Age-dependent regulation of synaptic connections by dopamine D2 receptors

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.

Fig. 1

D2R regulates spine development in hippocampal neurons in vivo. Hippocampal slices were prepared for diolistic labeling from 3–week–old C57BL/6 mice intraperitoneally injected with vehicle or with agonists or antagonists of D2R and D1R (a, c), or 4–week–old C57BL/6 mice injected with lentivirus expressing EGFP, D1R, D2R, D1R siRNA or D2R siRNA (b, d, e, f). (a, b) Representative images of DiI–labeled (a) or virus transduced (b) basal dendrites of hippocampal CA1 neurons. (c) Quantification of spine density for (a). (d) Quantification of spine density for (b). n = 3 mice for each condition, and 15 neurons from 3 slices of each animal were imaged for spine analysis in (c) and (d). (e) Sample trace of mEPSCs. (f) Analysis of mEPSC frequency; 5–8 slices from 3–5 animals were used for each condition; n = the total number of recorded neurons. Quinpirole: 0.5 mg/kg; bromocriptine: 10 mg/kg; eticlopride: 0.5 mg/kg; SKF38393: 1 mg/kg; SCH23390: 1 mg/kg. Data are presented as mean ± SEM. Scale bars, 5 μm. Two–tailed Mann–Whitney test was used to calculate p–values.

Fig. 2

D2R regulates the morphogenesis and growth dynamics of dendritic spines in cultured hippcampal neurons. Cultured hippocampal neurons were transfected with the the Venus or indicated constructs at DIV14 and imaged at DIV17. (a) Representative images of transfected neurons (top) and dendrites at a higher magnification (bottom). (b, c) Quantification for (a). (d) Representative images of cultured hippocampal neurons. (e, f) Quantification for (d). (g). Representative time–laspse images of neurons (top) and dendrites at a higher magnification (bottom) before (0′) and 60 min (60′) after quinpirole treatment. (h) Quantification for the number of added and retracted spines (normalized to the total number of spines) during the 60–min–treatment period. (i) Quantification for the conversion between different types of dendritic protrusions during the 60–min–imaging period. M: mushroom spines; T: thin spines; F: filopodia; S: stubby spines. The results were replicated in 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. Data are presented as mean ± SEM. Two–tailed Mann–Whitney test was used to calculate p–values for comparison to vehicle treated cells in (b, c, h, i) and between vehicle and quinpirole treated cells transfected with the same construct in (e, f). Quinpirole: 1 μM; bromocriptine: 2.5 μM; eticlopride: 1 μM.

Fig. 3

GluN2B is required for D2R–mediated regulation of dendritic spines. (a–f) Cultured hippocampal neurons transfected with the Venus construct were treated with vehicle or chemicals as indicated. Representative images of transfected neurons (top) and dendrites at a higher magnification (bottom) are shown in (a) and (d). (b, c) Quantification for (a). (e, f) Quantification for (d); the results were replicated in three independent experiments; histograms show one of the three replicates (n = 15 neurons for each condition). (g, h) Hippocampal slices were prepared from C57BL/6 mice (P21) intraperitoneally injected with chemicals as indicated and diolistically labeled. Representative images are shown in (g), and quantification is in (h); n= 15 neurons from 3 slices of 3 animals for each condition. (i, j) Cultured cortical neurons (DIV17) were treated with quinpirole, then harvested for immunoblotting. Representative cropped immunoblots for GluN1, GluN2A and GluN2B are shown in (i); the full–length images are shown in Supplementary Fig. 7a, b. (j) Quantification for (i); n = 3 experiments for each condition. Scale bar, 20 μm for images of neurons and 5 μm for images of dendrites. Data are presented as mean ± SEM. Two–tailed Mann–Whitney test was used to calculate p–values for comparison between cells treated with indicated chemicals and those treated with the same chemical and quinpirole. Quinpirole: 1 μM; AP5: 50 μM; Ifenprodil: 3 μM; Ro 25–6981: 1μM; TCN201: 10 μM.

Fig. 4

cAMP mediates D2R’s effects on spine density. Cultured hippocampal neurons transfected with the Venus construct were treated with vehicle or chemicals as indicated. (a) Representative images of transfected neurons (top) and dendrites at a higher magnification (bottom). (b, c) Quantification for (a). The results were replicated in 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. Quinpirole: 1μM; forskolin: 50 μM; 740Y–P: 50 μg/ml. Two–tailed Mann–Whitney test was used to calculate p–values for comparison between cells treated with indicated chemicals and those treated with the same chemical and quinpirole. Data are presented as mean ± SEM.

