a b c Nature Neuroscience: doi: /nn.3632

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1 c Supplementry Figure 1. The reltion etween stndrd devition (STD) nd men of inter-press intervls (IPIs) under different schedules. -c, Disproportionlly fster decrese of the stndrd devition compred to the men for IPI1 (), IPI2 () nd IPI3 (c) s trining progressed. Note tht the liner fit line (red, ppers distorted under logrithmic scle) devited from the 1:1 line (lck) in ll three cses.

2 FR4/8s FR4/8s CT KO c CT FR4/8s d KO FR4/8s e f g h Sequence length Action efficiency Within-sequence press rte Inter-press intervl

3 Supplementry Figure 2. Impired sequence lerning, decresed ction efficiency nd incresed pressing speed in RGS9L-Cre/Nr1f/f mutnts.,, Exmple of the ehviorl microstructure of of RGS9L-Cre/Nr1f/f mutnt (KO) nd littermte control (CT) mouse under the schedule FR4/8s. This mutnt () exhiited longer lever-press sequences thn its littermte controls (). Ech dot indictes lever press, with red nd lck indicting the first nd finl press of ech individul sequence. The lck nd red solid lines on the X xis represent mgzine entries nd licks, respectively. The lck dshed lines indicte the time of rewrd nd corresponding lever press 2. c, d, Another exmple of microstructure of ehvior of RGS9L-Cre/Nr1f/f mutnt (d), who exhiited shorter lever-press sequences thn littermte controls (c). e-h, Sequence length difference to four (e), ction efficiency (f, percentge of lever presses ctully rewrded), withinsequence press rte (g) nd inter-press intervl (h) for RGS9L-Cre/Nr1f/f mutnts nd controls under FR4/8s. For dt nd sttistics: (e) CT 0.79 ± 0.10 vs. KO 1.50 ± 0.15, t 44 = 3.94, P < 0.001; (f) CT 78.1 ± 2.2 % vs. KO 57.7 ± 4.0 %, t 44 = 4.82, P < 0.001; (g) CT ± 10.2 times/min vs. KO ± 27.7 times/min, t 44 = 6.76, P < ; (h) IPI 1 : CT ± 86.4 ms vs. KO ± ms, t 44 = 3.98, P < 0.001; IPI 2 : CT ± 88.2 ms vs. KO ± ms, t 44 = 2.72, P < 0.01; IPI 3 : CT ± 77.9 ms vs. KO ± ms, t 44 = 3.93, P < 0.001; See Methods for detils.

4 DS d 2 x 8 rry coronl 0.50mm SNr e 2 x 8 rry coronl -3.52mm c GPe f 4 x 4 rry sgittl 2.16mm coronl -0.46mm Supplementry Figure 3. Depiction of electrode plcement confirmed y cresyl violet stining. -f, The electrode rry nd plcement in dorsl stritum (), SNr () nd GPe (c), which were further confirmed y post experimentl stining (d-f). Atls dpted from Pxinos & Frnklin 50.

5 FSI MSN D1-MSN D2-MSN TAN Supplementry Figure 4. Stritl neurons clssifiction., The 3-D plot of stritl units, nd ech dot indictes single unit. Puttive prvlumin-expressing fst-spiking interneurons (FSIs, red), medium-spiny neurons (MSNs. lck) nd choline cetyltrnsferse-expressing toniclly ctive interneurons (TANs, lue) were seprted sed on cell wveform width, pekto-trough rtio nd firing rte. Optogeneticlly identified D1-MSNs (drk green) nd D2-MSNs (drk red) overlpped with the cluster of puttive MSNs recorded from C57BL/6J mice. There were no significnt differences etween D1-MSNs nd D2-MSNs in terms of spike mplitude (t 261 = 0.07, P = 0.94), spike hlf-width (t 261 = 1.30, P = 0.20), spike pek-to-trough rtio (t 261 = 0.19, P = 0.85) or seline firing rte (D1-MSNs 4.5 ± 0.4 Hz, D2-MSNs 4.4 ± 0.5 Hz; t 261 = 0.45, P = 0.63)., Exmple wveforms of FSI (red, left pnel), MSN (lck, left second pnel) nd TAN (lue, right pnel) recorded from C57BL/6J mice, nd D1-MSN (drk green, middle pnel) nd D2-MSN (drk red, right second pnel) recorded from D1-ChR2 nd D2- ChR2 mice respectively. Among the totl stritl units recorded from ll trining sessions of C57BL/6J mice, 3.4% were clssified s puttive FSIs, 5.0% were clssified s TANs nd 91.6% were clssified s MSNs (lso see detiled dt in Supplementry Tles 2-4).

