Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( C , D ) ( C ) Diagram showing the extent of ratchet - like constrictions . The mean difference in apical area between adjacent peaks ( relaxed cell surface ) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse . Cells in the non - activated region ( NR ) constricted on average more than 5 µm2 , which is characteristic for ratchet - like constriction . Non - constricting cells in the activated region ( AR , non - con . ) displayed a mean difference in apical area per pulse of about 0 , reflecting non - ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size . Constricting cells in the activated region ( AR , con . ) show an intermediate behavior with less efficient constrictions than in the NR . Data points are significantly different as determined by ANOVA ( F ( 2 , 539 ) = 72 . 04 , p = 1 . 9e - 28 ) and post - hoc Tukey test . ( D ) The pulsation period ( T ) . All cells pulse with a period of about 90 s . There were no statistically significant differences between group means as determined by one - way ANOVA ( F ( 2 , 540 ) = 1 . 438 , p = 0 . 24 ) . ( C and D ) Red dots indicate the mean values and the error bars represent the standard deviation . More than 500 pulses ( nNR = 119 , nAR , non - con . = 128 , nAR , con = 295 ) of more than 100 cells were analyzed . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (C, D) (C) Diagram showing the extent of ratchet-like constrictions. The mean difference in apical area between adjacent peaks (relaxed cell surface) is indicated by a red dot and reflects the extent to which cells shrink in a single pulse. Cells in the non-activated region (NR) constricted on average more than 5 µm2, which is characteristic for ratchet-like constriction. Non-constricting cells in the activated region (AR, non-con.) displayed a mean difference in apical area per pulse of about 0, reflecting non-ratchet constrictions meaning that pulsing cells constricted but relaxed subsequently back to their initial size. Constricting cells in the activated region (AR, con.) show an intermediate behavior with less efficient constrictions than in the NR. Data points are significantly different as determined by ANOVA (F(2,539)=72.04, p=1.9e-28) and post-hoc Tukey test. (D) The pulsation period (T). All cells pulse with a period of about 90 s. There were no statistically significant differences between group means as determined by one-way ANOVA (F(2,540)=1.438, p=0.24). (C and D) Red dots indicate the mean values and the error bars represent the standard deviation. More than 500 pulses (nNR = 119, nAR, non-con. = 128, nAR, con = 295) of more than 100 cells were analyzed. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( E ) Diagram showing the prevalence of cells in the photo - activated region that constricted ( con . ) and that did not constrict ( Non - con . ) . More than 4 / 5 of the cells did not constrict to half of the initial area ( N = 5 embryos ; n : 407 cells ; Cohen ' s D : d = 10 ) . In each box plot , the central mark , the bottom , and the top edge of each box indicates the median , the 25th and 75th percentile , respectively . Whiskers extent to the most extreme data point . Data information : In all panels , * * * * indicates p ≤ 0 . 0001 , and n . s . indicates no statistically significant differences according to two - sample t - Test .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (E) Diagram showing the prevalence of cells in the photo-activated region that constricted (con.) and that did not constrict (Non-con.). More than 4/5 of the cells did not constrict to half of the initial area (N = 5 embryos; n: 407 cells; Cohen's D: d = 10). In each box plot, the central mark, the bottom, and the top edge of each box indicates the median, the 25th and 75th percentile, respectively. Whiskers extent to the most extreme data point. Data information: In all panels, **** indicates p ≤ 0.0001, and n.s. indicates no statistically significant differences according to two-sample t-Test.


Embryos expressing RhoGEF2 - CRY2 / CIBN : : GFPpm and the membrane marker GAP43 : : mCherry were mounted with the ventral epithelium facing the objective . To analyze apical dynamics at each activation cycle , the photo - activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm - sized apical image stack resulting in a total time resolution of 35 s . The basal surface of the anterior ( left ) half of the embryo was photo - activated . ( F , G ) Graphs showing the average apical area ( F ) and anisotropy ( G ) of cells in the non - activated region ( green ) , in the activated region that constricted ( purple ) and that did not constrict ( red ) . The straight line indicates the mean value at a given time - point , the semi - transparent colored area indicated the respective standard deviation . Cells in the non - activated region constricted efficiently and progressively acquired anisotropy . Constricting cells in the activated region constricted slower in an isotropic manner . Non - constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy ( nNR = 34 , nAR , non - con . = 20 , nAR , con = 59 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Embryos expressing RhoGEF2-CRY2/CIBN::GFPpm and the membrane marker GAP43::mCherry were mounted with the ventral epithelium facing the objective. To analyze apical dynamics at each activation cycle, the photo-activation time was limited to ~ 20 s and was followed by the acquisition of a 5 µm-sized apical image stack resulting in a total time resolution of 35 s. The basal surface of the anterior (left) half of the embryo was photo-activated. (F,G) Graphs showing the average apical area (F) and anisotropy (G) of cells in the non-activated region (green), in the activated region that constricted (purple) and that did not constrict (red). The straight line indicates the mean value at a given time-point, the semi-transparent colored area indicated the respective standard deviation. Cells in the non-activated region constricted efficiently and progressively acquired anisotropy. Constricting cells in the activated region constricted slower in an isotropic manner. Non-constricting cells in the activated region did not show net decrease in apical area and gain of anisotropy (nNR = 34, nAR, non-con. = 20, nAR, con = 59).


( C - E ) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2 - CRY2 : : mCherry were imaged during late cellularization . ( C ) z - projections of confocal image stacks show the localization of the respective GFP - tagged optogenetic anchor . Please note that the construct PatJ - CIBN ( indicated by asterisk ) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2 - CRY2 : : mCh . All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation . Scale bars , 20 μm . ( D ) A region of interest ( right side of the dashed line ) in a 10 µm - sized stack was photo - activated using two - photon illumination for 30 s before acquiring the RhoGEF2 - CRY2 : : mCherry signal using confocal microscopy . All anchor proteins were able to recruit RhoGEF2 - CRY2 : : mCherry specifically to the activated region . Scale bars , 10 μm . ( E ) Although all basal anchors were effective at recruiting RhoGEF2 - CRY2 : : mCherry to the cell base , only PatJ - CIBN : : GFP - pm resulted in effective myosin - II recruitment and actomyosin ring constriction

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C-E) Embryos expressing either of the optogenetic anchor proteins and RhoGEF2-CRY2::mCherry were imaged during late cellularization. (C) z-projections of confocal image stacks show the localization of the respective GFP-tagged optogenetic anchor. Please note that the construct PatJ-CIBN (indicated by asterisk) lacks a fluorescent marker and its localization was thus inferred from the recruited RhoGEF2-CRY2::mCh. All anchor proteins showed predominantly basal localization during cellularization and at the onset of ventral furrow formation. Scale bars, 20 μm. (D) A region of interest (right side of the dashed line) in a 10 µm-sized stack was photo-activated using two-photon illumination for 30 s before acquiring the RhoGEF2-CRY2::mCherry signal using confocal microscopy. All anchor proteins were able to recruit RhoGEF2-CRY2::mCherry specifically to the activated region. Scale bars, 10 μm. (E) Although all basal anchors were effective at recruiting RhoGEF2-CRY2::mCherry to the cell base, only PatJ-CIBN::GFP-pm resulted in effective myosin-II recruitment and actomyosin ring constriction


( A ) Localization of the basal - specific optogenetic anchor CIBN - PatJ : : GFP - pm during ventral furrow formation . Embryos expressing CIBN - PatJ : : GFP - pm were imaged using two - photon excitation during ventral furrow formation . Transverse cross section ( z - projection ) at a stage when cells were already apically constricted and elongated , and the ventral tissue already internalized .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Localization of the basal-specific optogenetic anchor CIBN-PatJ::GFP-pm during ventral furrow formation. Embryos expressing CIBN-PatJ::GFP-pm were imaged using two-photon excitation during ventral furrow formation. Transverse cross section (z-projection) at a stage when cells were already apically constricted and elongated, and the ventral tissue already internalized.


Embryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY2 and Sqh : : mCherry ( B , C were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation . ( B ) myosin - II distribution in a cross section ( z - projection ) 3 min after photo - activation . myosin - II was locally recruited to the base as indicated by the red arrowheads in the activated region ( red line ) . ( C ) Top view ( sum of slice of 4 µm - sized image stack ) showing basal myosin - II recruitment in the photo - activated region ( red dashed square ) Data information : Scale bars , 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY2 and Sqh::mCherry (B,C were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation. (B) myosin-II distribution in a cross section (z-projection) 3 min after photo-activation. myosin-II was locally recruited to the base as indicated by the red arrowheads in the activated region (red line). (C) Top view (sum of slice of 4 µm-sized image stack) showing basal myosin-II recruitment in the photo-activated region (red dashed square) Data information: Scale bars, 20 µm


Embryos co - expressing CIBN - PatJ : : GFP - pm , RhoGEF2 - CRY an the membrane marker GAP43 : : mCherry ( D - I ) were mounted with the ventral tissue facing the objective . After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo - activated using two - photon illumination and the myosin - II signal was recorded in alternation ( D ) Cross section ( z - projection ) showing the plasma membrane and ( E ) top view showing the apical plasma membrane at the initial time point before photo - activation . At this stage , the central mesodermal tissue has started to internalize and cells were constricted at the apical surface . In an alternating fashion , the base of the cells in the anterior half of the embryo ( left ) was photo - activated for 100 sec and the GAP43 : : mCherry signal in the entire field of view was recorded . The furrow further ingressed and closed completely 3 min ( F ) and 6 min ( G ) after initial activation , respectively . In the activated region , the tissue failed to internalize and to form a closed furrow both at 3 min ( H ) and 6 min ( I ) after initial photo - activation . The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm ( D ) . In the non - activated region 6 min after initial activation the width of the tissue shrank to about 25 µm ( G ) , whereas it remained constant at 40 µm in the activated region ( I ) . Data information : Scale bars , 20

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Embryos co-expressing CIBN-PatJ::GFP-pm, RhoGEF2-CRY an the membrane marker GAP43::mCherry (D-I) were mounted with the ventral tissue facing the objective. After cells underwent apical constriction and cell elongation the basal volume of the ventral cells was photo-activated using two-photon illumination and the myosin-II signal was recorded in alternation (D) Cross section (z-projection) showing the plasma membrane and (E) top view showing the apical plasma membrane at the initial time point before photo-activation. At this stage, the central mesodermal tissue has started to internalize and cells were constricted at the apical surface. In an alternating fashion, the base of the cells in the anterior half of the embryo (left) was photo-activated for 100 sec and the GAP43::mCherry signal in the entire field of view was recorded. The furrow further ingressed and closed completely 3 min (F) and 6 min (G) after initial activation, respectively. In the activated region, the tissue failed to internalize and to form a closed furrow both at 3 min (H) and 6 min (I) after initial photo-activation. The width of the central tissue forming the ventral furrow at the initial point of the experiment is 40 µm (D). In the non-activated region 6 min after initial activation the width of the tissue shrank to about 25 µm (G), whereas it remained constant at 40 µm in the activated region (I). Data information: Scale bars, 20 µm


( A ) Immunoblot showing the activation of PI3K / AKT pathway ( by assessing the levels of p - AKT Ser473 ) of ECs 24h after induction of Pik3caH1047R expression .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Immunoblot showing the activation of PI3K/AKT pathway (by assessing the levels of p-AKT Ser473) of ECs 24h after induction of Pik3caH1047R expression.


( B ) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression . Differentially expressed genes ( DEGs ) ( p adj < 0 . 05 ) in Pik3caH1047R over Pik3caWT ECs at 24h ( grey , unchanged ; orange , downregulated ; purple , upregulated ) . n = 4 biological replicates per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Volcano plot of genes analysed by RNAseq in ECs upon Pik3caH1047R expression. Differentially expressed genes (DEGs) (p adj < 0.05) in Pik3caH1047R over Pik3caWT ECs at 24h (grey, unchanged; orange, downregulated; purple, upregulated). n=4 biological replicates per genotype.


( D ) Immunoblot showing the activation of PI3K / AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation ( - ) and 30 min stimulation with 20 % fetal bovine serum ( FBS ) ( + ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunoblot showing the activation of PI3K/AKT pathway of Pik3caWT and Pik3caH1047R ECs after 2h starvation (-) and 30 min stimulation with 20% fetal bovine serum (FBS) (+).


( E , F ) ( E ) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum ( - ) or 20 % serum ( + ) for 24h . ( F ) Percentage of proliferative ECs ( in S and G2M phases ) from Fig 1E . Error bars are s . e . m . n ≥ 3 biological replicates per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 and * * * * p < 0 . 0001 were considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E, F) (E) Cell cycle analysis by flow cytometry of Pik3caWT and Pik3caH1047R ECs after cultured either without serum (-) or 20% serum (+) for 24h. (F) Percentage of proliferative ECs (in S and G2M phases) from Fig 1E. Error bars are s.e.m. n ≥ 3 biological replicates per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 and ****p < 0.0001 were considered statistically significant.


( B ) Representative images of control and EC - Pik3caH1047R / WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels . Scale bars : 150 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative images of control and EC-Pik3caH1047R/WT mouse retinas isolated at indicated time points stained with IB4 for blood vessels. Scale bars: 150 μm.


( D ) Quantification of IB4 - positive areas per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Quantification of IB4-positive areas per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype.


( E ) Representative images of EC - mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP . Scale bars : 150 μm . ( F ) Quantification of GFP / IB4 ratio per retina . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E) Representative images of EC-mTmG mouse retinas isolated at indicated time points stained with IB4 and GFP. Scale bars: 150 μm. (F) Quantification of GFP/IB4 ratio per retina. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( A , B ) Representative images of EC - Pik3caH1047R / WT ( A ) and EC - mTmG ( B ) P6 retinas from mice treated with decreasing doses of 4 - OHT on P1 . Retinas were stained for blood vessels ( IB4 ) and GFP as indicated . Scale bars : 150 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A, B) Representative images of EC-Pik3caH1047R/WT (A) and EC-mTmG (B) P6 retinas from mice treated with decreasing doses of 4-OHT on P1. Retinas were stained for blood vessels (IB4) and GFP as indicated. Scale bars: 150 μm.


( C ) Quantification of GFP / IB4 ratio of EC - mTmG retinas . Error bars are s . e . m . n ≥ 3 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Quantification of GFP/IB4 ratio of EC-mTmG retinas. Error bars are s.e.m. n ≥ 3 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( D ) Quantification of IB4 - positive area per retina in Pik3caH1047R / WT retinas . Data presented as a percentage of the control for each 4 - OHT dose . Error bars are s . e . m . n = 4 retinas per genotype . Data information : Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 was considered statistically significant .

(D) Quantification of IB4-positive area per retina in Pik3caH1047R/WT retinas. Data presented as a percentage of the control for each 4-OHT dose. Error bars are s.e.m. n = 4 retinas per genotype. Data information: Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 was considered statistically significant.


( A ) Representative images of EC - Pik3caH1047R / WT P6 retinas isolated from mice treated with 0 . 125 mg / kg of 4 - OHT on P1 . Retinas were immunostained for blood vessels ( IB4 ) . Red asterisks show arteries and arterioles and yellow arrowheads veins . Scale bars : 150 μm ( left panel ) and 30 μm ( right , high magnification panels ) . ( B ) Representative images of P6 retinas from control and EC - Pik3caH1047R / WT mice treated with 0 . 125 mg / kg 4 - OHT on P1 , following immunostaining for p - S6 ( S235 / 236 ) and blood vessels ( IB4 ) . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( C ) Representative control and EC - Pik3caH1047R / WT P6 retinas immunostained for blood vessels ( IB4 ) , EC nuclei ( Erg ) and EdU . Scale bars : 150 μm ( left panels ) and 30 μm ( right panels , high magnification ) . ( D - G ) Quantification of ( D ) p - S6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) , ( E ) EC proliferation by EdU staining , ( F ) EC number by Erg positive cells and ( G ) retinal vascularity by IB4 - positive area in control and EC - Pik3caH1047R / WT retinas . Error bars are s . e . m . n > 5 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * * p < 0 . 01 was considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A) Representative images of EC-Pik3caH1047R/WT P6 retinas isolated from mice treated with 0.125 mg/kg of 4-OHT on P1. Retinas were immunostained for blood vessels (IB4). Red asterisks show arteries and arterioles and yellow arrowheads veins. Scale bars: 150 μm (left panel) and 30 μm (right, high magnification panels). (B) Representative images of P6 retinas from control and EC-Pik3caH1047R/WT mice treated with 0.125 mg/kg 4-OHT on P1, following immunostaining for p-S6 (S235/236) and blood vessels (IB4). Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (C) Representative control and EC-Pik3caH1047R/WT P6 retinas immunostained for blood vessels (IB4), EC nuclei (Erg) and EdU. Scale bars: 150 μm (left panels) and 30 μm (right panels, high magnification). (D-G) Quantification of (D) p-S6 (S235/236) intensity (presented as a fold change of vehicle-treated control), (E) EC proliferation by EdU staining, (F) EC number by Erg positive cells and (G) retinal vascularity by IB4-positive area in control and EC-Pik3caH1047R/WT retinas. Error bars are s.e.m. n > 5 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. **p < 0.01 was considered statistically significant.


