Nature Neuroscience: doi: /nn Supplementary Figure 1. Amygdaloid complex and evoked synaptic currents recorded in CeM amygdala neurons.
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- Shona Moore
- 5 years ago
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1 Supplementary Figure 1 Amygdaloid complex and evoked synaptic currents recorded in CeM amygdala neurons. (a) Left: Schematic representation (modified from: Allen Brain Atlas) of coronal sections containing the amygdala subnuclei inside the square and antero/posterior coordinates from bregma in mm. CeM, represented in green, CeL in blue and BLA in red. Right: Schematic representation of amygdala subnuclei, cellular types and connectivity (b) Top: Images showing neuronal recordings from CeM (green) and stimulating electrodes in BLA (red) or CeL (blue). Scale bar, 500 µm Bottom: BLA-evoked glutamatergic EPSCs (bottom, left; isolated in the presence of GABA A R and GABA B R antagonists picrotoxin and CGP) and CeL-evoked GABAergic IPSCs (bottom, right; isolated in the presence of glutamate receptor AMPAR and NMDAR antagonists CNQX and AP5). Scale bars 8 pa and 25 ms. (c) Image showing SR101 staining CeM astrocytes and blood vessels (arrow). Scale bars: top image 90 µm and bottom image 30 µm.
2 Supplementary Figure 2 Synaptic potency of CeL-evoked IPSCs and BLA-evoked EPSCs recorded in the heteroneuron after homoneuron ND in different experimental conditions. (a) Synaptic potency of CeL-evoked IPSCs before and after homoneuron ND (time=0, n=22). (b) Synaptic potency of CeL-evoked IPSCs before and after homoneuron ND in control conditions (n=22, p=0.88), in the presence of AM251 (n=11, p=0.087), in GFAP- CB1R WT (n=7, p=0.12), GFAP-CB1R -/- (n=7, p=0.56) and IP3R2 -/- (n=7, p=0.92) mice, in the presence of MPEP+LY (n=10, p=0.42), SCH (n=7, p=0.37) and CPT (n=11, p=0.82). (c) Synaptic potency of BLA-evoked EPSCs before and after homoneuron ND (time=0; n=24). (d) Synaptic potency of BLA-evoked EPSCs before and after homoneuron ND in control conditions (n=24, p=0.2), in the presence of AM251 (n=9, p=0.089), and in GFAP-CB1R WT (n=8, p=0.16), GFAP-CB1R -/- (n=10, p=0.24) and IP3R2 -/- (n=10, p=0.21) mice, in the presence of MPEP+LY (n=13, p=0.13), CPT (n=9, p=0.75) and SCH (n=12, p=0.56). *P < 0.05, **P < 0.01, ***P < 0.001; Student s paired t-test. Error bars indicate SEM.
3 Supplementary Figure 3 Basal probability of release of CeL-evoked IPSCs and BLA-evoked EPSCs. (a) Basal probability of release of CeL-evoked IPSCs in control conditions (n=22), in the presence of AM251 (n=11), in GFAP-CB1R WT (n=7), GFAP-CB1R -/- (n=7) and IP3R2 -/- (n=10) mice, in the presence of MPEP+LY (n=10), SCH (n=7) and CPT (n=13). No differences were found between the different experimental conditions (One-Way ANOVA, F 7, 79 =1.96, p=0.071). (b) Basal probability of release BLA-evoked EPSCs in control conditions (n=24), in the presence of AM251 (n=12), and in GFAP-CB1R WT (n=11), GFAP-CB1R -/- (n=9) and IP3R2 -/- (n=10) mice, in the presence of MPEP+LY (n=13), SCH (n=12) and CPT (n=9). No differences were found between the different experimental conditions (One-Way ANOVA, F 7, 91 =1.93, p=0.073). Box-whisker plots indicate median, interquartile range and 10th-90th percentiles of the distribution.