6 c Supplementry Figure 5. Procedure for clssifiction of different types of sequence-relted neuronl ctivity., Decision tree for determining sequence-relted strt/stop, sustined nd inhiited types of neuronl ctivity, s well s potentil sutypes with overlpping response, sed on the firing rte modultion during ech lever press within ction sequence. -c, A typicl exmple of 3-D clssifiction nd isoltion of different response types sed on the principle component nlysis (PCA) of individul-lever-press relted firing rte modultion vector [r1, r2, r3, r4] (see Methods for more detils). The sequence-relted strt, stop nd oundry ctivity were clerly clssified s individul clusters (). The sequence-relted inhiited nd sustined ctivity were further seprted nd highlighted under different view ngle for etter visuliztion (c).

7 MSN: tsk-relted SNr/GPe: tsk-relted c MSN: strt/stop & inhiited d SNr/GPe: strt/stop & inhiited e MSN: strt/stop & sustined f SNr/GPe: strt/stop & sustined g MSN: within-strt/stop h SNr: within-strt/stop i GPe: within-strt/stop Supplementry Figure 6. Sttistics for neurons with complex ctivity in Stritum, SNr nd GPe cross lerning.,, Percentge of generl tsk-relted MSNs () nd SNr/GPe neurons (). c, d, Percentge of sequence-relted neurons displying oth strt/stop nd inhiited ctivity for MSNs (c) nd SNr/GPe neurons (d). e, f, Percentge of sequence-relted neurons showing oth strt/stop nd sustined ctivity for MSNs (e) nd SNr/GPe neurons (f). g-i, Percentge of sequence-relted strt only, stop only nd oundry (i.e. oth strt nd stop) types of neurons for MSNs (g), SNr (h) nd GPe neurons (i).

8 DS SNr c GPe Supplementry Figure 7. Sptil distriution of neurons recorded on different electrodes exhiiting vrious types of sequence-relted ctivity cross regions. -c, Proportion of sequence-relted strt, stop, oundry, inhiited nd sustined ctivity in dorsl stritum (), SNr () nd GPe (c) roughly cross electrodes 1 more lterl to 8 more medil (2x8 rry for DS nd GPe or 1 4, 4x4 rry, for GPe), corresponding to the lterl medil grdient. GPe ppers to hve more even distriution cross electrodes thn SNr nd DS, where there is trend towrd more lterl electrodes to hve higher proportion of sequence-relted ctivity. Note tht the most lterl electrode of SNr is proly t the order of SNr s few cells re recorded.

9 D1-/D2-MSN sequence ctivity D1-/D2-MSN strt/stop ctivity c D1-MSN strt/stop ctivity d D2-MSN strt/stop ctivity e Timing of Strt ctivity

10 Supplementry Figure 8. (-) Percentge of sequence-relted ctivity in puttive D1- nd D2-MSNs using identifiction criteri of light response ltency of less thn or equl to 10ms., Sequence-relted ctivity in D1- vs. D2-MSNs. The results consistently showed tht sequence-relted strt/stop ctivity ws similrly oserved in D1- vs. D2-MSNs (t 23 = 1.02, P = 0.32), ut tht sequence-relted inhiited ctivity ws dominnt in D2- over D1-MSNs (t 23 = 3.89, P < 0.001), while sequence-relted sustined ctivity ws preferentilly implemented in D1- rther thn D2-MSNs (t 23 = 2.24, P < 0.05)., Within strt/stop ctivity in D1- vs. D2- MSNs. While similr percentge of D1-MSNs signled sequence strt vs. stop (t 15 = 0.68, P = 0.50), there ws higher proportion of D2-MSNs signling sequence strt thn sequence stop (t 8 = 3.18, P < 0.01). (c-d) Positive nd negtive firing rte modultion in D1-/D2-MSNs for sequence-relted strt/stop ctivity. The percentge of strt/stop neurons showing positive vs. negtive firing rte modultion, for D1-MSNs (c) nd D2-MSNs (d). (e) Timing of sequencerelted strt ctivity in D1-/D2-MSNs in reltion to sequence initition. The percentge of timing distriution of strt neurons relted to sequence initition for D1-MSNs (red rs, on verge ± 43.7 ms) nd D2-MSNs (lue rs, on verge ± 36.3 ms). There ws no significnt difference in the timing of strt ctivity etween the two neuronl popultion (t 70 = 0.52, P = 0.60).

