The overview of the experimental approach to describe the activity patterns across striatal functional domains.

A. Schematic illustration of the experimental timeline, related to Figures 1-4. D1-Cre/Ai14 and A2A-Cre/Ai14 mice, expressing reporter proteins, were used at different ages for the characterization of the spontaneous activity, intrinsic excitability and passive membrane properties.

B. Immunostaining of dorsal striatum of D1-Cre and A2A-Cre mice of different ages. Striosomes are visible during early postnatal development by denser expression of the tdTomato after Cre recombination under control of Drd1 and A2A promoters. Red: tdTomato; green: µ-opioid receptor staining. Scale bar 100 µm.

Spontaneous activity in striosomal and matrix dSPNs and iSPNs.

A. Example traces of spontaneous APs, recorded by whole-cell patch-clamp from striosomal dSPN of P4 D1-Cre/Ai14 animal and iSPN of P7 A2a-Cre/Ai14 animal.

B. Percent’s of spontaneously active (with AP firing) and silent (without AP firing) dSPNs and iSPNs in striosomes and matrix of P4-P14 animals. *p<0.05, **p<0.01, Chi-square test. Number of cells is indicated on the bars. Numbers of animals used are: dSPN P4 N=6, P7 N=9, P10 N=4; iSPN P7 N=4, P10 N=5, P14 N=3. Frequency of spontaneous APs in striosomal and matrix dSPNs of P4 animals and striosomal and matrix iSPNs of P7 animals from the same data set as the previous graph.

C,D Spontaneous Ca2+ oscillations in striosomal and matrix dSPNs (C) and iSPNs (D), recorded in striatal slices of P4 D1-Cre/Ai95D and P7 A2a-Cre/Ai95D animals. Striosomal compartment border was marked on the maximal intensity projection of the movie, and ROIs corresponding to the active cells were selected on the standard deviation projection of the movie. Example traces show spontaneous Ca2+ oscillations in striosomal and matrix cells of D1-Cre (C) and A2a-Cre (D) slices. Percent’s of spontaneously active cells in striosomes and matrix from active cells in 8 KCl conditions are presented as single values per slice and mean±SEM. dSPNs: 17 slices for striosomes, 17 slices for matrix, 3 animals. iSPNs: 20 slices for striosomes, 20 slices for matrix, 5 animals. *p<0.05, **p<0.01, ***p<0.001, unpaired t-test.

Intrinsic excitability properties of dSPNs during the first three weeks of life.

A. Example traces of the membrane potentials after injection of current steps with 10 pA increments of striosomal and matrix dSPNs of dorsal striatum, recorded from P4-P10 D1-Cre/Ai14 animals. Action potentials of P21 dSPN are presented for the comparison (it was not possible to detect the compartment location of the recorded neuron at this age point).

B. Example traces of APs at the expanded scale, striosomal and matrix APs are overlayed. C. Membrane properties and AP characteristics, which were significantly different for striosomal and matrix dSPNs. All measured parameters and sample size are presented in Tables S1-2. Data are presented as individual values, mean±SEM, and mean of the difference with 95% CI. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA.

Intrinsic excitability properties of iSPNs during the first three weeks of life.

A. Example traces of the membrane potentials after injection of current steps with 10 pA increments of striosomal and matrix iSPNs of dorsal striatum, recorded from P7-P14 A2A-Cre/Ai14 animals. Action potentials of P21 iSPN are presented for the comparison (it was not possible to detect the compartment location of the recorded neuron at this age point).

B. Example traces of APs at the expanded scale, striosomal and matrix APs are overlayed. C. Membrane properties and AP characteristics, which were significantly different for striosomal and matrix dSPNs. All measured parameters and sample size are presented in Tables S3-4. Data are presented as individual values, mean±SEM, and mean of the difference with 95% CI. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, two-way ANOVA.

Descriptive statistics of the membrane properties of dSPNs

Two-way ANOVA of the dSPN membrane properties (P4-P10)

Descriptive statistics of the membrane properties of iSPNs

Two-way ANOVA of the iSPN membrane properties (P4-P10)

Cholinergic modulation of the neonatal spontaneous activity.

A. Immunostaining for ChAT of the dorsal striatal slices of Oprm1-Cre/Ai14 animals at the age of P7, P21 and P44. Green: ChAT staining, Red: tdTomato. Scale bar 100 µm. The density of fluorescent signal in striosomal domains was normalized to the density of fluorescent signal in the matrix domain of the same image. Data are presented as individual values and mean±SEM; P7: 37 images/5 animals; P21: 49 images/7 animals; P44: 44 images from 3 animals. Statistical analysis: Non-normalized fluorescence density of green signal in striosomal regions was compared to matrix ones, ****p<0.0001, Wilcoxon test.

B. Example traces of APs, recorded in the cell-attached mode from the striosomal iSPN of the P7 A2A-Cre/Ai14 animal during baseline, application of the inhibitor of muscarinic M1 receptor VU0255035 (5 µM) and wash-out.

C. Quantification of AP frequency before and during application of VU0255035 in P4-5 striosomal and matrix dSPNs and P6-7 striosomal and matrix iSPNs. Sample size, dSPNs: 14 striosomal cells, 5 matrix cells, 5 animals; iSPNs: 11 striosomal cells, 10 matrix cells, 8 animals. Data are presented as individual values and mean of the difference with 95% CI, *p<0.05, paired t-test.

