Abstract
Dopamine D2 receptors (D2Rs) are densely expressed in the striatum and have been linked to neuropsychiatric disorders such as schizophrenia1,2. High-affinity binding of dopamine suggests that D2Rs detect transient reductions in dopamine concentration (the dopamine dip) during punishment learning3,4,5. However, the nature and cellular basis of D2R-dependent behaviour are unclear. Here we show that tone reward conditioning induces marked stimulus generalization in a manner that depends on dopamine D1 receptors (D1Rs) in the nucleus accumbens (NAc) of mice, and that discrimination learning refines the conditioning using a dopamine dip. In NAc slices, a narrow dopamine dip (as short as 0.4 s) was detected by D2Rs to disinhibit adenosine A2A receptor (A2AR)-mediated enlargement of dendritic spines in D2R-expressing spiny projection neurons (D2-SPNs). Plasticity-related signalling by Ca2+/calmodulin-dependent protein kinase II and A2ARs in the NAc was required for discrimination learning. By contrast, extinction learning did not involve dopamine dips or D2-SPNs. Treatment with methamphetamine, which dysregulates dopamine signalling, impaired discrimination learning and spine enlargement, and these impairments were reversed by a D2R antagonist. Our data show that D2Rs refine the generalized reward learning mediated by D1Rs.
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Data availability
Source Data for Figs. 1–5 and Extended Data Figs. 1–10 are provided with the paper. All data are available from the corresponding author upon reasonable request.
Code availability
All code is available from the corresponding author upon reasonable request.
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Acknowledgements
We thank T. Yoshikawa for support of the MAP model, Y. Li for the GRABDA1m plasmid, N. Uchida, J. Johansen, M. Lazarus, H. Urakubo, K. Kasai, S. Koike, and S. Kondo for discussions, and A. Kurabayashi, M. Asaumi, R. Nakazato and A. Nishikawa for technical assistance. This work was supported by CREST (JPMJCR1652 to H.K.) from JST, SRPBS (JP19dm0107120 to H.K.), Brain/MINDS (19dm0207069h0001 to S.Y.) from AMED, Grants-in-Aid (No. 26221001 to H.K.; 19K16249, 16H06395, 16H06396 and 16K21720 to S.Y.) from JSPS, the World Premier International Research Center Initiative (WPI) from MEXT, Takeda Science Foundation (to S.Y.), and The Naito Foundation (to Y.I.). M.T. and T.S. are Research Fellows for Young Scientists of JSPS.
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S.Y., Y.I. and H.K. designed the experiments. T.S. and K.Y. performed the behavioural experiments. Y.I. and S.Y. performed the slice experiments. M.T. provided technical assistance for histology and immunohistochemistry. Y.I., T.S., K.Y. S.I., H.K. and S.Y. analysed and interpreted the data. S.I. provided theoretical predictions. S.Y., Y.I. and H.K. wrote the manuscript and all authors edited the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Discrimination learning.
a, Schematic of a behavioural setup for the tone–reward discrimination task in classical conditioning in head-restrained mice. A tube monitored licking responses. b, Numbers of trials during the discrimination task. For sessions with multiple task types, the order of presentation was pseudo-randomized. c, The time course of lick scores. n = 7 mice. d, Peristimulus time histograms (PSTHs) of licking responses plotted against time from CS onset on days 1, 2, 3, and 4. The grey bar indicates the period of tone presentation. n = 7 mice. e, Lick scores for CS+ and CS− on day 4. Two-sided Wilcoxon signed-rank test (n = 7 mice, W = 0, *P = 0.016). f, Schematic of fibre photometry of DA activities in the lateral NAc. g, Averaged z-scores of green fluorescence during discrimination on days 1, 2, and 3. Orange bars indicate integration periods. n = 7 mice. h, Plots of photometry signals in US timing (1.5–3 s after CS onset). Two-sided Wilcoxon signed-rank test with Bonferroni correction (n = 7 mice, day 1, W = 0, *P = 0.016; day 2, W = 0, *P = 0.016; day 3, W = 0, *P = 0.016). Error bars and shading indicate s.e.m. Vertical grey shading in d, g indicates CS periods. Samples of mice are biological replicates.
