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Age-dependent regulation of synaptic connections by dopamine D2 receptors

Nature Neuroscience volume 16, pages 1627–1636 (2013)Cite this article

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Abstract

Dopamine D2 receptors (D2R) are G protein–coupled receptors that modulate synaptic transmission and are important for various brain functions, including learning and working memory. Abnormal D2R signaling has been implicated in psychiatric disorders such as schizophrenia. Here we report a new function of D2R in dendritic spine morphogenesis. Activation of D2R reduced spine number via GluN2B- and cAMP-dependent mechanisms in mice. Notably, this regulation occurred only during adolescence. During this period, D2R overactivation caused by mutations in the schizophrenia risk gene Dtnbp1 led to spine deficiency, dysconnectivity in the entorhinal-hippocampal circuit and impairment of spatial working memory. Notably, these defects could be ameliorated by D2R blockers administered during adolescence. Our findings suggest an age-dependent function of D2R in spine development, provide evidence that D2R dysfunction during adolescence impairs neuronal circuits and working memory, and indicate that adolescent interventions to prevent aberrant D2R activity protect against cognitive impairment.

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Figure 1: D2R regulates spine development in hippocampal neurons in vivo.
Figure 2: D2R regulates the morphogenesis and growth dynamics of dendritic spines in cultured hippocampal neurons.
Figure 3: GluN2B is required for D2R-mediated regulation of dendritic spines.
Figure 4: cAMP mediates D2R's effects on spine density.
Figure 5: Spine deficiency in sandy mice is caused by D2R hyperactivity.
Figure 6: The critical period for D2R to regulate dendritic spine number and reversal of the spine deficiency by antipsychotics treatment in sandy mice.
Figure 7: The age dependency of D2R-mediated regulation of dendritic spines requires the developmental change in GluN2B expression.
Figure 8: D2R hyperactivity during adolescence impairs the entorhinal-hippocampal circuit and spatial working memory.

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Acknowledgements

We thank H. Arnheiter (NINDS/NIH) for critical discussion of the manuscript and E.J. Sherman for editing the manuscript. This work was supported by the Intramural Research Program of the National Institute of Mental Health (ZIA MH002281 to Z.L.).

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  1. Unit on Synapse Development and Plasticity, National Institute of Mental Health, US National Institutes of Health, Bethesda, Maryland, USA

    Jie-Min Jia, Jun Zhao, Zhonghua Hu, Daniel Lindberg & Zheng Li

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Contributions

J.-M.J. conducted the experiments and data analysis. J.Z. analyzed mEPSCs. D.L. contributed to the CTB experiment. Z.L. and J.-M.J. designed the experiments and wrote the manuscript. Z.H. generated the constructs and lentivirus expressing Drd1, Drd2 and the siRNAs against them.

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Correspondence to Zheng Li.

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Integrated supplementary information

Supplementary Figure 1 The D2R and D1R siRNAs are efficient and specific.

Cultured hippocampal neurons were transfected with D2R siRNA constructs along with constructs expressing HA–tagged D2R, siRNA–resistant D2R (D2R–M), Beclin1 or ATG5, and stained with anti–HA antibody at 3 d after transfection in (a–d). Representative images of transfected neurons are shown in (a, c). Quantification of expressed proteins is shown in (b, d); n = 15 neurons for each condition. (e) Cultured cortical neurons (DIV5) were infected with lentivirus expressing D2R siRNA, and lysed at DIV12 for RT–PCR of D1R and D2R. (f) mEPSC amplitude recorded in hippocampal slices prepared from 4–week–old C57BL/6 mice injected with lentivirus expressing EGFP, D2R or D2R siRNA; n= the total number of recorded neurons from 5–8 slices of 3–5 mice for each condition. Scale bar, 20 μM. Mann Whitney test was used for statistical analysis. Data are presented as mean ± SEM.

Supplementary Figure 2 D1R does not regulate spine number.

(a, b) Cultured hippocampal neurons (DIV14) were transfected with the Venus construct along with either the empty vector or the D1R construct, and treated with vehicle or agonists and antagonists of D1R for 24 h. Representative images of transfected neurons (top) and dendrites at a higher magnification (bottom) are shown in (a). Quantification for (a) is shown in (b). (c) Quantification of total spine density in primary hippocampal neurons (DIV17) treated with vehicle, D2R agonist or D2R antagonist. The results were replicated by three independent experiments. Histograms show one of the three replicates (n = 15 neurons for each condition). Scale bar, 20 μM for images of neurons and 5 μM for images of dendrites. Mann Whitney test was used for statistical analysis. Data are presented as mean ± SEM.

Supplementary Figure 3 Quinpirole induced GluN2B endocytosis is required for the effects of quinpirole on spines.