( B , C ) Representative images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panel ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Representative images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panel) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 3 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 and * * p < 0 . 01 were considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 3 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05 and **p < 0.01 were considered statistically significant.


( B , C ) Representative confocal images of P6 retinas isolated from control and EC - Pik3caH1047R / WT mouse littermates . Blood vessels were stained with IB4 . Lower panels showing high magnification images of the representative areas showing ( B ) blood vessels ( IB4 ) , EC nuclei ( Erg ) , EdU incorporation and ( C ) pS6 ( S235 / 236 ) . Scale bars : 150 μm ( upper panels ) and 30 μm ( lower panels ) . ( D - G ) Quantification of ( D ) retinal vascularity by IB4 staining , ( E ) EC number by Erg immunostaining , ( F ) EC proliferation by EdU staining , and ( G ) pS6 ( S235 / 236 ) intensity ( presented as a fold change of vehicle - treated control ) . Bars represent the mean ± s . e . m . n ≥ 4 retinas per genotype . Statistical analysis was performed by nonparametric Mann - Whitney test . * p < 0 . 05 , * * p < 0 . 01 and * * * p < 0 . 001 were considered statistically significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B, C) Representative confocal images of P6 retinas isolated from control and EC-Pik3caH1047R/WT mouse littermates. Blood vessels were stained with IB4. Lower panels showing high magnification images of the representative areas showing (B) blood vessels (IB4), EC nuclei (Erg), EdU incorporation and (C) pS6 (S235/236). Scale bars: 150 μm (upper panels) and 30 μm (lower panels). (D-G) Quantification of (D) retinal vascularity by IB4 staining, (E) EC number by Erg immunostaining, (F) EC proliferation by EdU staining, and (G) pS6 (S235/236) intensity (presented as a fold change of vehicle-treated control). Bars represent the mean ± s.e.m. n ≥ 4 retinas per genotype. Statistical analysis was performed by nonparametric Mann-Whitney test. *p < 0.05, **p < 0.01 and ***p < 0.001 were considered statistically significant.


( B ) Representative sequencing chromatograms of PIK3CA and TEK mutant VM - derived ECs . Arrows show the detected point mutations .

O O O O O O O O O O O O O O O O O O O O O O O

(B) Representative sequencing chromatograms of PIK3CA and TEK mutant VM-derived ECs. Arrows show the detected point mutations.


( C ) Representative confocal images of PIK3CA and TEK patient - derived ECs immunostained for VE - cadherin ( EC - specific junctional protein ) and ERG ( EC - specific transcription factor ) . Cell nuclei were visualised with DAPI . Scale bars : 30 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Representative confocal images of PIK3CA and TEK patient-derived ECs immunostained for VE-cadherin (EC-specific junctional protein) and ERG (EC-specific transcription factor). Cell nuclei were visualised with DAPI. Scale bars: 30 μm.


( D ) Immunoblot showing the activation of PI3K / AKT / mTORC1 pathway ( by assessing the levels of p - AKT and p - S6 ) among different PIK3CA and TEK patient - derived ECs . Primary HUVEC were used as wild - type control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D) Immunoblot showing the activation of PI3K/AKT/mTORC1 pathway (by assessing the levels of p-AKT and p-S6) among different PIK3CA and TEK patient-derived ECs. Primary HUVEC were used as wild-type control.


( E - F ) Immunoblot showing the impact of miransertib at increasing doses on PI3K / AKT / mTORC1 pathway ( by assessing p - AKT and p - S6 levels ) in ( E ) PIK3CA and ( F ) TEK mutant patient - derived ECs .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(E-F) Immunoblot showing the impact of miransertib at increasing doses on PI3K/AKT/mTORC1 pathway (by assessing p-AKT and p-S6 levels) in (E) PIK3CA and (F) TEK mutant patient-derived ECs.


( G ) Wild - type ( HUVECs ) , PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay . Fitting curves and 95 % CI IC50 values for wild - type ( HUVECs ) , PIK3CA and TEK ECs are shown . For PIK3CA and TEK - mutant ECs n = 3 biological replicates ( patients ) ; for wild - type ECs ( HUVECs ) n = 4 technical replicates PIK3CA - mutant vs . TEK - mutant p = 0 . 501 ; PIK3CA - mutant vs . HUVECs p = 0 . 0815 ; TEK - mutant vs . HUVECs : p = 0 . 4085 . Statistical analysis was performed by comparison of best - fit values using the extra sum - of - squares F test .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Wild-type (HUVECs), PIK3CA and TEK mutant EC viability upon the treatment with miransertib for 72h at different doses assessed by MTS assay. Fitting curves and 95% CI IC50 values for wild-type (HUVECs), PIK3CA and TEK ECs are shown. For PIK3CA and TEK-mutant ECs n = 3 biological replicates (patients); for wild-type ECs (HUVECs) n = 4 technical replicates PIK3CA-mutant vs. TEK-mutant p = 0.501; PIK3CA-mutant vs. HUVECs p = 0.0815; TEK-mutant vs. HUVECs: p = 0.4085. Statistical analysis was performed by comparison of best-fit values using the extra sum-of-squares F test.


A . Experimental demonstration . A1 . Illustration of the mapping principle between ChR2 - expressed PV neurons ( green ) and regular pyramidal neuron ( Pyr , orange ) . A2 . Acute slices were obtained and recorded at ZT0 or ZT12 . A3 . Coronal brain atlas showing the primary visual cortex ( Top left panel , modified from MBL mouse brain atlas ) . Yellow box highlights the region of experiment . Top right panel , the reference image taken with a 10x objective from a slice prepared from a PV : Ai32 mouse . The 0 . 45 mm2 shaded cubic area was divided into 15 x 15 grids for independent light stimulation . The white arrowhead indicates the position of the recording electrode . Bottom panel , representative raw traces of light - evoked IPSCs ( LEIPSCs ) recorded from one pyramidal neuron . A4 . The spatial distribution of the LEIPSCs from the example in A3 . A5 . The heatmap of the LEIPSCs in A4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Experimental demonstration. A1. Illustration of the mapping principle between ChR2-expressed PV neurons (green) and regular pyramidal neuron (Pyr, orange). A2. Acute slices were obtained and recorded at ZT0 or ZT12. A3. Coronal brain atlas showing the primary visual cortex (Top left panel, modified from MBL mouse brain atlas). Yellow box highlights the region of experiment. Top right panel, the reference image taken with a 10x objective from a slice prepared from a PV:Ai32 mouse. The 0.45 mm2 shaded cubic area was divided into 15 x 15 grids for independent light stimulation. The white arrowhead indicates the position of the recording electrode. Bottom panel, representative raw traces of light-evoked IPSCs (LEIPSCs) recorded from one pyramidal neuron. A4. The spatial distribution of the LEIPSCs from the example in A3. A5. The heatmap of the LEIPSCs in A4.


B . The average heatmaps at ZT0 and ZT12 after soma alignment . Soma position was indicated by the blue arrow head . C . Comparison of the cumulative distribution ( Kolmogorov - Smirnov test [ K - S test ] ; P = 0 . 002 ) and the mean ( Inset . ZT0 : 813 . 3 ± 53 . 13 pA ; ZT12 : 1306 ± 93 . 73 pA ; P < 0 . 0001 ; Mann - Whitney U test ) of the max LEIPSC amplitude between ZT0 and ZT12 . D . Comparison of the cumulative distribution ( K - S test , P = 0 . 0016 ) and the mean ( Inset . ZT0 : 10 . 44 ± 1 . 44 nA ; ZT12 : 18 . 87 ± 1 . 44 nA ; P < 0 . 0001 ; Mann - Whitney U test ) of the sum LEIPSC amplitude between ZT0 and ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 cells from 9 mice ; ZT12 : 51 cells from 9 mice . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. The average heatmaps at ZT0 and ZT12 after soma alignment. Soma position was indicated by the blue arrow head. C. Comparison of the cumulative distribution (Kolmogorov-Smirnov test [K-S test]; P = 0.002) and the mean (Inset. ZT0: 813.3 ± 53.13 pA; ZT12: 1306 ± 93.73 pA; P < 0.0001; Mann-Whitney U test) of the max LEIPSC amplitude between ZT0 and ZT12. D. Comparison of the cumulative distribution (K-S test, P = 0.0016) and the mean (Inset. ZT0: 10.44 ± 1.44 nA; ZT12: 18.87 ± 1.44 nA; P < 0.0001; Mann-Whitney U test) of the sum LEIPSC amplitude between ZT0 and ZT12. Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


