Differential Alterations in Striatal Direct and Indirect Pathways Mediate Two Autism-like Behaviors in Valproate-Exposed Mice

Abstract

Autism is characterized by two key diagnostic criteria including social deficits and repetitive behaviors. Although recent studies implicated ventral striatum in social deficits and dorsal striatum in repetitive behaviors, here we revealed coexisting and opposite morphologic and functional alterations in the dorsostriatal direct and indirect pathways, and such alterations in these two pathways were found to be responsible, respectively, for the two abovementioned different autism-like behaviors exhibited by male mice prenatally exposed to valproate. The alteration in direct pathway was characterized by a potentiated state of basal activity, with impairment in transient responsiveness of D1-MSNs during social exploration. Concurrent alteration in indirect pathway was a depressed state of basal activity, with enhancement in transient responsiveness of D2-MSNs during repetitive behaviors. A causal relationship linking such differential alterations in these two pathways to the coexistence of these two autism-like behaviors was demonstrated by the cell type-specific correction of abnormal basal activity in the D1-MSNs and D2-MSNs of valproate-exposed mice. The findings support those differential alterations in two striatal pathways mediate the two coexisting autism-like behavioral abnormalities, respectively. This result will help in developing therapeutic options targeting these circuit alterations.SIGNIFICANCE STATEMENT Autism is characterized by two key diagnostic criteria including social deficits and repetitive behaviors. Although a number of recent studies have implicated ventral striatum in social deficits and dorsal striatum in repetitive behaviors, but social behaviors need to be processed by a series of actions, and repetitive behaviors, especially the high-order repetitive behaviors such as restrictive interests, have its scope to cognitive and emotional domains. The current study, for the first time, revealed that prenatal valproate exposure induced coexisting and differential alterations in the dorsomedial striatal direct and indirect pathways, and that these alterations mediate the two coexisting autism-like behavioral abnormalities, respectively. This result will help in developing therapeutic options targeting these circuit alterations to address the behavioral abnormalities.

Keywords: D1-MSNs; D2-MSNs; autism; striatum; valproate.

Figure 1.

VPA mice exhibit core autistic-like behaviors including social interaction deficits and repetitive behaviors. A, Representative traces during three-chamber test in Control and VPA mice. B, C, Control mice spent more time in stranger compartment (B), and displayed higher social index (C), but these were not detected in VPA mice. D, Behavioral paradigm (left), VPA mice displayed less social time in home-cage social interaction test. E, Behavioral paradigm (left). Summary data showing that VPA mice displayed more grooming time. F, Representative photograph after marble burying test. G, The numbers of marbles buried were increased in VPA mice compared with Controls. H, Representative traces during OFT. I, Summary data showing no significant changes in locomotor activities of VPA mice. J–L, Summary data showing that time duration in center area during OFT (J), time spent in open arm (K), and the percentage of open arm time to total time (L) in EPM were decreased in VPA mice compared with Control mice (Control: n = 11 mice; VPA: n = 9 mice, two-way ANOVA with Bonferroni post hoc for B, two-tailed unpaired t test for C–E, G, I–L). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2.