11 finl finl unit #16 c d FSI tsk-relted FSI sequence ctivity FSI within-strt/stop e finl finl unit #32 f g h TAN tsk-relted TAN sequence ctivity TAN within-strt/stop

12 Supplementry Figure 9. Action sequence-relted ctivity in puttive stritl interneurons., PETH of fst-spiking interneuron - puttive stritl prvlumin-expressing neuron (FSI) showing sustined firing ctivity throughout the whole ction sequence. The FSI exhiited high seline firing rte with nrrow wveform (downright pnel). -d, Sttistics of percentge of stritl FSIs shows generl tsk-relted ctivity (), or sequence-relted ctivity (c), or sequence-relted strt only, stop only nd oth strt nd stop ctivity (d). e, PETH of tonicllyctive interneurons - puttive stritl choline cetyltrnsferse-expressing (TAN) showing inhiited firing ctivity throughout the whole ction sequence. The TANs exhiited moderte seline firing with very wide wveform (downright pnel). f-h, Proportion of stritl TANs showing generl tsk-relted ctivity (f), sequence-relted ctivity (g), nd sequence-relted strt only, stop only nd oth strt nd stop ctivity (h). Different types of puttive stritl interneurons showed distinct sequence-relted ctivity; while significnt proportion of oth FSIs nd TANs exhiited sequence-relted strt/stop ctivity, FSIs showed minly sustined ctivity nd TANs mostly displyed inhiited ctivity throughout the whole sequence.

13 M1 sequence ctivity M1 strt/stop ctivity c Sequence Initition/termintion d Sequence Execution strt/stop strt/stop > inhiited > sustined strt/stop > sustined strt/stop _ Inhiition Excittion > inhiited Supplementry Figure 10. (-) Sequence-relted neuronl ctivity in primry motor cortex (M1)., Percentge of tsk-relted M1 neurons showing sequence-relted strt/stop, inhiited or sustined ctivity., Distriution of percentge of strt only, stop only nd oundry (oth strt nd stop) sutypes of ctivity in M1 sequence-relted strt/stop neurons. (c-d) Digrm of two different opertion modes of sl gngli direct/indirect pthwys during sequence initition/termintion vs. sequence execution. c, Phsic co-ctivtion of D1- nd D2-MSNs during sequence initition/termintion. d, Sustined ctivity dominted in D1-MSNs nd inhiited ctivity dominted in D2-MSNs during sequence execution. The downstrem nuclei SNr nd GPe demonstrted corresponding ctivity in consistent wy.

14 Suppl Tle 1. Numer of C57BL/6J mice with electrode rrys implnttion Are side DS + SNr L + L DS + SNr R + R SNr L DS + GPe L + R GPe L + R DS L + R DS + M1 L + R N Suppl Tle 1. Numer of cells recorded per session in different regions in C57BL/6J mice Are Averge numer of cells per session/schedule Electrodes M Mice DS SNr GPe Suppl Tle 1c. Numer of puttive interneurons recorded in C57BL/6J mice Puttive cell type Cells Sessions Electrodes Mice FSI 33* TAN 48* * Only count the numer of cells recorded from different electrodes in ny given mouse. Suppl Tle 1d. Numer of MSNs identified in D1-/D2-cre mice Cre line Cells Sessions Electrodes Mice D * 80 5 D * * Only include those sessions successfully identified >3 cre-positive neurons per session. Supplementry Movie 1. Mice cn lern to perform very rpid ction sequences. An exmple of electrode-implnted mouse performing rpid ction sequence under FR4/1s with simultneous neurl recording.