E . The average heatmaps of LEIPSCs after pia aligned to the reference image in A3 . Dark and light blue overlay traces on the side of the map depicted the marginal distribution of the sum LEIPSCs for ZT0 ( Dark blue ) and ZT12 ( Light blue ) . F . Comparison of the cumulative distribution ( K - S test , P = 0 . 0021 ) and the mean ( Inset , ZT0 : 0 . 067 ± 0 . 004 mm2 ; ZT12 : 0 . 089 ± 0 . 0036 mm2 ; P = 0 . 0003 ; Mann - Whitney U test ) inhibitory input size between ZT0 and ZT12 . Data information : Sample sizes used in this figure : ZT0 : 48 cells from 9 mice ; ZT12 : 51 cells from 9 mice . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. The average heatmaps of LEIPSCs after pia aligned to the reference image in A3. Dark and light blue overlay traces on the side of the map depicted the marginal distribution of the sum LEIPSCs for ZT0 (Dark blue) and ZT12 (Light blue). F. Comparison of the cumulative distribution (K-S test, P = 0.0021) and the mean (Inset, ZT0: 0.067 ± 0.004 mm2; ZT12: 0.089 ± 0.0036 mm2; P = 0.0003; Mann-Whitney U test) inhibitory input size between ZT0 and ZT12. Data information: Sample sizes used in this figure: ZT0: 48 cells from 9 mice; ZT12: 51 cells from 9 mice. Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


A . mEPSC frequency was higher at ZT12 than ZT0 . Top : example traces of mEPSC recordings . Bottom left : mEPSC frequency ( ZT0 : 12 . 17 ± 0 . 94Hz ; ZT12 : 17 . 03 ± 1 . 20Hz ; P = 0 . 0036 ; Mann - Whitney U test ) . Bottom right : cumulative probability histogram of the inter - event intervals ( K - S test , P < 0 . 0001 ) . B . mIPSC frequency was lower at ZT12 than ZT0 . Top : example traces . Bottom left : mIPSC frequency ( ZT0 : 19 . 15 ± 0 . 99Hz ; ZT12 : 15 . 19 ± 0 . 63Hz ; P = 0 . 0042 ; Mann - Whitney U test ) . Bottom right : cumulative probability histogram of the inter - event intervals of mIPSCs ( K - S test , P < 0 . 0001 ) . C . mEPSC amplitude and kinetics did not differ between ZT0 and ZT12 . Top : average traces of the well - isolated events . Bottom left : mEPSC amplitude ( ZT0 : 20 . 51 ± 0 . 78pA ; ZT12 : 21 . 26 ± 0 . 75pA ; P = 0 . 34 ; Mann - Whitney U test ) . Bottom right : The distribution of mEPSC amplitude of all events ( K - S test , P < 0 . 01 ) . D . mIPSC amplitude and kinetics did not differ between ZT0 and ZT12 . Top : average traces of the well - isolated mIPSCs . Bottom left : mIPSC amplitude ( ZT0 : 41 . 31 ± 1 . 91pA ; ZT12 : 44 . 39 ± 1 . 31pA ; P = 0 . 35 ; Mann - Whitney U test ) . Bottom right : The cumulative distribution of mIPSC amplitudes ( K - S test , P < 0 . 0001 Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. mEPSC frequency was higher at ZT12 than ZT0. Top: example traces of mEPSC recordings. Bottom left: mEPSC frequency (ZT0: 12.17 ± 0.94Hz; ZT12: 17.03 ± 1.20Hz; P = 0.0036; Mann-Whitney U test). Bottom right: cumulative probability histogram of the inter-event intervals (K-S test, P < 0.0001). B. mIPSC frequency was lower at ZT12 than ZT0. Top: example traces. Bottom left: mIPSC frequency (ZT0: 19.15 ± 0.99Hz; ZT12: 15.19 ± 0.63Hz; P = 0.0042; Mann-Whitney U test). Bottom right: cumulative probability histogram of the inter-event intervals of mIPSCs (K-S test, P < 0.0001). C. mEPSC amplitude and kinetics did not differ between ZT0 and ZT12. Top: average traces of the well-isolated events. Bottom left: mEPSC amplitude (ZT0: 20.51 ± 0.78pA; ZT12: 21.26 ± 0.75pA; P = 0.34; Mann-Whitney U test). Bottom right: The distribution of mEPSC amplitude of all events (K-S test, P < 0.01). D. mIPSC amplitude and kinetics did not differ between ZT0 and ZT12. Top: average traces of the well-isolated mIPSCs. Bottom left: mIPSC amplitude (ZT0: 41.31 ± 1.91pA; ZT12: 44.39 ± 1.31pA; P = 0.35; Mann-Whitney U test). Bottom right: The cumulative distribution of mIPSC amplitudes (K-S test, P < 0.0001 Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01.


B . Charges of sEPSCs recorded at light phase were higher compared to those at dark phase ( ZT0 : 4 . 21 ± 0 . 28 nC ; ZT4 : 7 . 05 ± 0 . 61 nC ; ZT8 : 7 . 74 ± 0 . 63 nC ; ZT12 : 7 . 81 ± 0 . 59 nC ; ZT18 : 5 . 25 ± 0 . 53 nC ; One - way ANOVA , F ( 4 , 88 ) = 8 . 78 , P < 0 . 0001 , Holm - Sidak post - hoc analysis ) . A copy of the 24 - h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm . C . Charges of sIPSCs oscillate during the light / dark cycle ( ZT0 : 45 . 73 ± 2 . 99 nC ; ZT4 : 28 . 85 ± 1 . 76 nC ; ZT8 : 28 . 25 ± 2 . 77 nC ; ZT12 : 28 . 04 ± 2 . 15 nC ; ZT18 : 43 . 37 ± 2 . 42 nC ; One - way ANOVA , F ( 4 , 106 ) = 13 . 65 , P < 0 . 0001 , Holm - Sidak post - hoc analysis ) . A copy of the 24 - h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Charges of sEPSCs recorded at light phase were higher compared to those at dark phase (ZT0: 4.21 ± 0.28 nC; ZT4: 7.05 ± 0.61 nC; ZT8: 7.74 ± 0.63 nC; ZT12: 7.81 ± 0.59 nC; ZT18: 5.25 ± 0.53 nC; One-way ANOVA, F(4,88) = 8.78, P <0.0001, Holm-Sidak post-hoc analysis). A copy of the 24-h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm. C. Charges of sIPSCs oscillate during the light/dark cycle (ZT0: 45.73 ± 2.99 nC; ZT4: 28.85 ± 1.76 nC; ZT8: 28.25 ± 2.77 nC; ZT12: 28.04 ± 2.15 nC; ZT18: 43.37 ± 2.42 nC; One-way ANOVA, F(4,106) = 13.65, P <0.0001, Holm-Sidak post-hoc analysis). A copy of the 24-h sEPSCs was drawn with a dashed line to demonstrate the daily rhythm. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


E . Dark exposure prevented the increase in sEPSCs that normally occurs at ZT12 ( ZT12 as reported in B ; Dark - ZT12 : 5 . 46 ± 0 . 43 nC ; P = 0 . 0082 ; Mann - Whitney U test ) . F . Dark exposure did not significantly affect the reduction of sIPSCs ( ZT12 as reported in C ; Dark - ZT12 : 33 . 43 ± 1 . 74 nC ; P = 0 . 07 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Dark exposure prevented the increase in sEPSCs that normally occurs at ZT12 (ZT12 as reported in B; Dark-ZT12: 5.46 ± 0.43 nC; P = 0.0082; Mann-Whitney U test). F. Dark exposure did not significantly affect the reduction of sIPSCs (ZT12 as reported in C; Dark-ZT12: 33.43 ± 1.74 nC; P = 0.07; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


