Article Figures & Data
Figures
- Figure 1.
Corticostriatal cocultures from drd1a-tdTomato; drd2-EGFP mice. A, Left, Dissected brain at postnatal day 0. Arrowheads indicate EGFP-positive striata in both hemispheres allowing for precise dissection. Right, Schematic of the culture preparation in which cells are dissociated and combined at a 1:3 ratio of cortical (nonfluorescing) to striatal (tdTomato and EGFP fluorescing) neurons. B, A representative D2 MSN at 15 DIV shows typical development of dendritic arbor (top) and formation of spines (insets, bottom). Calibration bars, 20 μm. C, Voltage traces from an example D2 MSN in vitro illustrates responses to step current injections. D, Spontaneous membrane potential fluctuations at resting membrane potential reveal the regular occurrence of up and down states that were blocked by TTX (data not shown).
- Figure 2.
GABAergic synaptic connectivity between MSNs is sensitive to D2R activation. A, Superimposed GFP, RFP, and phase-contrast image of a D1–D2 MSN pair. Scale bar, 30 μm. B, Example voltage-clamp traces from reciprocally coupled MSNs at 24 DIV. Yellow arrows mark the stimulation pulse. Dashed purple lines highlight both autaptic (1 and 4) and synaptic (2 and 3) responses. Local perfusion of 25 μm BMR (blue trace, superimposed) abolished all evoked and most spontaneous responses. C, Schematic illustrating that a maximum of four synapses can be studied in a paired recording, as labeled, if the MSNs in the pair are both reciprocally connected and have autapses. D, E, Rates of connectivity between specific mature MSN pairs (21–24 DIV) at synapses (D) and autapses (E) exhibit an increased rate of synapse formation with 72 h 10 μm quinpirole treatment, exclusively when the postsynaptic neuron is a D2 MSN. *p < 0.05, ***p < 0.001; χ2 test (total number of cells per pair tested in parentheses).
- Figure 3.
Chronic D2R activation increases the strength of D2 eIPSCs. A, eIPSC traces with superimposed averages from representative pairs of MSN combinations in nontreated and treated conditions. B, Summary plots compare charge transfer, fitted decay time constant (τw) and rate of failure, separated by experimental condition (n = 13 and 15 for D1–D1 nontreated and treated MSNs, respectively; n = 19 and 16 for D2–D1 nontreated and treated MSNs, respectively; n = 16 and 21 for D1–D2 nontreated and treated MSNs, respectively; n = 22 and 33 for D2–D2 nontreated and treated MSNs, respectively). *p < 0.05, **p < 0.01, ***p < 0.001; Mann–Whitney test.
- Figure 4.
D2R activation increases dendritic complexity. A, B, Biocytin-injected (left) and traced (right) nontreated (A) and treated (B) D2 MSNs. Scale bar, 50 μm. C, Analysis of Sholl crossings and dendrite lengths reveals an increase in primary dendrites and enhanced number of crossings with no change in dendritic length (inset). There was a significant main effect of the treatment (p < 0.0001) and of distance from the soma (p < 0.0001). Sholl crossings were analyzed with two-way ANOVA and Bonferroni's post-test, and individual points and dendritic length were analyzed with unpaired t tests (n = 11–12/condition from 3 cultures). D, Dendrite counts from phase-contrast images confirms a significant increase in number of primary dendrites in treated D2 MSNs (Mann–Whitney test). E, Soma diameter of D2, but not D1, MSNs decreases slightly in the treated condition (n > 28 cells/group, *p < 0.05; unpaired t test).
- Figure 5.
