN, n = 1902 mCherry-hPMCA2w/b(αRFP)+ cells from four mice, 12 slices. Data are represented as mean ± SEM.
Extended Data Fig. 8 Area, amplitude and duration of Ca2+ events extracted by AQuA before and after DA neuron burst.
a) Cumulative distributions of the area (μm2), amplitude (∆F/F0) and duration (s) of Ca2+ events extracted by AQuA, before and after DA neuron burst in IP3R2+/+ mice (before burst, 6942 events; after burst, 10760 events; area, p = 0.218; amplitude, p < 0.0001; duration, p = 0.968; two-tailed Kolmogorov-Smirnov test). b) Same as in a), but from IP3R2−/− mice (before burst, 2483 events; after burst, 4483 events; area, p = 0.083; ∆F/F0, p < 0.0001; duration, p = 0.967; two-tailed Kolmogorov-Smirnov test).
Extended Data Fig. 9 Targeted expression of mCherry-hM3D in VTA astrocytes from adult male mice.
a) Confocal microscope fluorescence images of the VTA from an adult mouse injected with AAV-9/2-hGFAP-hM3D(Gq)_mCherry-WPRE-hGHp(A), showing the red fluorescence of mCherry-hM3D (red), nuclear Top-Ro3 (blue) and the specific green staining for either neurons (α-NeuN) or astrocytes (α-GFAP). Merged images, localization of hM3D in astrocytes (GFAP-positive cells) and not in neurons (NeuN-positive cells). Scale bars, 50 μm. b) High magnifications of the VTA from a mouse injected with AAV-9/2-hGFAP-hM3D(Gq)_mCherry-WPRE-hGHp(A), illustrating the colocalization of mCherry-hM3D with the astrocyte marker GFAP in astrocyte processes. Scale bars, 10 μm. c) Bar chart showing the percentage of mCherry-hM3D positive cells that are astrocytes (GFAP positive) or neurons (NeuN positive). αGFAP; n = 683 mCherry-hM3D+ cells from 3 mice, 6 slices; αNeuN, n = 1127 mCherry-hM3D+ cells from five mice, 10 slices. Data are represented as mean ± SEM.
Extended Data Fig. 10 DA neuron burst firing modulation of excitatory synapses onto adjacent DA neurons in adult IP3R2+/+ and IP3R2−/− littermates and non-littermates female and male mice.
a, b) Time course and bar chart of the mean amplitude of normalized EPSCs in adult female and male C57BL/6J (a) and IP3R2−/− (b) non littermates mice (C57BL/6J; female mice, n = 7 from 6 mice, p = 0.019; male mice, n = 7 from 5 mice, p = 0.01; IP3R2−/−; female mice, n = 9 from 7 mice, p = 0.108; male mice, n = 9 from 9 mice, p = 0.194; two-tailed One sample t-test). c, d) Same as in a, b) but from adult female and male IP3R2+/+ (c) and IP3R2−/− (d) littermate mice (IP3R2+/+; female mice, n = 8 from 7 mice, p = 0.028; male mice, n = 7 from 5 mice, p = 0.021; IP3R2−/−; female mice, n = 8 from 7 mice, p = 0.087; male mice, n = 9 from 7 mice, p = 0.112; two-tailed One sample t-test). Data are represented as mean ± SEM.
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Requie, L.M., Gómez-Gonzalo, M., Speggiorin, M. et al. Astrocytes mediate long-lasting synaptic regulation of ventral tegmental area dopamine neurons. Nat Neurosci 25, 1639–1650 (2022). https://doi.org/10.1038/s41593-022-01193-4
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DOI: https://doi.org/10.1038/s41593-022-01193-4
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