H . Sleep deprivation further increased sEPSCs compared to ad lib sleep mice ( ZT4 as reported in B ; SD - ZT4 : 11 . 22 ± 0 . 92 nC ; P = 0 . 0013 ; Mann - Whitney U test ) . I . Sleep deprivation abolished the reduction of sIPSC ( ZT4 as reported in C ; SD - ZT4 : 47 . 13 ± 3 . 39 nC ; P < 0 . 0001 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H. Sleep deprivation further increased sEPSCs compared to ad lib sleep mice (ZT4 as reported in B; SD-ZT4: 11.22 ± 0.92 nC; P = 0.0013; Mann-Whitney U test). I. Sleep deprivation abolished the reduction of sIPSC (ZT4 as reported in C; SD-ZT4: 47.13 ± 3.39 nC; P < 0.0001; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


B - E . Slices were obtained at ZT12 ( B - C ) or ZT0 ( D - E ) and pre - incubated with 50 µM CARB for more than 1h before recording mIPSCs in the presence of drug . Slices from the same animals were used as controls . CARB increased mIPSC frequency at ZT12 ( CTL : 7 . 74 ± 0 . 55 Hz ; CARB : 12 . 41 ± 1 . 03 Hz ; P = 0 . 0001 ; Mann - Whitney U test ) , but not ZT0 ( CTL : 11 . 79 ± 1 . 00 Hz ; CARB : 11 . 47 ± 1 . 60 Hz ; P = 0 . 39 ; Mann - Whitney U test ) . mIPSC amplitude was unchanged at either time ( C , ZT12 : CTL : 40 . 94 ± 2 . 08 pA ; CARB : 40 . 64 ± 1 . 66 pA ; P = 0 . 99 ; E , ZT0 : CTL : 34 . 73 ± 2 . 14 pA ; CARB : 33 . 61 ± 2 . 39 pA ; P = 0 . 85 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B-E. Slices were obtained at ZT12 (B-C) or ZT0 (D-E) and pre-incubated with 50 µM CARB for more than 1h before recording mIPSCs in the presence of drug. Slices from the same animals were used as controls. CARB increased mIPSC frequency at ZT12 (CTL: 7.74 ± 0.55 Hz; CARB: 12.41 ± 1.03 Hz; P = 0.0001; Mann-Whitney U test), but not ZT0 (CTL:11.79 ± 1.00 Hz; CARB: 11.47 ± 1.60 Hz; P = 0.39; Mann-Whitney U test). mIPSC amplitude was unchanged at either time (C, ZT12: CTL: 40.94 ± 2.08 pA; CARB: 40.64 ± 1.66 pA; P = 0.99; E, ZT0: CTL: 34.73 ± 2.14 pA; CARB: 33.61 ± 2.39 pA; P = 0.85; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


F - G . mIPSC frequency ( F , CTL : 9 . 82 ± 0 . 74 Hz ; Atr : 7 . 70 ± 0 . 57 Hz ; P = 0 . 0009 ; Wilcoxon signed - rank test ) and amplitude ( G , CTL : 45 . 88 ± 2 . 87 pA ; Atr : 43 . 64 ± 2 . 40 pA ; P = 0 . 70 ; Wilcoxon signed - rank test ) recorded at ZT0 with acute atropine ( Atr ) wash on . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-G. mIPSC frequency (F, CTL: 9.82± 0.74 Hz; Atr: 7.70 ± 0.57 Hz; P = 0.0009; Wilcoxon signed-rank test) and amplitude (G, CTL: 45.88 ± 2.87 pA; Atr: 43.64 ± 2.40 pA; P = 0.70; Wilcoxon signed-rank test) recorded at ZT0 with acute atropine (Atr) wash on. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


H - I . Effect of acute wash - on and wash - off of pirenzepine ( Pir ) on mIPSC frequency ( H , CTL : 9 . 00 ± 1 . 24 Hz ; Pir : 7 . 94 ± 1 . 07 Hz ; Washout : 8 . 99 ± 1 . 14 Hz ; One - way repeated measures ANOVA , F ( 2 , 20 ) = 7 . 483 , P = 0 . 0037 ; Holm - Sidak ' s multiple comparison test ) and amplitude ( I , CTL : 61 . 23 ± 3 . 72 pA ; Pir : 58 . 85 ± 4 . 06 pA ; Washout : 58 . 25 ± 4 . 20 pA ; One - way repeated measures ANOVA , F ( 2 , 20 ) = 3 . 197 , P = 0 . 0624 ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

H-I. Effect of acute wash-on and wash-off of pirenzepine (Pir) on mIPSC frequency (H, CTL: 9.00 ± 1.24 Hz; Pir: 7.94 ± 1.07 Hz; Washout: 8.99 ± 1.14 Hz; One-way repeated measures ANOVA, F(2, 20) = 7.483, P = 0.0037; Holm-Sidak's multiple comparison test) and amplitude (I, CTL: 61.23 ± 3.72 pA; Pir: 58.85 ± 4.06 pA; Washout: 58.25 ± 4.20 pA; One-way repeated measures ANOVA, F(2, 20) = 3.197, P = 0.0624). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


J - K . Effect of pirenzepine or mecamylamine ( MEC ) on carbachol - induced elevation in mIPSC frequency at ZT12 ( J , CARB : 10 . 02 ± 0 . 64 Hz ; CARB + MEC : 10 . 95 ± 0 . 78 Hz ; CARB + Pir : 7 . 75 ± 0 . 79 Hz ; One - way ANOVA , F ( 2 , 25 ) = 4 . 848 , P = 0 . 0166 ; Holm - Sidak ' s multiple comparison test ) and amplitude ( K , CARB : 39 . 85 ± 2 . 60 pA ; CARB + MEC : 42 . 17 ± 1 . 65 pA ; CARB + Pir : 40 . 67 ± 4 . 34 pA ; One - way ANOVA , F ( 2 , 25 ) = 0 . 1501 , P = 0 . 8614 ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J-K. Effect of pirenzepine or mecamylamine (MEC) on carbachol-induced elevation in mIPSC frequency at ZT12 (J, CARB: 10.02 ± 0.64 Hz; CARB+MEC: 10.95 ± 0.78 Hz; CARB+Pir: 7.75 ± 0.79 Hz; One-way ANOVA, F(2, 25) = 4.848, P = 0.0166; Holm-Sidak's multiple comparison test) and amplitude (K, CARB: 39.85 ± 2.60 pA; CARB+MEC: 42.17 ± 1.65 pA; CARB+Pir: 40.67 ± 4.34 pA; One-way ANOVA, F(2, 25) = 0.1501, P = 0.8614). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


A1 . Diagram illustrating the in vivo two - photon imaging timeline . A2 . GCaMP6s ( green ) was expressed in V1 of the PV : Ai9 mice ( red : td - Tomato ) . Scale : 500 µm . A3 . Schematics of the calcium imaging showing a mouse that was head - fixed and free running on an air - suspended arena during the imaging section . Locomotor activity was simultaneously monitored ( bottom left ) along with Ca2 + imaging ( Top right . Scale : 100 µm ) . Example spontaneous ΔF / F0 traces ( bottom right ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O

A1. Diagram illustrating the in vivo two-photon imaging timeline. A2. GCaMP6s (green) was expressed in V1 of the PV:Ai9 mice (red: td-Tomato). Scale: 500 µm. A3. Schematics of the calcium imaging showing a mouse that was head-fixed and free running on an air-suspended arena during the imaging section. Locomotor activity was simultaneously monitored (bottom left) along with Ca2+ imaging (Top right. Scale: 100 µm). Example spontaneous ΔF/F0 traces (bottom right).


B . Top panel , Example spontaneous ΔF / F0 traces from three PV neurons repetitively imaged at ZT0 and ZT12 . Bottom panels , Heatmaps showing the spontaneous ΔF / F0 time - series traces of all PV neurons imaged at ZT0 and ZT12 . The color code represents the magnitude of ΔF / F0 ( % ) . C . Cumulative probability distribution of the sum ΔF / F0 of all PV neurons imaged at ZT0 and ZT12 ( K - S test ; P = 0 . 02 ) . Inset : the mean sum ΔF / F0 ( ZT0 : 401 . 6 ± 19 . 55 ; ZT12 : 559 . 1 ± 35 . 29 ; P = 0 . 0002 ; Mann - Whitney U test ) . D . Left panel , diagram depicting repetitive imaging protocol . Right panel , mean activity of PV neurons indicated by Z score imaged at four timepoints . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Top panel, Example spontaneous ΔF/F0 traces from three PV neurons repetitively imaged at ZT0 and ZT12. Bottom panels, Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at ZT0 and ZT12. The color code represents the magnitude of ΔF/F0(%). C. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at ZT0 and ZT12 (K-S test; P = 0.02). Inset: the mean sum ΔF/F0 (ZT0: 401.6 ± 19.55; ZT12: 559.1 ± 35.29; P = 0.0002; Mann-Whitney U test). D. Left panel, diagram depicting repetitive imaging protocol. Right panel, mean activity of PV neurons indicated by Z score imaged at four timepoints. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