Extended Data Fig. 2 Generalization of cues after reward conditioning and SCH23390 effects.
a, Schematic of tone–reward classical conditioning task in classical conditioning in head-restrained mice. A tube monitored licking responses. b, Raster plots of licking responses before (left), during (middle) and after (right) conditioning in a representative mouse. Grey shading indicates the periods of CS presentation. c–f, Generalization to pure tones (c, d), and light (e, f). PSTHs in c, e show licking responses before (left), during (middle) and after (right) conditioning. Lick scores are plotted against time (d, f). n = 11 (c, d) and 7 mice (e, f). g, Schematic of drug infusion into the bilateral NAc. h, i, Effect of drug infusion on generalized conditioned responses. Top, plots of lick scores; bottom, PSTHs at generalization tests. Grey shading indicates periods of CS presentation. Black and red bars indicate infusion periods. n = 6 (h) and 6 mice (i). Error bars and shading indicate s.e.m. Vertical grey shading in c, e, h, i indicates CS periods. Samples of mice are biological replicates.
Extended Data Fig. 3 Photometry of DA terminal activity from sub-regions of the NAc during discrimination learning.
a, Schematic of four-site (anterior or posterior/medial or lateral) photometry. b, Representative confocal images showing locations of four fibres in the NAc. Signals were stably obtained from the medial NAc for anterior position (left) and the lateral NAc core for posterior position (right). The locations of the tips of fibres were plotted on the reference atlas. Grey areas indicate the NAc core. c, Schematics of behavioural tasks. CS− trials during discrimination consisted of 40 trials at 10 kHz and 40 trials at 16 kHz. d, e, Representative raster plots (d) and averaged PSTHs (e) of lick responses during generalization, discrimination and extinction. n = 9 mice, as in Fig. 1h–p. f, g, Averaged photometry signals obtained from the lateral (f) and medial NAc (g). n = 9 mice. h, Plots of photometry signals in the lateral NAc in CS timing (0–1 s after CS onset). Two-sided Wilcoxon signed-rank test (n = 9 mice, generalization tests, W = 3, *P = 0.020; discrimination test, W = 0, **P = 0.004). i, Plots of photometry signals in US timing (1.5–3 s after CS onset). Green boxes indicate the periods of sampling indicated in f. Two-sided Wilcoxon signed-rank test (vs baseline) with Bonferroni correction for multiple comparisons (n = 9 mice, day 2 discrimination, W = 0, **P = 0.004; day 3 discrimination, W = 1, **P = 0.008; day 4 extinction early, W = 0, **P = 0.004; day 4 extinction late, W = 15, P = 0.43; day 5 extinction early, W = 17, P = 0.57; day 5 extinction late, W = 25, P = 0.82). Error bars and shading indicate s.e.m. Vertical grey shading in e–g indicates CS periods. Samples of mice are biological replicates.
Extended Data Fig. 4 Generalization and discrimination learning for various combinations of tones for CS+ and CS−.
a, b, Schematics for assignment of tones to CS+ with US and CS− trials in variations of generalization–discrimination learning tasks. c, d, Lick scores plotted against time. Trial numbers for each trial type are indicated above the plots. In CS− trials in c, equal numbers of trials were pseudo-randomly allocated to presentation of 10-kHz or 16-kHz tone. For example, there were 40 trials at 6 kHz and 40 trials at 10 kHz during discrimination. In CS+ trials in d, only 6-kHz tones were presented during conditioning and the discrimination test and 36 pseudo-randomly mixed trials of 6 kHz and 10 kHz were presented during discrimination. n = 6 mice per group. e, f, Averaged lick traces at generalization tests (left) or discrimination tests (right). n = 6 mice per group. g, h, Generalization indices at generalization tests and discrimination tests. Two-sided Wilcoxon signed-rank test (g, n = 6 mice, W = 0, *P = 0.03; h, n = 6 mice, W = 0, *P = 0.03). Error bars and shading indicate s.e.m. Vertical grey shading in e, f indicate CS periods. Samples of mice are biological replicates.
