Extended Data Fig. 8: Optogenetics of DA fibres and spine plasticity in slices.
From: Dopamine D2 receptors in discrimination learning and spine enlargement
a–c, Schematics of virus injections and confocal images of mCherry expression in the NAc for histological verification of virus expression in the NAc in A2A-Cre/DAT-Cre double transgenic mice. a, mCherry was expressed in soma and neuropil (a, n = 3 mice) under the conditions used in slice experiments. b, A condition for the test of DAT-Cre-derived non-target expression in the NAc shows no DIO-mCherry expression in the NAc of DAT-Cre mice (n = 3 mice). c, A condition for the test of anterograde or retrograde infection to D2-SPNs in the NAc. Although AAV5s are known to retrogradely infect51, DIO-mCherry was not expressed in the NAc of A2A-Cre mice when injected in the VTA (n = 3 mice), consistent with no direct input from D2-SPN to the VTA. These data also demonstrate no anterograde transfection from the VTA DA neurons to the NAc. d, Confocal images of ChR2-mCherry expression and chart of the proportion of TH+ cells among ChR2-mCherry positive cells in the VTA DA neurons from A2A-Cre/DAT-Cre double transgenic mice. Data from four mice were aggregated. e, Individual spine volume changes in D2-SPNs in response to tonic DA or various durations of DA dips (left) and D1-SPNs in response to tonic DA or various durations of DA bursts (right). n = 13 (CGS), 6 (dip 2 s), 5 (dip 0.6 s), 7 (dip 0.4 s) and 7 slices (tonic) for D2-SPNs. n = 5 (tonic), 5 (burst 0.5 s) and 7 slices (burst 1 s) for D1-SPNs. Spearman correlation coefficients (shown) were tested by two-sided Student’s t-test. P = 7.1 × 10−12 (left) and P = 6.3 × 10−5 (right). f, Testing the effects of DA burst stimulation on spine plasticity in D2-SPNs. Red circle shows the two-photon uncaging spot. Scale bar, 2 μm. g, Time course of volume changes and plots of spine volume changes. For reference, a plot with 5-Hz tonic stimulation from Fig. 3h is shown. n = 7 slices for tonic, n = 5 slices for burst 1 s. Two-sided Mann–Whitney U test (U = 12, P = 0.37). h, i, Stimulation protocols for measuring 2pEPSCs before and after STDP stimulation with (h) and without (i) a DA dip. j, k, Time courses of the 2pEPSC amplitudes of stimulated and neighbouring spines with (j) and without (k) DA dip. Insets show 2pEPSC for stimulated and neighbouring spines before (black) and after the stimulation (red). n = 11 spines from 5 slices (j) and 14 spines from 6 slices (k). l, Average changes in 2pEPSCs. n = 11 spines from 5 slices (dip 2 s) and 14 spines from 6 slices (tonic). Two-sided Mann–Whitney U test (U = 37, *P = 0.029). m, n, Time course of volume changes of stimulated and neighbouring spines with DA dip (j) and without DA dip (k). n = 11 spines from 5 slices (m) and 14 spines from 6 slices (n). o, Average changes in spine volume. n = 11 spines from 5 slices (dip 2 s) and 14 spines from 6 slices (tonic). Two-sided Mann–Whitney U test (U = 10, **P = 4.0 × 10−4). Scale bars in a–d indicate 10 μm. Error bars indicate s.e.m. Samples of slices are biological replicates and spines are technical replicates (different spines from the same dendrite).