4 Supplementary Figure 4 Differential astrocyte-mediated regulation of BLA-evoked EPSCs and CeL-evoked IPSCs in the same CeM neurons. (a) DIC images and schemes showing neuronal recordings in CeM (green) and stimulating electrodes in BLA (red) or CeL (blue). Scale bar 500 µm. (b) IPSCs and EPSCs in basal conditions and after ND. IPSCs were evoked by stimulating the CeL and were isolated in the presence of CNQX and AP5; after the washout of CNQX and AP5, EPSCs evoked by stimulation of BLA were isolated with picrotoxin and CGP. Scale bars, 25 ms and 8 pa. (c) Left: CeL-evoked IPSCs Pr (blue) and BLA-evoked EPSCs Pr (red) before and after homoneuron ND (at time=0 and 50, respectively). Right: CeL-evoked IPSCs Pr before and after ND (n=6, p=0.02) and BLAevoked EPSCs Pr before and after ND (n=6, p=0.04). (d) Left: Synaptic potency of CeL-evoked IPSCs (blue) and synaptic potency of BLA-evoked EPSCs (red) before and after homoneuron ND (at time=0 and 50, respectively). Right: Synaptic potency of CeL-evoked IPSCs before and after ND (n=6, p=0.1) and synaptic potency of BLA-evoked EPSCs before and after ND (n=6, p=0.96). *P < 0.05, **P < 0.01, ***P < 0.001; Student s paired t-test. Error bars indicate SEM.
5 Supplementary Figure 5 Synaptic potency of CeL-evoked IPSCs and BLA-evoked EPSCs after CNO application in different experimental conditions. (a) Left: Synaptic potency of CeL-evoked IPSCs before and after CNO pressure pulse application (time=0; n=7). Right: Synaptic potency of BLA-evoked EPSCs before and after CNO pressure pulse application (time=0; n=8). (b) Synaptic potency of CeL-evoked IPSCs and BLA-evoked EPSCs before and after CNO pressure pulse application in control (IPSCs n=6, p=0.93; EPSCs n=8, p=0.48), in AM251 (IPSCs n=8, p=0.87; EPSCs n=6, p=0.77) and in the presence of SCH (n=7, p=0.66) and CPT (n=7, p=0.12). (c) Left: Synaptic potency of CeL-evoked IPSCs before and after CNO bath application (time=0, n=9) and SCH application (time=30). Right: Synaptic potency of BLA-evoked EPSCs before and after CNO bath application (time=0; n=6) and CPT application (time=30). (d) Synaptic potency of CeL-evoked IPSCs in control conditions and after CNO bath application (n=8, p=0.3) and SCH bath application (n=3, p=0.91) and synaptic potency of BLA-evoked EPSCs before and after CNO bath application (n=6, p=0.59) and CPT bath application (n=4, p=0.24). *P < 0.05, **P < 0.01, ***P < 0.001; Student s paired t-test. Error bars indicate SEM.
6 Supplementary Figure 6 Sustained activation of CeM astrocytes by bath application of CNO. (a) Left: Fluorescence image showing mcherry and fluo4 pseudocolor images representing fluorescence intensities in CeM astrocytes before and after CNO bath application. Scale bar, 10µm. Right: Astrocytic calcium levels before and after CNO bath application. Scale bars, 45% and 60s. (b) Left: Time course of the calcium event frequency in basal condition and during CNO (at time=0). Right: Calcium event frequency in basal condition and during CNO (n=6, p=0.009) (c) Schematic representation and CeL-evoked IPSCs recorded in CeM neurons before and after CNO application. Scale bars, 15 pa and 25 ms. (d) Left: Time course of CeL-evoked IPSCs Pr before and after CNO application (time 0) and SCH application (time=30). Right: CeL-evoked IPSCs Pr before and after CNO application (n=9, p=0.002) and SCH application (n=3, p=0.88). (e) Schematic representation and BLA-evoked EPSCs recorded in CeM neurons before and after CNO application. Scale bars, 15 pa and 25 ms. (f) Left: Time course of BLA-evoked EPSCs Pr before and after CNO application (time 0) and CPT application (time=30). Right: BLA-evoked EPSCs Pr before and after CNO application (n=6, p=0.0001) and CPT application (n=4, p=0.23). *P < 0.05, **P < 0.01, ***P < 0.001; Student s paired t-test. Error bars indicate SEM.