Fig. 5

Spine deficiency in sandy mice is caused by D2R hyperactivity. (a–c) Primary hippocampal neurons were prepared from wild–type and sandy mice, transfected with the Venus construct at DIV14, and imaged at DIV17. Representative images of transfected neurons and dendrites at a higher magnification are shown in (a), and quantification is shown in (b, c); the results were replicated by three independent experiments, and one of the three replicates (n = 15 for each condition) is shown in the histograms. (d–i) At 3 weeks of age, sandy mice and their wild–type littermates were injected with lentivirus expressing EGFP or D2R siRNA, or intraperitoneally injected with vehicle or forskolin (4 mg/kg), then used for preparation of hippocampal slices for spine analysis (d, e), mEPSC analysis (f, g) or diolistic labeling (h, i). (d, h) Representative images of basal dendrites from CA1 neurons. (e) Quantification of spine density for (d); 15 neurons from 3 slices of each animal and 3 mice for each condition were imaged and analyzed. (i) Quantification of spine density for (h); 15 neurons from 3 slices of each animal and 3 mice for each condition were imaged and analyzed. (f) Sample trace of mEPSCs. (g) Quantificaiton for (f); 5–8 slices of 3–5 animals for each condition; n= the total number of recorded neurons. Scale bar, 20 μm for images of neurons and 5 μm for images of dendrites. Data are presented as mean ± SEM. Mann–Whitney test was used for statistical analysis.

Fig. 6

The critical period for D2R to regulate dendritic spine number and reversal of the spine deficiency by antipsychotics treatment in sandy mice. Hippocampal slices for diolistic labeling were prepared from C57BL/6 mice of various ages intraperitoneally injected with vehicle or with the D2R agonist 0.5 mg/kg of quinpirole (a, b), sandy mice and their wild–type littermates of various ages (c, d), or sandy mice and their wild–type littermates at 3 weeks of age intraperitoneally injected with vehicle, 5 mg/kg of loxapine or 8 mg/kg of clozapine (e, f). Representative images of DiI–labeled basal dendrites of hippocampal CA1 neurons are shown in (a, c, e). Quantification of spine density is shown in (b, d, f). 15 neurons from 3 slices of each animal were imaged and analyzed. n = 3 mice for each condition. Scale bar, 5 μm. Data are presented as mean ± SEM. Mann–Whitney test was used for comparison between wild–type and sandy mice in (b, d), and between vehicle and drug treated mice of the same genotype in (f).

Fig. 7

The age–dependency of D2R–mediated regulation of dendritic spines requires the developmental change in GluN2B expression. Cultured young (DIV18) or mature (DIV56) hippocampal neurons were transfected with the Venus construct alone or along with GFP–GluN2A or GFP–GluN2B and treated with quinpirole (1 μM) alone or along with GluN2A inhibitor TCN201 (10 μM). (a) Represenative images of mature neurons. (b, c) Quantification for (a). (d) Representative images of young neurons. (e, f) Quantification for (d). n = 15 neurons for each condition. Scale bar, 20 μm for images of neurons and 5 μm for images of dendrites. Data are presented as mean ± SEM. Mann–Whitney test was used for comparison between vehicle and quinpirole treated cells.

Fig. 8

D2R hyperactivity during adolescence impairs the entorhinal–hippocampal circuit and spatial working memory. Sandy mice were treated with eticlopride (5 μg/ml) during either adolescence (P21–35) or adulthood (P56–70). Wild–type mice were treated with quinpirole (2.5 μg/ml) from P21 to P28. At 3–4 weeks after treatment, mice were used for the retrograde tracing experiment and behavioral tests. (a) Representative images of retrogradely labeled neurons in the EC. Scale bar, 100 μm. (b) The ratio of retrogradely labeled MEC to LEC neurons. (c) The number of retrogradely labeled neurons in each section (150 μm in length along the MEC–LEC axis) of the EC normalized to the total number of labeled neurons in the EC. 0 in the x–axis indicates the border between the MEC and LEC. n = 5 mice for each condition in (b) and (c). (d) Alternation scores from the Y–maze test. (e) Total distance traveled during 30 min in the open field test. Pooled data from two independent experiments are shown in (d) and (e). Data are presented as mean ± SEM. Mann–Whitney test was used for statistical analysis. p–values less than 0.05 for comparison between untreated and eticlopride–fed sandy mice (blue), between sandy and wild–type mice (yellow), and between untreated and quinpirole–fed wild–type mice (orange) are shown in (c); p–values less than 0.05 for comparison between sandy and wild–type mice are shown in (e); the complete list of p–values for (c, e) is provided in Supplementary Table 1.