B . Heatmaps showing the spontaneous ΔF / F0 time - series traces of all PV neurons imaged at ZT4 and SD _ ZT4 . The color code represents the magnitude of ΔF / F0 ( % ) . C . Cumulative probability distribution of the sum ΔF / F0 of all PV neurons imaged at ZT4 and SD _ ZT4 ( K - S test ; P = 0 . 0001 ) . Inset : the mean sum ΔF / F0 ( ZT4 : 322 . 4 ± 18 . 64 ; SD _ ZT4 : 424 . 0 ± 21 . 54 ; P = 0 . 0004 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at ZT4 and SD_ZT4. The color code represents the magnitude of ΔF/F0 (%). C. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at ZT4 and SD_ZT4 (K-S test; P = 0.0001). Inset: the mean sum ΔF/F0 (ZT4: 322.4 ± 18.64; SD_ZT4: 424.0 ± 21.54; P = 0.0004; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


D . Heatmaps showing the spontaneous ΔF / F0 time - series traces of all PV neurons imaged at SD _ ZT4 and Dark _ SD . The color code represents the magnitude of ΔF / F0 ( % ) . E . Cumulative probability distribution of the sum ΔF / F0 of all PV neurons imaged at SD _ ZT4 and Dark _ SD ( K - S test ; P = 0 . 015 ) . Inset : the mean sum ΔF / F0 ( SD _ ZT4 : 375 . 1 ± 20 . 19 ; Dark _ SD : 293 . 8 ± 16 . 55 ; P = 0 . 0022 ; Mann - Whitney U test ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Heatmaps showing the spontaneous ΔF/F0 time-series traces of all PV neurons imaged at SD_ZT4 and Dark_SD. The color code represents the magnitude of ΔF/F0(%). E. Cumulative probability distribution of the sum ΔF/F0 of all PV neurons imaged at SD_ZT4 and Dark_SD (K-S test; P = 0.015). Inset: the mean sum ΔF/F0 (SD_ZT4: 375.1 ± 20.19; Dark_SD: 293.8 ± 16.55; P = 0.0022; Mann-Whitney U test). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001.


A . Top panel , Example spontaneous ΔF / F0 traces from three putative pyramidal neurons repetitively imaged at ZT0 and ZT12 . Bottom panels , Heatmaps showing the spontaneous ΔF / F0 time - series traces of all putative pyramidal neurons imaged at ZT0 and ZT12 . B . The cumulative distribution of the sum ΔF / F0 of putative pyramidal neurons judged by skewness ( K - S test ; P < 0 . 0001 ) . Inset : the means of sum ΔF / F0 ( ZT0 : 679 . 5 ± 38 . 22 ; ZT12 : 485 . 8 ± 32 . 22 ; P < 0 . 0001 ; Mann - Whitney U test ) . C . Mean activity of putative pyramidal neurons indicated by Z score imaged at four timepoints . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at P < 0 . 05 . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Top panel, Example spontaneous ΔF/F0 traces from three putative pyramidal neurons repetitively imaged at ZT0 and ZT12. Bottom panels, Heatmaps showing the spontaneous ΔF/F0 time-series traces of all putative pyramidal neurons imaged at ZT0 and ZT12. B. The cumulative distribution of the sum ΔF/F0 of putative pyramidal neurons judged by skewness (K-S test; P < 0.0001). Inset: the means of sum ΔF/F0 (ZT0: 679.5 ± 38.22; ZT12: 485.8 ± 32.22; P < 0.0001; Mann-Whitney U test). C. Mean activity of putative pyramidal neurons indicated by Z score imaged at four timepoints. Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at P < 0.05. *P < 0.05; **P < 0.01; ***P < 0.001; ****P<0.0001.


D1 . Schematic showing in vivo dLGN - evoked Ca2 + reponses in V1 . D2 . Immunofluorescent staining showing the GCaMP6s ( green ) and ChR2 - mCherry ( red ) expression in V1 and dLGN respectively . The optical fiber track was also labeled . ( Scale : 500 µm ) . D3 . Response profile of an example neurons for dLGN stimulation during the light / dark cycle . Horizontal blue brief lines represent stimulus frequency .

O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D1. Schematic showing in vivo dLGN-evoked Ca2+ reponses in V1. D2. Immunofluorescent staining showing the GCaMP6s (green) and ChR2-mCherry (red) expression in V1 and dLGN respectively. The optical fiber track was also labeled. (Scale: 500 µm). D3. Response profile of an example neurons for dLGN stimulation during the light/dark cycle. Horizontal blue brief lines represent stimulus frequency.


E - F . Top : color - coded calcium signals of all responsive neurons ( % ΔF / F0 ) . Bottom : The mean Ca2 + response waveform for individual mouse ( dotted color lines ) and all mice ( thick black and gray lines ) . Trials were aligned to LED onset time ( gray solid lines ) . Data information : Sample size is indicated as ( mice , cells ) .

O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-F. Top: color-coded calcium signals of all responsive neurons (% ΔF/F0). Bottom: The mean Ca2+ response waveform for individual mouse (dotted color lines) and all mice (thick black and gray lines). Trials were aligned to LED onset time (gray solid lines). Data information: Sample size is indicated as (mice, cells).


G . The fraction of neurons responsive to dLGN stimulus in ZT0 and ZT12 . Each gray line represents one imaging mouse . Black and white circles and error bars represent the mean and SEM ( ZT0 : 8 . 51 ± 1 . 86 ; ZT12 : 5 . 96 ± 1 . 39 ; P = 0 . 015 ; Wilcoxon signed - rank test ; n = 9 mice ) . H . The Response fidelity of each cell in ZT0 and ZT12 . Black and white circles and error bars represent the mean and SEM ( ZT0 : 0 . 42 ± 0 . 022 ; ZT12 : 0 . 31 ± 0 . 023 ; P < 0 . 0001 ; Wilcoxon signed - rank test ; n = 195 cell ) . I . Comparison of peak evoked response of each cell in ZT0 and ZT12 . Black and white circles and error bars represent the mean and SEM ( ZT0 : 81 . 01 ± 4 . 79 ; ZT12 : 48 . 42 ± 3 . 82 ; P < 0 . 0001 ; Wilcoxon signed - rank test ; n = 195 cell ) . Data information : Sample size is indicated as ( mice , cells ) . Error bars in all figures indicate standard error of mean ( s . e . m ) . The level of significance was set at * P < 0 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. The fraction of neurons responsive to dLGN stimulus in ZT0 and ZT12. Each gray line represents one imaging mouse. Black and white circles and error bars represent the mean and SEM (ZT0: 8.51 ± 1.86; ZT12: 5.96 ± 1.39; P = 0.015; Wilcoxon signed-rank test; n = 9 mice). H. The Response fidelity of each cell in ZT0 and ZT12. Black and white circles and error bars represent the mean and SEM (ZT0: 0.42 ± 0.022; ZT12: 0.31 ± 0.023; P < 0.0001; Wilcoxon signed-rank test; n = 195 cell). I. Comparison of peak evoked response of each cell in ZT0 and ZT12. Black and white circles and error bars represent the mean and SEM (ZT0: 81.01 ± 4.79; ZT12: 48.42 ± 3.82; P < 0.0001; Wilcoxon signed-rank test; n = 195 cell). Data information: Sample size is indicated as (mice, cells). Error bars in all figures indicate standard error of mean (s.e.m). The level of significance was set at *P < 0.05


A . Protein lysates from the melanocytic - like melanoma cells 501mel , MM011 , MM074 and MM117 or the mesenchymal - like melanoma cells MM029 , MM047 and MM099 were immuno - blotted for proteins as indicated . Molecular mass of the proteins is indicated ( kDa ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Protein lysates from the melanocytic-like melanoma cells 501mel, MM011, MM074 and MM117 or the mesenchymal-like melanoma cells MM029, MM047 and MM099 were immuno-blotted for proteins as indicated. Molecular mass of the proteins is indicated (kDa).


B . Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . IC50 for each cell line is indicated . Melanocytic - type ( MITF - High , proliferative ) melanoma cells are shown in red , while mesenchymal - like ( MITF - low , invasive ) melanoma cells are shown in blue . Data information : data are presented as mean values + Standard Deviation ( SD ) for three replicates ( n = 3 ) .