D2R activation increases number of presynaptic terminals. A, Confocal images of nontreated (left) and treated (right) D2 MSN segments, stained against GFP (green) and VGAT (red). Scale bar, 5 μm. B, VGAT puncta density (left) and puncta intensity (right) in identified GFP+ and GFP− dendrites as quantified in ImageJ (*p < 0.05; Mann–Whitney U test). C, D, Representative sIPSC (C) and mIPSC (D) traces from D2 MSNs recorded from nontreated (left) and treated (middle) D2 MSNs. Plots of cumulative interevent intervals in D2 MSNs (right) show a reduction in time between events in treated D2 neurons. E–G, Summary plots compare frequency (E), peak amplitude (F), and τw (G) of sIPSCs and mIPSCs in D2 MSNs in both experimental conditions (n = 38 and 55 nontreated and treated cells/group). D1 MSNs similarly exhibited no change in kinetics or amplitude (data not shown). *p < 0.05 when comparing mIPSCs or sIPSCs between treatment condition (Mann–Whitney U test). ++p < 0.01 when comparing mIPSCs and sIPSCs within treatment group (Wilcoxon signed-rank test).
- Figure 6.
V–M analysis of eIPSC amplitudes. A, B, V–M analyses of eIPSCs from a single representative nontreated (A) and treated (B) D2 MSNs. A1, B1, Superimposed eIPSCs illustrate amplitude fluctuations with changes in external calcium ([Ca2+]o) concentration. A2, B2, The time course of individual responses is plotted with four [Ca2+]o concentrations as indicated. A3, B3, Mean peak values are overlayed per concentration. A4, B4, V–M plots generated per cell highlight differences in release probabilities between conditions. C, D, Summary data show contrasting V–M trends between nontreated (C) and treated (D) conditions, suggesting higher release probability in D2 MSNs treated with quinpirole. V–M is normalized to the mean value of 2 mm Ca2+ in each cell (n = 13 and 10 cells/group from 4 culture preparations). Scale bars: x-axis, 150 ms; y-axis, 100 pA.
- Figure 7.
Enhanced GABAA sensitivity in D2 MSNs following D2R activation. A, Confocal images of D2 MSN segments with GFP (green) and gephyrin (red) staining in nontreated (left) and quinpirole-treated (right) conditions. Scale bar, 5 μm. B, Summary plots of gephyrin puncta density (left) and puncta intensity (right) of identified GFP+ and GFP− dendrites (**p < 0.01; Mann–Whitney U test). C, D, Representative currents in response to direct Y-tube application of GABA, THIP (1 μm), and etomidate (5 μm) in nontreated (C) and treated (D) D2 MSNs. E, Summary of direct drug effects in D1 and D2 MSNs in each experimental condition (n = 16–24 cells/group; Kruskal–Wallis test with Dunn's post test: *p < 0.05, **p < 0.01, ***p < 0.001 comparing the same cell type between conditions; #p < 0.05, ##p < 0.01 comparing different cell types within conditions).
Tables
Nontreated Treated D1 MSN D2 MSN D1 MSN D2 MSN Resting membrane potential (mV) −73.9 ± 1.8 (n = 22) −75.5 ± 1.5 (n = 43) −75.1 ± 5.2 (n = 9) −73.9 ± 1.4 (n = 38) Input resistance (mΩ) 265 ± 18 (n = 19) 215 ± 23 (n = 42) 268 ± 27 (n = 18) 209 ± 20 (n = 42) Capacitance (pF) 54 ± 5 (n = 16) 43 ± 4 (n = 23) 52 ± 4 (n = 13) 67 ± 5 (n = 19)***,+ Inward rectification index 1.7 ± 0.1 (n = 8) 1.7 ± 0.1 (n = 22) 2.0 ± 0.2 (n = 8) 2.1 ± 0.1 (n = 13) ↵aWhole-cell recordings from identified MSNs in nontreated and treated conditions showed that chronic D2R activation increased the capacitance and inward rectification index in only D2 MSNs. D1 MSNs remained unchanged despite the treatment, serving as an internal control (data derived from at least 4 culture preparations).
↵***p < 0.001, comparing same cell type between treatment conditions using an unpaired t test;
↵+p < 0.05 comparing different cell types within a treatment condition using an unpaired t test.