Prenatal VPA exposure generates opposing effects on glutamatergic synaptic transmission and intrinsic excitability in DMS D1-MSNs and D2-MSNs, respectively. A, D, Representative recordings of mEPSCs from D1-MSNs (A) and D2-MSNs (D) in the Control and VPA groups. B, E, Summary data for mEPSC frequency with cumulative probability plots of interevent intervals in D1-MSNs (B) and D2-MSNs (E). C, F, Summary data for mEPSC amplitude with cumulative probability plots in D1-MSNs (C) and D2-MSNs (F; D1-MSNs: Control, n = 15 cells from eight mice; VPA, n = 18 cells from 6 mice. D2-MSNs: Control, n = 15 cells from seven mice; VPA, n = 17 cells from 5 mice, Mann–Whitney U test for B, two-tailed unpaired t test for C, E, F). G, K, Representative recording traces of PPR of D1-MSNs (G) and D2-MSNs (K) from the Control and VPA groups. H, L, Summary data for PPR recording at 50-ms time interval in D1-MSNs (H) and D2-MSNs (L) from the Control and VPA groups (D1-MSNs: Control, n = 10 cells from 6 mice; VPA, n = 7 cells from 4 mice; D2-MSNs: Control, n = 10 cells from 6 mice; VPA, n = 9 cells from 5 mice, two-tailed unpaired t test for H, Mann–Whitney U test for L). I, M, Representative traces of NMDAR EPSCs at +40 mV (upper traces) and AMPAR EPSCs at –80 mV (lower traces) in D1-MSNs (I) and D2-MSNs (M) from the Control and VPA groups. J, N, Statistics of the ratio of AMPAR to NMDAR EPSCs in D1-MSNs (J) and D2-MSNs (N; D1-MSNs: Control, n = 18 cells from 5 mice; VPA, n = 18 cells from 5 mice; D2-MSNs: Control, n = 19 cells from 6 mice; VPA, n = 16 cells from eight mice, two-tailed unpaired t test). O, S, Representative AMPAR-mediated and NMDAR-mediated currents determined by pharmacological isolation at +40 mV in D1-MSNs (O) and D2-MSNs (S) from the Control and VPA groups. P, T, Statistics of the ratio of AMPAR to NMDAR EPSCs determined by pharmacological isolation in D1-MSNs (P) and D2-MSNs (T; D1-MSNs: Control, n = 13 cells from 4 mice; VPA, n = 12 cells from 3 mice; D2-MSNs: Control, n = 10 cells from 3 mice VPA, n = 12 cells from 3 mice, two-tailed unpaired t test). Q, U, Example mean amplitude traces of evoked NMDAR-mediated EPSCs from D1-MSNs (Q) and D2-MSNs (U) in Control and VPA groups. R, V, Summary data showing no significant changes in input–output curve between VPA and Control group in D1-MSNs (R) and D2-MSNs (V; D1-MSNs: Control, n = 9 from 3 mice; VPA, n = 11 from 4 mice, D2-MSNs: Control, n = 9 from 4 mice; VPA, n = 8 from 3 mice, Friedman's M test). W, Y, Summary data for AP Rheobase from D1-MSNs (W) and D2-MSNs (Y) in Control and VPA groups. X, Z, Average instant firing frequency-current (F/I) curves for D1-MSNs (X) and D2-MSNs (Z) in the Control and VPA groups (D1-MSNs: Control, n = 15 cells from 4 mice; VPA, n = 16 cells from 5 mice; D2-MSNs: Control, n = 16 cells from 4 mice; VPA, n = 17 cells from 6 mice, two-tailed unpaired t test for W, Y, Friedman's M test for X, Z). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 3.

Prenatal VPA exposure blunts dopamine receptor agonist-mediated effects on synaptic transmission and intrinsic excitability of MSNs in the DMS. A, Representative traces of mEPSCs in D1-MSNs. B, C, Summary data showing that SKF81297 incubation (20 µm) enhanced the mEPSC frequency (B) and amplitude (C) of D1-MSNs in Control mice, but not in VPA mice (Control, n = 12 cells from 5 mice; VPA, n = 14 cells from 6 mice, two-way ANOVA with Bonferroni post hoc). D, Representative averaged traces of eEPSC in D2-MSNs. Light traces represent the baseline EPSC average from 0 to 10 min (labeled with light color number 1), dark traces represent the average EPSCs from the last 10 min after LTD induction (dark color number 2). E, Normalized EPSC amplitude of D2-MSNs showing a less reduction of eEPSCs in VPA mice than those in Control mice by quinpirole incubation. F, Summary of the eEPSC magnitude in D2-MSNs (Control, n = 9 cells from 4 mice; VPA, n = 12 cells from 6 mice, Friedman's M test for E, two-tailed unpaired t test for F). G, I, K, M, Representative current-response curves of D1-MSNs (G, I) and D2-MSNs (K, M) in brain slices in Control and VPA mice. H, J, L, N, Average instant firing frequency-current (F/I) was increased by SKF81297 perfusion (5 µm) in D1-MSNs (H) and reduced by quinpirole perfusion (10 µm) in D2-MSNs (L) from Control mice, but not in VPA mice (J, N; D1-MSNs: Control, n =12 cells from 3 mice; VPA: n = 13 cells from 4 mice; D2-MSNs: Control, n =18 cells from 6 mice; VPA: n = 13 cells from 5 mice, Friedman's M test). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; **p < 0.01, ***p < 0.001.

Figure 4.

Prenatal VPA exposure differentially alters the morphology of D1-MSNs and D2-MSNs in the DMS. A, Schematic representation and injecting site of AAV virus in DMS to sparse labeling MSNs. B, C, K, Representative confocal stacks and three-dimensional reconstruction images (right in C, K) of D1-MSNs (B, C) and D2-MSNs (B, K) in the DMS in the Control and VPA D2-eGFP mice. Arrowheads indicate cell body in B. Scale bars, 50 µm. D–G, L–O, Statistical results showing that dendritic branching complexity (D, L), the total dendrite length (E, M), branch points (F, N) and tips (G, O) were increased in D1-MSNs and decreased in D2-MSNs from VPA D2-eGFP mice (D1-MSNs: Control, n = 21 neurons from 3 mice; VPA, n = 21 neurons from 4 mice; D2-MSNs: Control, n = 14 neurons from 4 mice; VPA, n = 15 neurons from 5 mice, Friedman's M test for D, L, two-tailed unpaired t test for E–G, M–O). H, P, Representative confocal stacks and three-dimensional reconstruction images of dendrites in D1-MSNs (H) and D2-MSNs (P) from the Control and VPA D2-eGFP mice. Scale bar, 3 µm. I, J, Q, R, Statistical results showing that total spine density (I, Q) and density of different subtypes of spines (J, R) were increased in D1-MSNs, but not in D2-MSNs in VPA D2-eGFP mice (D1-MSNs: Control, n = 34 dendrites from 3 mice; VPA, n = 38 dendrites from 4 mice; D2-MSNs: Control, n = 28 dendrites from 4 mice; VPA, n = 35 dendrites from 5 mice, two-tailed unpaired t test for I, J, Q, R). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; *p < 0.05, ***p < 0.001.