O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Melanoma cells were treated with increasing concentrations of THZ1 as indicated for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. IC50 for each cell line is indicated. Melanocytic-type (MITF-High, proliferative) melanoma cells are shown in red, while mesenchymal-like (MITF-low, invasive) melanoma cells are shown in blue. Data information: data are presented as mean values + Standard Deviation (SD) for three replicates (n=3).


D . Protein lysates from MM074 , MM074BRAFi - R or MM074CDK7i - R were immuno - blotted for indicated proteins . Molecular sizes of the proteins are indicated ( kDa ) . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Protein lysates from MM074, MM074BRAFi-R or MM074CDK7i-R were immuno-blotted for indicated proteins. Molecular sizes of the proteins are indicated (kDa). The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized to each relative Vinculin control.


B . Protein lysates from the indicated cells were immuno - blotted for the indicated proteins . Molecular sizes of the proteins are indicated in kDa . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Protein lysates from the indicated cells were immuno-blotted for the indicated proteins. Molecular sizes of the proteins are indicated in kDa. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software normalized relative to each relative ACTIN control.


E - H . qRT - PCR analysis showing average TBP - normalized expression of GATA6 ( E ) , AMIGO2 ( F ) , SERPINE1 ( G ) and ABCG2 ( H ) in the indicated cells treated with either siCTL or siGATA6 for 72h . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * * < 0 . 01 , * * * < 0 . 005 and ns = non -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E-H. qRT-PCR analysis showing average TBP-normalized expression of GATA6 (E), AMIGO2 (F), SERPINE1 (G) and ABCG2 (H) in the indicated cells treated with either siCTL or siGATA6 for 72h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant


J . MM029 ( left ) and MM099 ( right ) were pre - treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * * < 0 . 01 , * * * < 0 . 005 and ns = non -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J. MM029 (left) and MM099 (right) were pre-treated with either siCTL or siGATA6 for 48h and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, **<0.01, ***<0.005 and ns=non-significant


C - D . MM099 ( c ) and MM029 ( d ) were pre - treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . E - F . MM099 ( e ) and MM029 ( f ) were pre - treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h . Mean growth is shown relative to vehicle ( DMSO ) - treated cells . D Data information : data are presented as mean values + SD for three replicates ( n = 3 ) . IC50 for each cell line is indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-D. MM099 (c) and MM029 (d) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing concentrations of THZ1 for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. E-F. MM099 (e) and MM029 (f) were pre-treated with either siCTL or siABCG2 as indicated and treated with increasing doses of Vemurafenib for 72h. Mean growth is shown relative to vehicle (DMSO)-treated cells. D Data information: data are presented as mean values + SD for three replicates (n=3). IC50 for each cell line is indicated.


C - E . qRT - PCR analysis showing average TBP - normalized fold expression of MITF ( C ) , SOX10 ( D ) and GATA6 ( E ) in 501mel treated either with DMSO / THZ1 ( 50nM ) ( upper ) or DMSO / JQ1 ( 10μM ) ( lower ) for 24h . Data information : data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C-E. qRT-PCR analysis showing average TBP-normalized fold expression of MITF (C), SOX10 (D) and GATA6 (E) in 501mel treated either with DMSO/THZ1 (50nM) (upper) or DMSO/JQ1 (10μM) (lower) for 24h. Data information: data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.


A . qRT - PCR analysis showing average TBP - normalized fold expression of SOX10 , MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h . Data information : , data are presented as mean values + SD for six replicates ( n = 6 ) . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. qRT-PCR analysis showing average TBP-normalized fold expression of SOX10, MITF and GATA6 in 501mel treated with either siCTL or siSOX10 for 48h. Data information: , data are presented as mean values + SD for six replicates (n=6). The p-value (Student's t-test) is indicated, *<0.05.


D . MM099MITF - SOX10 - PAX3 expressing inducible MITF - SOX10 - PAX3 genes was treated or not with Doxycycline ( 1μg / ml ) for 24h and protein lysates were immuno - blotted for the indicated protein . The numbers below the gel lanes represent relative protein level , which was determined from the band intensity using ImageJ software , and normalized relative to VINCULIN .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. MM099MITF-SOX10-PAX3 expressing inducible MITF-SOX10-PAX3 genes was treated or not with Doxycycline (1μg/ml) for 24h and protein lysates were immuno-blotted for the indicated protein. The numbers below the gel lanes represent relative protein level, which was determined from the band intensity using ImageJ software, and normalized relative to VINCULIN.


E . qRT - PCR analysis showing average TBP - normalized fold expression of GATA6 , ABCG2 , AMIGO2 or SERPINE1 in MM099MITF - SOX10 - PAX3 treated or not with Doxycycline ( 1μg / ml ) for 24h . Data information : data are presented as mean values + SD for three biological triplicates . The p - value ( Student ' s t - test ) is indicated , * < 0 . 05 , * * < 0 . 01 ; * * * < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. qRT-PCR analysis showing average TBP-normalized fold expression of GATA6, ABCG2, AMIGO2 or SERPINE1 in MM099MITF-SOX10-PAX3 treated or not with Doxycycline (1μg/ml) for 24h. Data information: data are presented as mean values + SD for three biological triplicates. The p-value (Student's t-test) is indicated, *<0.05, **<0.01; ***<0.001.


A . Tumor sections were immuno - labelled ( IHC ) with anti - GATA6 ( red ) and anti - SOX10 ( green ) antibodies and images were captured by confocal microscopy at the indicated magnification . We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them . Scale bar 250 μM for 40x , 100μM for 100x .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Tumor sections were immuno-labelled (IHC) with anti-GATA6 (red) and anti-SOX10 (green) antibodies and images were captured by confocal microscopy at the indicated magnification. We analysed six tumor sections of metastases and observed significant GATA6 expression in only one of them. Scale bar 250 μM for 40x, 100μM for 100x.


D - E . Graphs showing the average expression of GATA6 ( d ) and MITF ( e ) ( RPKM ) for each phenotype cluster in T0 ( drug naïve ) ( blue ) , phases 1 - 2 ( MDR ) ( green and yellow ) and phase 3 ( drug - resistance ) ( red ) . Data information : data are presented as mean values + SEM ( n = 6 , 574 cells from 5 PDX ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D-E. Graphs showing the average expression of GATA6 (d) and MITF (e) (RPKM) for each phenotype cluster in T0 (drug naïve) (blue), phases 1-2 (MDR) (green and yellow) and phase 3 (drug-resistance) (red). Data information: data are presented as mean values + SEM (n=6,574 cells from 5 PDX).


A , Live images of neurons co - transfected with SyGCaMP5 and MtDsRed before and during 10 Hz field stimulation . The full arrow indicates a mitochondrially occupied terminal , and the empty arrow a terminal without a mitochondrion . Scale bar , 10 m .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Live images of neurons co-transfected with SyGCaMP5 and MtDsRed before and during 10 Hz field stimulation. The full arrow indicates a mitochondrially occupied terminal, and the empty arrow a terminal without a mitochondrion. Scale bar, 10 m.


B , Average ΔF / F0 SyGCaMP5 traces from n = 11 neurons ( 98 terminals ) plotting the average of terminals without a mitochondrion ( black trace ) and with a mitochondrion ( red trace ) . Stimulation occurred for 10s ( t = 20 - 30 ) at 10 Hz .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B, Average ΔF/F0 SyGCaMP5 traces from n=11 neurons (98 terminals) plotting the average of terminals without a mitochondrion (black trace) and with a mitochondrion (red trace). Stimulation occurred for 10s (t=20-30) at 10 Hz.


C , Average Ca2 + response following stimulation in terminals with or without a mitochondrion ( average of ΔF measurement taken for a t = 20 - 30s of B ) , ΔF / F0 = 1 . 9 0 . 3 with and 3 . 5 0 . 4 without mitochondria , paired t - test , * * * p < 0 . 0001 .

O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Average Ca2+ response following stimulation in terminals with or without a mitochondrion (average of ΔF measurement taken for a t=20-30s of B), ΔF/F0 = 1.9 0.3 with and 3.5 0.4 without mitochondria, paired t-test, ***p<0.0001.


D E , However , there is no significant difference in the presynaptic Ca2 + response in terminals with and without a mitochondrion in response to single action potentials . D , Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 . 1 ms stimulation pulses were applied every 5 s . The red trace represents an average of the terminals occupied with a mitochondrion in this neuron , whereas the black trace represents the averaged response of terminals without a mitochondrion . E , Maximal fluorescence intensity in terminals without a mitochondrion ( left ) or with a mitochondrion ( right ) within the same axon ( dots connected by a line ) ( n = 8 neurons , 44 terminals , paired t - test , p = 0 . 55 ) .