Extended Data Fig. 5 Optogenetic manipulation of DA neurons in the VTA.
a, Schematic of experimental setup. b, Representative confocal images of virus expression and fibre position. The locations of fibre tips were plotted on the reference atlas. TH, tyrosine hydroxylase immunostaining. White bars indicate traces of optical fibres. Grey areas in the atlas indicate the NAc core. c, Photometry of DA neurons stimulated with red LED. Top, schema for stimulus presentation; middle, photometry responses at NAc; bottom, plots of peak photometry responses. Friedman’s test for mCherry (n = 8 mice, χ2(3) = 15.8, P = 0.001; post hoc two-sided Wilcoxon signed rank test with Bonferroni correction, no stimulation versus 5 Hz, W = 10, P = 0.26; no stimulation versus 20 Hz, W = 17, P = 0.89; no stimulation versus sucrose, W = 0, **P = 0.008) and for CsChrimsonR (n = 7 mice, χ2(3) = 18.9, P = 0.0003; post hoc two-sided Wilcoxon signed rank test with Bonferroni correction, no stimulation versus 5 Hz, W = 0, P = 0.093; no stimulation versus 20 Hz, W = 0, *P = 0.016; no stimulation versus sucrose, W = 0, *P = 0.016). d, Schematic of reinforcement of licking behaviour by optogenetic self-stimulation of DA neurons. Licking behaviour triggered optogenetic self-stimulation of DA neurons in the VTA without water delivery. e, Cumulative lick number against time (top) and plots across the conditions (bottom). Friedman’s test for mCherry (n = 8 mice, χ2(2) = 1, P = 0.60) and for CsChrimson (n = 7 mice, χ2(2) = 11.1, P = 0.004; post hoc two-sided Wilcoxon signed rank test with Bonferroni correction, no stimulation versus 5 Hz, W = 8, P = 0.31; no stimulation versus 20 Hz in CsChrimsonR, W = 0, *P = 0.016). f, Schematic of experimental setup for optogenetic enhancement of DA dips during discrimination. AAVs were injected to express eNpHR3.0 and GCaMP6f in VTA-DA neurons. Optical fibres were implanted at the VTA for stimulation of eNpHR3.0 and at the right NAc for photometry of GCaMP. g, Locations of the fibre tips plotted on the reference atlas. h, Averaged z-scores of photometry responses during CS− trials in the discrimination period for eNpHR3.0 (red) and mCherry (blue). i, Lick scores plotted against time. Red bar indicates the period of optogenetic stimulation. n = 6 (h, i, mCherry) and 5 (h, i, eNpHR3.0) mice. Light green bar indicates sampling periods for the fibre photometry data shown in h. j, Locations of the tips of fibres plotted on the reference atlas. k, Averaged z-scores of photometry responses with 20-Hz optogenetic stimulation during extinction. l, Lick scores plotted against time with 20-Hz DA stimulation during extinction. Red bar indicates the period of optogenetic stimulation. Green bar indicates sampling periods for the fibre photometry data shown in k. m, Averaged lick traces during CT and extinction test (top) and lick score at CT and extinction test (bottom). n = 5 mice (k–m). n, Plots of extinction indices across conditions. n = 6 mice for mCherry 5 Hz, n = 4 mice for CsChrimsonR 5 Hz, n = 4 mice for CsChrimsonR 20 Hz. Kruskal–Wallis test (χ2(2) = 9.5, P = 0.009; post hoc Steel’s test, CsChrimsonR 5 Hz versus mCherry, t = 1.28, P = 0.34; CsChrimsonR 20 Hz versus mCherry, t = −2.6, *P = 0.02). Data for CsChrimsionR 5 Hz and mCherry 5 Hz are from Fig. 2m. Scale bars in b indicate 500 μm. Scale bars in h, k indicate 1 s and 1 z-score. Scale bars in m indicate 1 s and 2 Hz. Error bars and shading indicate s.e.m. Vertical grey shading in h, k indicates CS periods. Samples of mice are biological replicates.