Figure 5.

Prenatal VPA exposure induces different responsiveness of DMS D1-MSNs and D2-MSNs during social interaction and marble burying tasks. A, Photometry setup used to record calcium signaling in GCaMP-expressing neurons. Light path for fluorescence excitation and emission is through a fiber optic implanted in DMS. B, Viral expression of GCaMP6m and placement of the fiber-optic probe in DMS. C, Schematic of viral injection and following behavioral tests. D, H, Heatmap illustrating the calcium responses (ΔF/F, %) for D1-MSNs (D) and D2-MSNs (H) during social interaction. E, I, Peri-event plots of averaged calcium signals (ΔF/F, %) during social behavior in D1-MSNs (E) and D2-MSNs (I). Solid line and shaded regions are the mean ± SEM. F, J, The calcium responses on social interaction were weaker in D1-MSNs (F), but not in D2-MSNs (J) of VPA mice compared with Control mice. G, K, Summary data of social interaction in D1-Cre (G) and D2-Cre (K) mice (D1-Cre: Control, n = 13 mice; VPA, n = 13 mice, D2-Cre: Control, n = 14 mice; VPA, n = 11 mice, Mann–Whitney U test for F, G, J, K). L, P, Heatmap of the calcium response (ΔF/F, %) in D1-MSNs (L) and D2-MSNs (P) during marble burying behaviors. M, Q, Peri-event plots of averaged calcium signals (ΔF/F, %) during marble burying in D1-MSNs (M) and D2-MSNs (Q). N, R, The calcium responses during marble burying were higher in D2-MSNs (R), but not in D1-MSNs (N) in VPA mice compared with Control mice. O, S, Summary data of the numbers of marbles in D1-Cre (O) and D2-Cre (S) mice (D1-Cre: Control, n = 10 mice; VPA, n = 11 mice, D2-Cre: Control, n = 13 mice; VPA, n = 13 mice, two-tailed unpaired t test). T, U, V, W, Representative baseline calcium traces and peak analysis from a D1-Cre (T, U) and D2-Cre (V, W) mouse. Baseline D1-MSN activity was higher in VPA mice, illustrated by an increased calcium transient frequency, whereas baseline D2-MSN activity was lower in VPA mice, illustrated by a decreased calcium transient frequency (D1-Cre: Control, n = 10 mice; VPA, n = 11 mice, D2-Cre: Control, n = 11 mice; VPA, n = 12 mice, Mann–Whitney U test). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; *p < 0.05, **p < 0.01.

Figure 6.

Cell-specific inhibition of DMS D1-MSNs improves social deficits, whereas activation of D2-MSNs alleviates repetitive behaviors in VPA offspring. A, Schematic representation and injecting site of virus in the DMS. B, Current-voltage relationship of representative D1-MSNs and D2-MSNs recorded before and after 10 µmol/l CNO perfusion in brain slices. Gray traces represent the rheobase of APs from D1-MSNs and D2-MSNs. C, I, Representative traces during three-chamber test in hM4Di-expressing VPA D1-Cre mice (C) and hM3Dq-expressing VPA D2-Cre mice (I). D, E, After CNO injection, hM4Di-expressing VPA D1-Cre mice spent more time in the stranger compartment (D), and showed higher social index during three-chamber test (E). F, Summary data showing that social time in home-cage social interaction test was increased in hM4Di-expressing VPA D1-Cre mice compared with mCherry-expressing VPA D1-Cre mice after CNO injection. G, H, Summary data showing no significant changes of self-grooming time (G) and numbers of marbles buried (H) both in hM4Di-expressing VPA D1-Cre mice and in mCherry-expressing VPA D1-Cre mice after CNO injection. J, K, Summary data showing no significant changes of time duration in stranger compartments (J) and social index (K) in hM3Dq-expressing VPA D2-Cre mice during three-chamber test after CNO injection. L, Summary data showing similar social time during home-cage social interaction test in hM3Dq-expressing VPA D2-Cre mice and mCherry-expressing VPA D2-Cre mice after CNO injection. M, N, Summary data showing that self-grooming time (M) and numbers of marbles buried (N) were significantly decreased in hM3Dq-expressing VPA D2-Cre mice compared with mCherry-expressing VPA D2-Cre mice after CNO injection (VPA: D1-mCherry, n = 9 mice; D1-hM4Di, n = 11 mice; D2-mCherry, n = 10 mice; D2-hM3Dq, n = 13 mice, two-way ANOVA with Bonferroni post hoc for D, J, two-tailed unpaired t test for E–H, K–N). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7.