O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D E, However, there is no significant difference in the presynaptic Ca2+ response in terminals with and without a mitochondrion in response to single action potentials. D, Example trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5. 1 ms stimulation pulses were applied every 5 s. The red trace represents an average of the terminals occupied with a mitochondrion in this neuron, whereas the black trace represents the averaged response of terminals without a mitochondrion. E, Maximal fluorescence intensity in terminals without a mitochondrion (left) or with a mitochondrion (right) within the same axon (dots connected by a line) (n=8 neurons, 44 terminals, paired t-test, p=0.55).


F , Average ΔF / F0 SyGCaMP5 traces for 80 stimuli ( n = 7 neurons , 21 terminals , t = 20 - 28s , ΔF / F0 = 1 . 3 0 . 2 with and 2 . 2 0 . 5 without mitochondria , paired t - test , * p < 0 . 05 ) , 40 stimuli ( n = 5 neurons , 25 terminals , t = 20 - 24s , ΔF / F0 = 0 . 9 0 . 3 with and 2 . 0 0 . 4 without mitochondria , paired t - test , * p < 0 . 05 ) , 20 stimuli ( n = 10 neurons , 55 terminals , t = 20 - 22s , ΔF / F0 = 0 . 8 0 . 4 with and 1 . 8 0 . 4 without mitochondria , paired t - test , * p < 0 . 05 ) and 10 stimuli ( n = 4 neurons , 21 terminals , t = 20 - 21s , ΔF / F0 = 0 . 4 0 . 3 with and 0 . 4 0 . 1 without mitochondria , paired t - test , p = 0 . 90 ) , all delivered at 10 Hz . Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, Average ΔF/F0 SyGCaMP5 traces for 80 stimuli (n=7 neurons, 21 terminals, t= 20-28s, ΔF/F0 = 1.3 0.2 with and 2.2 0.5 without mitochondria, paired t-test, *p<0.05), 40 stimuli (n=5 neurons, 25 terminals, t= 20-24s, ΔF/F0 = 0.9 0.3 with and 2.0 0.4 without mitochondria, paired t-test, *p<0.05), 20 stimuli (n=10 neurons, 55 terminals, t= 20-22s, ΔF/F0 = 0.8 0.4 with and 1.8 0.4 without mitochondria, paired t-test, *p<0.05) and 10 stimuli (n=4 neurons, 21 terminals, t= 20-21s, ΔF/F0 = 0.4 0.3 with and 0.4 0.1 without mitochondria, paired t-test, p=0.90), all delivered at 10 Hz. Experiments performed in E18 or P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min . Neurons were stimulated using field stimulation for 10s at 10 Hz . Red trace = average of the terminals occupied with a mitochondrion , black trace = averaged response of terminals without a mitochondrion . B , as in A , but after 30 min treatment with 10 µM Ru360 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O

A, Average trace of hippocampal neurons transfected with MtDsRed and SyGCaMP5 imaged after incubation in ACSF for 30 min. Neurons were stimulated using field stimulation for 10s at 10 Hz. Red trace = average of the terminals occupied with a mitochondrion, black trace = averaged response of terminals without a mitochondrion. B, as in A, but after 30 min treatment with 10 µM Ru360.


C , Summary bar graph of the control neurons ( corresponds to the traces shown in panel A ) . Average Ca2 + response ( t = 20 - 30s ) following stimulation for terminals occupied by a mitochondrion ( ΔF / F0 = 3 . 5 0 . 4 ) and unoccupied terminals ( ΔF / F0 = 4 . 9 0 . 5 , n = 15 neurons , 96 terminals , * * * p < 0 . 001 , paired t - test ) . D , Summary bar graph of the Ru360 - treated neurons ( corresponds to the traces shown in panel B ) . Average Ca2 + response ( t = 20 - 30s ) following stimulation for terminals occupied by a mitochondrion ( ΔF / F0 = 4 . 1 0 . 5 ) and unoccupied terminals ( ΔF / F0 = 3 . 8 0 . 9 , n = 13 neurons , 79 terminals , p = 0 . 53 , paired t - test ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C, Summary bar graph of the control neurons (corresponds to the traces shown in panel A). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 3.5 0.4) and unoccupied terminals (ΔF/F0 = 4.9 0.5, n=15 neurons, 96 terminals, ***p<0.001, paired t-test). D, Summary bar graph of the Ru360-treated neurons (corresponds to the traces shown in panel B). Average Ca2+ response (t=20-30s) following stimulation for terminals occupied by a mitochondrion (ΔF/F0 = 4.1 0.5) and unoccupied terminals (ΔF/F0 = 3.8 0.9, n=13 neurons, 79 terminals, p=0.53, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , Example images of hippocampal neurons co - transfected with Mito - mKate2 and VGlut1pHluorin before and during field stimulation . The white arrow indicates a terminal occupied with a mitochondrion , while the empty arrow indicates a terminal without a mitochondrion . B , Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito - mKate2 and stimulated using field stimulation at 10 Hz for 20s ( 40 - 60s ) . Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace . C , Summary graph corresponds to the traces shown in panel B ) . Mean stimulation fluorescence ( time points 60 - 70s ) comparing terminals of the same axon which contain mitochondria ( right , ΔF / F0 = 2 . 3 0 . 9 ) compared to terminals without mitochondria ( left , ΔF / F0 = 3 . 2 0 . 7 ) ( n = 9 neurons , 66 terminals , * p < 0 . 05 , paired t - test ) . Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, Example images of hippocampal neurons co-transfected with Mito-mKate2 and VGlut1pHluorin before and during field stimulation. The white arrow indicates a terminal occupied with a mitochondrion, while the empty arrow indicates a terminal without a mitochondrion. B, Average traces of presynaptic terminals of hippocampal neurons transfected with VGlut1pHluorin and Mito-mKate2 and stimulated using field stimulation at 10 Hz for 20s (40-60s). Terminals with a mitochondrion are represented by the red trace and terminals without are represented by the black trace. C, Summary graph corresponds to the traces shown in panel B). Mean stimulation fluorescence (time points 60-70s) comparing terminals of the same axon which contain mitochondria (right, ΔF/F0 = 2.3 0.9) compared to terminals without mitochondria (left, ΔF/F0 = 3.2 0.7) (n=9 neurons, 66 terminals, *p<0.05, paired t-test). Experiments performed in P0 rat hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.


A , C E , Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN - GFP and C , myc - Miro1 ( Miro OE ) or E , myc - Miro1ΔEF ( Miro ΔEF ) . Neurons are either non - treated ( NT ) , TTX treated ( 1 M , 48h ) , PTX treated ( 100 M , 48h ) , or DMSO treated ( 1 : 2000 , as PTX ) . Scale bar , 10 m . B , D F , Fraction of SYN - GFP clusters co - localising with mitochondria in cultures in B , control conditions , DMSO 28 . 7 % 3 . 9 , TTX 16 . 6 % 3 . 1 , PTX 50 . 0 % 7 . 2 , ANOVA * * p0 . 001 , n = 8 - 9 neurons , D , with the expression of myc - Miro1 , DMSO 33 . 9 % 4 . 7 , TTX 35 . 9 % 5 . 4 , PTX 46 . 7 % 4 . 0 , ANOVA , * p0 . 05 , n = 8 - 9 , F , with the expression of myc - Miro1ΔEF , ANOVA p0 . 05 , n = 9 - 12 . Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10 - 12 . Error bars represent SEM .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A, C E, Confocal images of neuronal processes of fixed neurons transfected with MtDsRed and SYN-GFP and C, myc-Miro1 (Miro OE) or E, myc-Miro1ΔEF (Miro ΔEF). Neurons are either non-treated (NT), TTX treated (1 M, 48h), PTX treated (100 M, 48h), or DMSO treated (1:2000, as PTX). Scale bar, 10 m. B, D F, Fraction of SYN-GFP clusters co-localising with mitochondria in cultures in B, control conditions, DMSO 28.7% 3.9, TTX 16.6% 3.1, PTX 50.0% 7.2, ANOVA **p0.001, n=8-9 neurons, D, with the expression of myc-Miro1, DMSO 33.9% 4.7, TTX 35.9% 5.4, PTX 46.7% 4.0, ANOVA, *p0.05, n=8-9, F, with the expression of myc-Miro1ΔEF, ANOVA p0.05, n=9-12. Experiments performed in E16 mouse hippocampal neuronal cultures at DIV 10-12. Error bars represent SEM.