Extended Data Fig. 6 Pharmacological properties of spine plasticity in D2-SPNs.
a, Example trace of 2pEPSC (average of five traces with stimulation of four spines from one slice). b, Membrane potential during one train of stimulation (top) and command of uncaging (red) and current injection (black) (bottom). To mimic the up state, constant current (~200 pA) was injected for 1.1 s and APs were evoked by pulsed current injections (0.6–2.0 nA, 2 ms). c, Magnified traces of membrane potentials. Note the 2pEPSP, right. d, Schematic of virus injection to label D2-SPNs e, Schematic of stimulation and representative images of dendritic spines in mCherry-expressing D2-SPNs. Red circles show two-photon uncaging spots. f, g, Time course of volume changes of stimulated and neighbouring spines in the absence of CGS (f) and in the absence of CGS with sulpiride (g). STDP stimulation was applied at 0 min. n = 5 (f) and 6 slices (g). h, Viral expression of AIP in D2-SPNs. i, Schematics of STDP stimulation of AIP-expressing D2-SPNs in the presence of CGS (top) and representative images of dendritic spines (bottom). j, Time courses of volume changes of stimulated and neighbouring spines in AIP-expressing D2-SPNs. A reference trace of mCherry is the same as in Fig. 3d. n = 6 slices. k–m, Electrophysiological properties of D2-SPNs expressing AIP. n = 10 and 6 slices for mCherry and AIP. Plots of resting membrane potential (RMP) (k), two-sided Mann–Whitney U test (U = 22, P = 0.39); 2pEPSC (l, right), two-sided Mann–Whitney U test (U = 23, P = 0.45); averaged traces of 2pEPSC (l, left); and membrane potentials during STDP stimulation (m) are shown. n–q, Spine plasticity of D2-SPNs in the presence of CGS with PKI (o, p, n = 6 slices) or AP5 (q, n = 6 slices). r, A schematic of virus injection to label D1-SPNs using Cre-off mCherry-expressing AAV (FAS-NLS-mCherry)44. s, Representative images of dendritic spines stimulated in the presence of CGS21680 (top) or puff-applied DA (bottom) in D1-SPNs. t, Time course of volume changes. n = 6 for CGS and 5 slices for DA (puff). u, Plots of volume changes. n = 6 for CGS, n = 5 slices for DA (puff). Two-sided Mann–Whitney U test (U = 0, **P = 0.006). Scale bars in e, i, o, s indicate 2 μm. Error bars indicate s.e.m. Samples of slices are biological replicates.
Extended Data Fig. 7 DA concentration profiles for various optogenetic stimulation protocols of DA fibres.
a, Schematic of virus injection (left) and amperometric detection of ChR2-driven DA release (right). A carbon fibre electrode was placed in the lateral NAc. b, c, Confocal images showing the expression of ChR2-mCherry in DA neurons in the VTA (b) and NAc (c). Scale bars indicate 500 μm for top parts and 5 μm for bottom parts of b, c. d, e, Replication of tonic DA levels in the NAc slice. Blue light pulses (2 ms) were applied at 5 Hz for 160 s with constant intensity (d, bottom) or modulated intensity (e, bottom). Amperometry signals for the evoked DA are shown (d, e, top). At the onset of stimulation, a strong but transient DA release which reflected long-term absence of autoinhibition was observed, whose peaks are omitted in the display. Insets show comparisons of averaged DA concentration between the first 10 s (1–11 s) and the last 10 s (150–160 s). Two-sided Wilcoxon signed-rank test for constant (n = 8 slices, W = 0, *P = 0.017) and for modulation (n = 9 