Primary hippocampal neurons (a, b, f–k) and cortical neurons (d, e) were treated with vehicle or quinpirole at DIV17 for 24 hrs. (a) Representative images of neurons stained for bassoon, synaptophysin, GluA1 or GluA2. (b) Quantification for (a). (c) The input–output relationship of EPSCNMDA recorded in hippocampal slices prepared from C57BL/6 mice intraperitoneally injected with D2R agonists (0.5 mg/kg of quinpirole or 10 mg/kg of bromocriptine). Since EPSCNMDA was comparable in quinpirole and bromocriptine treated slices (data not shown), data from these slices were pooled. n = 10 cells from 10 slices of 3 animals for each condition in (c). (d) Representative cropped immunoblots for phosphorylated GluN2B (Ser1303) and GluA1 (Ser845); the full–length images of western blots are shown in Supplementary Figure 7c. (e) Quantification for (d); n = 3 independent experiments. (f) Representative images of neurons transfected with the GFP–GluN2A construct. (g) Quantification for (f). (h) Representative images of GFP–GluN2B transfected neurons treated with 1 μM quinpirole alone or along with inhibitors for GluN2B (3 μM of ifenprodil or 1 μM of Ro 25–6891) or dynamin (20 μM). (i) Quantification for (h). (j) Representative images of neurons transfected with the Venus construct and treated with inhibitors as indicated. (k) Quantification of spine density for (k). For all images, n = 15 neurons for each condition. Scale bar, 20 μM. Mann Whitney test was used for statistical analysis. Data are presented as mean ± SEM.

Supplementary Figure 4 The amplitude of mEPSCs in CA1 neurons of wild–type and sandy mice At 3 weeks of age, sandy mice and their wild–type littermates were injected with lentivirus expressing EGFP or Drd2 siRNA, then used for mEPSC analysis.

n = the total number of neurons recorded from 5–8 slices of 3–5 animals each condition. Mann Whitney test was used for statistical analysis. Data are presented as mean ± SEM.

Supplementary Figure 5 The effect of D2R on spines in adult mice and the developmental change in the expression of NMDAR subunits in the mouse hippocampus.

In (a, b), 8–week–old wild–type or sandy mice were injected with lentivirus expressing EGFP, Drd2 or Drd2 siRNA, and analyzed for spines at 1 week after injection. In (c, d), the hippocampus of mice at 2–12 weeks were dissected and lysed for immunoblotting. (a) Representative images of dendrites from transduced CA1 neurons. (b) Spine analysis for (a); n = 15 neurons from 3 slices of 3 animals for each condition. (c) Representative cropped immunoblots; The full–length blots are shown in Supplementary Figure 7d. (d) Quantification for (c); n = the number of mice for each time point. Scale bar, 5 μm. Mann Whitney test was used for statistical analysis. p–values in (d) were calculated by comparing each GluN subunit at various postnatal weeks vs. that at 6 weeks. Data are presented as mean ± SEM.

Supplementary Figure 6 Entorhinal–hippocampal connectivity is changed by treating adolescent sandy mice with eticlopride, but not by treating adult wild–type mice with quinpirole.

(a) Sandy mice were fed water supplemented with eticlopride (5 μg/ml) or intraperitoneally injected with eticlopride (0.5 mg/kg) from P21 to P35, then injected with Alexa–555–conjugated CTB at 8 weeks of age to label entorhinal neurons projecting to the CA1 area. (b) Wild–type mice were fed water supplemented with 2.5 μg/ml quinpirole from P56 to P63, then injected with 1 mg/ml of CTB at 12 weeks of age. n = the number of animals. Mann Whitney test was used for statistical analysis. Data are presented as mean ± SEM.

Supplementary Figure 7 Full–length images of immunoblots.

For immunoblotting, after transfer, we cut each PVDF membrane into two parts. The top portion of the membrane was used to detect AMPA or NMDA receptors, and the bottom part was used to probe for actin or tubulin. (a) Full–length images for the cropped representative immunoblots for total NMDA receptors in Figure 3i. (b) Full–length images for the cropped blots for surface NMDA receptors in Supplementary Figure 3i. (c) Full–length images for the cropped blots in Supplementary Figure 3d. (d) Full–length images for the cropped blots in Supplementary Figure 5c. The bottom portion of the PVDF membrane in (c) was probed with the anti–actin antibody first, then with the anti-tubulin antibody.

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Jia, JM., Zhao, J., Hu, Z. et al. Age-dependent regulation of synaptic connections by dopamine D2 receptors. Nat Neurosci 16, 1627–1636 (2013). https://doi.org/10.1038/nn.3542

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