Cell-specific activation of D1-MSNs by DREADD induces social deficits, whereas inhibition of D2-MSNs activity induces repetitive behaviors in control offspring. A, Schematic representation and injecting site of virus in DMS. B, Current-voltage relationship of representative D1-MSNs and D2-MSNs recorded before and after 10 µmol/l CNO perfusion in brain slices. C, I, Representative traces during three-chamber test in hM3Dq-expressing D1-Cre mice (C) and hM4Di-expressing D2-Cre mice (I). D, E, After 0.3 mg/kg CNO injection, hM3Dq-expressing D1-Cre mice spent more time in the stranger compartment (D), and showed lower social index during three-chamber test (E). F, Summary data showing that social time in home-cage social interaction test was decreased in hM3Dq-expressing D1-Cre mice compared with mCherry-expressing D1-Cre mice after CNO injection. G, H, Summary data showing no significant changes of self-grooming time (G) and numbers of marbles buried (H) both in hM3Dq-expressing D1-Cre mice and in mCherry-expressing D1-Cre mice after CNO injection. J, K, Summary data showing no significant changes of time duration in stranger compartments (J) and social index (K) in hM4Di-expressing D2-Cre mice during three-chamber test after 0.3 mg/kg CNO injection. L, Summary data showing similar social time during home-cage social interaction test in hM4Di-expressing D2-Cre mice and mCherry-expressing D2-Cre mice after CNO injection. M, N, Summary data showing that self-grooming time (M) and numbers of marbles buried (N) were significantly increased in hM4Di-expressing VPA D2-Cre mice compared with mCherry-expressing D2-Cre mice after 0.3 mg/kg CNO injection (Control: D1-mcherry: n = 10 mice; D1-hM3Dq: n = 12 mice; D2-mcherry: n = 10 mice; D2-hM4Di: n = 11 mice, two-way ANOVA with Bonferroni post hoc for D, J, two-tailed unpaired t test for E–H, K–N). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; **p < 0.01, ***p < 0.001.

Figure 8.

Optogenetical inhibition of D1-MSNs improves social deficits, whereas activation of D2-MSNs alleviates repetitive behaviors in VPA offspring. A, Schematic representation and injecting site of virus in the DMS. B, C, Representative traces from in vitro slice recordings of DMS NpHR3.0-expressing D1-MSNs (B) illuminated by a 596-nm yellow light stimulation for 1 s, and ChR2-expressing D2-MSNs (C) illuminated by 10-Hz, 473-nm blue light stimulation. D, I, Representative traces of three-chamber test in the NpHR3.0-expressing VPA D1-Cre mice and EYFP-expressing VPA D2-Cre mice. E, F, Compared with the EYFP-expressing VPA mice, the NpHR3.0-expressing VPA D1-Cre mice spent significantly more time in the stranger compartment (E) and showed higher social index (F) during three-chamber test. G, Summary data showing that social time in home-cage social interaction test was increased in NpHR3.0-expressing VPA D1-Cre mice compared with EYFP-expressing VPA D1-Cre mice. H, Summary data showing no significant changes of self-grooming time both in NpHR3.0-expressing VPA D1-Cre mice and in EYFP-expressing VPA D1-Cre mice. J, K, Summary data showing no significant changes of time duration in stranger compartments (J) and social index (K) both in ChR2-expressing VPA D2-Cre mice and in EYFP-expressing VPA D2-Cre mice. L, Summary data showing similar social time during home-cage social interaction test in ChR2-expressing VPA D2-Cre mice and EYFP-expressing VPA D2-Cre mice. M, Summary data showing that self-grooming time was significantly decreased in ChR2-expressing VPA D2-Cre mice compared with EYFP-expressing VPA D2-Cre mice (VPA: D1-EYFP, n = 11 mice; D1-NpHR3.0, n = 10 mice; D2-EYFP, n = 10 mice; D2-ChR2, n = 12 mice, two-way RM measurement with Bonferroni post hoc for E–M). Data represent mean ± SEM. See Extended Data Table 1-1 for detailed statistical information; **p < 0.01.