slices, W = 14, P = 0.31). f, Time course averaged every 10 s over the stimulation period of 160 s. g, Plots of averaged tonic DA concentration. Each dot represents the averaged DA concentration indicated in the grey bar in f for each slice. h, Trace of DA concentration (top) and DA stimulation commands (bottom). During 5-Hz blue light stimulation, a 2-s pause was inserted 15 times in every 10 s. i, Averaged trace of the DA dip over 15 dips. To allow quick recovery to tonic levels, 5 pulses at 10 Hz were added after the pause. j, Plots of DA levels with various DA dips. Baselines indicate the average of 1 s before the dip onset and troughs indicate the average during the last 0.1 s of each dip. n = 14 (dip 2 s), 8 (dip 0.6 s) and 12 slices (dip 0.4 s). k, Plots of DA changes by various DA dips. Baseline values were subtracted from trough values. Two-sided Wilcoxon signed-rank test (dip 2 s, n = 14 slices, W = 0, **P = 4.0 × 10−4; dip 0.6 s, n = 8 slices, W = 0, **P = 0.003; dip 0.4 s, n = 12 slices, W = 0, **P = 7.0 × 10−4). l, Trace of DA concentration (top) and DA stimulation commands (bottom). During 5-Hz blue light stimulation, 20-Hz stimulation (dark blue) with an enhanced stimulation intensity was inserted 15 times every 10 s. m, Averaged trace of DA concentration over 15 bursts. n, Plots of DA levels by various DA bursts. Baselines indicate the average of 1 s before the burst onset and troughs indicate the average during the last 0.1 s of each burst. n = 11 (burst 1 s) and 7 slices (burst 0.5 s). o, Plots of DA changes by various DA bursts. Baseline values were subtracted from peak values. Two-sided Wilcoxon signed-rank test (burst 1 s, n = 11 slices, W = 0, **P = 0.003; burst 0.5 s, n = 7 slices, W = 0, **P = 0.003). Error bars and shading indicate s.e.m. Samples of slices are biological replicates.
Extended Data Fig. 8 Optogenetics of DA fibres and spine plasticity in slices.
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).
Extended Data Fig. 9 AIP expression and effects of A2AR and D2R antagonists on the time courses of lick scores.
a, Averaged intensity of AIP expression used in the experiment for Fig. 4c–f (n = 9 mice). Reference atlases are overlaid. b, Effects of AIP in conventional discrimination paradigm (Extended Data Fig. 1). n = 8 mice for mCherry, n = 6 mice for AIP. Two-sided Mann–Whitney U test (U = 10, *P = 0.04). c, Schematic of drug infusion to the bilateral NAc. Drugs were infused through implanted bilateral cannula 30 min before the task and infusion was continued throughout the task. d, e, Lick scores plotted against time for mice injected with SCH58261 (d) or sulpiride (e) during generalization–discrimination learning. Red bars indicate the periods of drug infusion (days 2 and 3). To control the experimental context, ACSF was infused during the discrimination test. Quantified results are presented in Fig. 4k–n. n = 6 mice for each group. f, Estimation of drug spreading. Averaged intensity of a fluorescent dye (Alexa 594, n = 3 mice) that was injected through the unilateral cannula over the same period as in the discrimination tasks with overlaid reference atlases. Scale bars in a, f indicate 500 μm, 1 s and 2 Hz. Scale bars in b indicate 1 s and 2 Hz. Error bars indicate s.e.m. Samples of mice are biological replicates.
Extended Data Fig. 10 Effects of MAP on locomotor activity and dopamine concentrations.