D. Quantification of the number of Ca2+ spikes per minute before and during application of VU0255035 in P4-5 striosomal and matrix dSPNs and P6-7 striosomal and matrix iSPNs. Sample size, dSPNs: 18 slices, 3 animals; iSPNs: 29 slices, 6 animals. Data are presented as individual values and mean of the difference with 95% CI, ***p<0.001, ****p<0.0001, paired t-test.

Suppression of the neonatal spontaneous activity during the second week of life causes the decrease in functional GABAergic innervation of SNpc.

A. Schematic illustration of the experimental timeline. Oprm1-Cre animals and wild-type littermates received cranial injection of AAV-hSyn-DIO-hM4D(Gi) alone or together with AAV-hSyn-DIO-EGFP into dorsal striatum at P1. At P6-P14 animals received s.c. injections of CNO or saline twice a day. Animals were sacrificed at the age P52-79 for the electrophysiology and imaging experiments.

B. Representative traces of mIPSCs recorded from SNpc DA neurons of experimental animals, treated either by CNO or by saline.

C. Amplitude and frequency of mIPSCs and capacitance and input resistance of recorded neurons. Sample size, cells/animals: WT CNO 14/9, Oprm1-Cre CNO 9/6, WT saline 7/4, Oprm1-Cre saline 11/7. Data are presented as individual values and mean ± SEM. *p<0.05, ***p<0.001, one-way ANOVA with Holm-Šídák’s multiple comparisons test.

D. Example images of striosome-dendrone bouquets of Oprm1-Cre animals after neonatal CNO or saline treatment. Red: TH staining, green: GFP. Scale bar 50 µm.

E. Example images of individual DA neurons at higher magnification from the same slices as in D. Red: TH staining; green: GFP; purple: gephyrin staining. Scale bar 10 µm. F. Quantification of the relative GFP and gephyrin volumes from the images of individual DA cells. Sample size, saline: 100 cells/20 slides/5 animals; CNO: 121 cell/24 slides/6 animals. Data are presented as violin plots, median: long-dashed line, quartiles: short-dashed lines. *p<0.05, ****p<0.0001, t-test.

Suppression of striosomal activity in juvenile age does not cause the decrease of the functional GABAergic innervation of SNpc DA neurons.

A. Schematic illustration of the experimental timeline. Oprm1-Cre animals and wild-type littermates received cranial injection of AAV-hSyn-DIO-hM4D(Gi) into dorsal striatum at P1. At P21-P29 animals received s.c. injections of CNO twice a day. Animals were sacrificed at the age P52-79 for the electrophysiology experiments.

B. Representative traces of mIPSCs recorded from SNpc DA neurons of Oprm1-Cre or wild type animals, treated by CNO. C. Amplitude and frequency of mIPSCs and capacitance and input resistance of recorded neurons. Sample size, cells/animals: WT CNO 12/3, Oprm1-Cre CNO 12/6. Data are presented as individual values and mean ± SEM. *p<0.05, **p<0.01, t-test.

Blockade of AMPA receptors does not suppress spontaneous neonatal activity.

A. Example traces of the AP firing, recorded in the cell-attached mode from the striosomal iSPN of the P7 A2A-Cre/Ai14 animal, during baseline and NBQX (20 µM) application.

B. Quantification of the AP frequency before and during application of NBQX in neonatal (P4-7) dSPNs and iSPNs. Data are presented as individual values and mean of the difference with 95% CI. dSPNs: 10 cells from 4 animals; iSPNSs: 10 cells from 5 animals. *p<0.05, paired t-test.

Characterization of the hM4D(Gi) expression at P10.

A. Schematic illustration of the experimental timeline. Oprm1-Cre animals received microinjection of AAV-hSyn-DIO-hM4D(Gi) to the dorsal striatum at P1. At P10 animals were sacrificed and used for the immunochemistry or patch-clamp.

B. Coronal slice of dorsal striatum with the expression of mCitrine in striosomes. Red: mCitrine staining; blue: DAPI. Scale bar 500 µm. C. Example trace of membrane potential recording of the striosomal SPN during application of 10 µM CNO.

The dendritic tree of DA neurons was not affected by chemogenetic suppression of the neonatal striosomal activity.

Oprm1-Cre animals and wild-type littermates received cranial injection of AAV-hSyn-DIO-hM4D(Gi) into dorsal striatum at P1. At P6-P14 animals received s.c. injections of CNO twice a day. Adult animals were used for whole-cell patch clamp experiments, where DA neurons in SNpc were filled by biocytin for the analysis of the dendritic tree.

A. Example images of DA cells filled by biocytin from SNpc of adult wild-type and Oprm1-Cre animals after viral transfection and neonatal injections of CNO.

B. Sholl analysis graph showing the number of intersections of the dendrites with different radiuses, starting from the soma. Sample size, cells/animals: WT CNO 11/8, Oprm1-Cre CNO 7/5, WT saline 5/3, Oprm1-Cre saline 9/6. Data are presented as mean ± SEM.