a, Experimental schedule for locomotor sensitization by MAP (1 mg kg−1). b, Representative locomotion traces during sensitization test 0–15 min after administration. Scale bar, 10 cm. c, Plots of average distances travelled in sensitization tests against time. n = 7 (control) and 7 mice (MAP). d, Plots of average distances travelled by control mice (n = 7) and MAP-treated mice (n = 7). Two-sided Mann–Whitney U test (U = 2, **P = 0.004). e, Schematic of virus injection. f, g, Confocal image of GRABDA1m expression (f) and locations of the tips of fibres plotted on the reference atlas (g). Grey areas indicate NAc core. Scale bar, 500 μm. h, GRABDA1m responses to MAP administration. Saline, 1 mg kg−1 MAP, or 4 mg kg−1 MAP were administered intraperitoneally in the home cage. n = 7 mice. Each experiment was conducted on a separate day and the order of drug administration was counter-balanced. i, Plots of GRABDA1m responses 30–90 min after MAP injection. n = 7 mice. Friedman’s test (χ2(2) = 14, P = 9.0 × 10−4.). Post hoc Steel–Dwass test (saline versus MAP 1 mg kg−1, t = 3.0, **P = 0.008; saline versus MAP 4 mg kg−1, t = 3.1, **P = 0.005; MAP 1 mg kg−1 versus MAP 4 mg kg−1, t = 2.4, *P = 0.048). j, Lick scores of mice injected with GRABDA1m and treated with MAP. MAP was administered in the same way as in Fig. 5a. n = 7 mice for saline, n = 6 mice for MAP. k, l, Discrimination learning by mice used for GRABDA1m recording. n = 7 mice for saline, n = 6 mice for MAP. Two-sided Mann–Whitney U test (U = 2, **P = 0.005). m, n, GRABDA1m responses to CS− (m) and CS+ with US (n) averaged over discrimination task on day 2 and 3 from mice injected with saline (black) or MAP (blue). n = 7 mice for saline, n = 6 mice for MAP. DA dips in CS− trials during discrimination task (m) were tested by two-sided Wilcoxon signed-rank test versus 0 (saline, W = 0, *P = 0.016; MAP, W = 5, P = 0.63). Dip size was evaluated by the z-score trough during 2–3 s after CS onset to avoid contamination by decay of CS responses. Effects of MAP on dip size (m) and phasic DA responses to US (n) were tested by two-sided Mann–Whitney U test (m, U = 6, *P = 0.035; n, U = 13, P = 0.25). Data were sampled during the discrimination sessions as indicated by the light green bar in j. o, p, GRABDA1m responses to CS+ and CS− at discrimination test from mice injected with saline (o) or MAP (p). Two-sided Wilcoxon signed-rank test (saline, n = 7 mice, W = 27, *P = 0.031; MAP, n = 6 mice, W = 14, P = 0.56). Data were sampled during the discrimination test as indicated by the green bar in j. q, Schematics of CPP protocol with delays after MAP administration. Allocation of chambers A and B and order of MAP and saline injection were counter-balanced. r, Plots of time spent in chambers. Two-sided Wilcoxon signed-rank test (saline, n = 10 mice, W = 24, P = 0.77; MAP 1 mg kg−1, n = 12 mice, W = 41, P = 0.91; MAP 4 mg kg−1, n = 12 mice, W = 8, *P = 0.012). s, Plots of lick scores of mice treated with MAP and infused with sulpiride in discrimination test. MAP was administered as in Fig. 5a. Pink bar indicates period of intra-NAc infusion of sulpiride. From days 2 to 4, either ACSF or sulpiride was infused for all the conditions to control the manipulation of infusion. n = 5 mice. t, Lick traces in discrimination test (top) and plots of generalization indices (bottom). n = 5 mice. u, Plots of Δgeneralization indices for infusion with ACSF (n = 7 mice, from Fig. 5f), sulpiride during the discrimination period (n = 5 mice, from Fig. 5f) or sulpiride during the discrimination test (n = 5 mice, green). Kruskal–Wallis test (χ2(2) = 10, P = 0.007; post hoc Steel’s test, ACSF versus sulpiride during discrimination, t = 2.8, **P = 0.009; ACSF versus sulpiride in discrimination test, t = 0.08, P = 0.99). v, Schematic of amperometric detection of ChR2-driven dopamine release in the NAc slice (top). Normalized time course of DA concentration in the presence and absence of MAP (3 μM) (bottom). Data shown in Fig. 3k and Fig. 4g are normalized to the baseline concentration. n = 15 slices for no MAP, n = 6 slices for MAP. w, Plots of decay time constant. n = 15 slices for no MAP, n = 6 slices for MAP. Two-sided Mann–Whitney U test (U = 13, *P = 0.012). Error bars and shading indicate s.e.m. Vertical grey shading in m–p indicates CS periods. Samples of mice and slices are biological replicates.
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Iino, Y., Sawada, T., Yamaguchi, K. et al. Dopamine D2 receptors in discrimination learning and spine enlargement. Nature 579, 555–560 (2020). https://doi.org/10.1038/s41586-020-2115-1
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DOI: https://doi.org/10.1038/s41586-020-2115-1
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