7 Supplementary Figure 7 CNO effects in mice lacking DREADDs expression. (a) Left: Fear response measured as percentage of freezing during the 15 sec cue presentation in sham-surgeries mice during fear conditioning, Right: Fear response measured as percentage of freezing during the 3 minutes of continuous CS presentation, data depicted in 1-minute time bins. One non-reinforced CS was presented in Test 1 and in Test 2 (n=10 mice injected with CNO, red, and 10 mice injected with saline, grey). No differences were observed between saline and CNO injected mice in any of the three time bins of Test 1 (p=0.88, p=0.39 and p=0.24) and Test 2 (p=0.99, p=0.81 and p=0.7). (b) Percentage of the time spent in the open arms in the elevated plus maze test (n=10 mice injected with CNO and 9 mice injected with saline). No differences were observed between saline and CNO injected mice (p=0.24). *P < 0.05, **P < 0.01, ***P < 0.001; Unpaired Student s t-test. Box-whisker plots indicate median, interquartile range and 10th-90th percentiles of the distribution.
8 Supplementary Figure 8 Schematic of the proposed model consistent with present findings. (a) The schematic shows that either an endogenous stimulus (ecbs; Figure 1 and 2) or a selective exogenous stimulus (DREADDs activation by CNO; Figure 3) are able to activate astrocytes and induce a regulation in CeL-CeM and BLA-CeM synapses. The astrocytic activity increases synaptic probability of release in CeL-CeM synapses through activation of A 2A receptors and decreases synaptic probability of release through activation of A 1 receptors in BLA-CeM synapses (Figures 1 and 3). (b) The main amygdala subnuclei are represented: the basolateral amygdala (BLA; red), the lateral portion of the central amygdala (CeL, Blue) and the medial portion of the central amygdala (CeM, green). GABAergic neurons are represented as blue circles and glutamatergic neurons as red circles. Consistently with the synaptic effects, upon astrocytic activation the firing rate of CeM neurons and the fear expression are decreased (Figure 4).
9 Supplementary Figure 9 GFAP-CB1R-/- mice present impairments in astrocytic CB1R effects but not in the neuronal CB1R dependent depolarization suppression of inhibition (DSI). (a) Left: Astrocytic calcium levels before and after ND (black) in the GFAP-CB1R-/- mice. Scale bars 20 s and 50 %. Right: Calcium event probability before and after ND at time=0 in GFAP-CB1R WT (n=10) and in GFAP-CB1R -/- (n=9) mice. (b) Left: Homoneuronal CeL-evoked IPSCs Pr before and after homoneuron ND (at time=0; n=6) in GFAP-CB1R -/- mice. Right: Homoneuronal CeL-evoked IPSCs Pr before and after homoneuron ND in in GFAP-CB1R WT (n=6, p=0.015) and GFAP-CB1R -/- (n=6, p=0.014). *P < 0.05, **P < 0.01, ***P < 0.001; paired Student s t-test. Error bars indicate SEM.
10 Two-Way ANOVAs values Source of variation F value DF P value Figure 1 d Figure 1 h Figure 1 l Figure 2c Figure 2e Figure 2g Figure 3 d Figure 3f Figure 3h Factor , 78 <0.001 Factor , 78 <0.001 Interactions , 78 <0.001 Factor , 158 <0.001 Factor , 158 <0.001 Interactions , 158 <0.001 Factor , Factor , 184 <0.001 Interactions , 184 <0.001 Factor , 28 <0.001 Factor , 28 <0.001 Interactions , Factor , Factor , Interactions , Factor , Factor , Interactions , Factor , 40 <0.001 Factor , 40 <0.001 Interactions , 40 <0.001 Factor , 38 <0.001 Factor , Interactions , Factor , 36 <0.001 Factor , Interactions , Supplementary table 1. Full report of Two-way ANOVA values. Full report of F values, degrees of freedom (DF) and P value for the Two-way ANOVA tests performed. For Factor 1 the variables are: Before ND and After ND for figure 1d, 1h, 1l, 2c, 2e and 2g; Before CNO and After CNO for figure 3d, 3f and 3h. For Factor 2 the variables are: Control, AM 251, GFAP-CB1R WT,GFAP-CB1R -/- and IP3R2 -/- in Figure 1d; Control, AM 251, GFAP-CB1R WT,GFAP-CB1R -/-,IP3R2 -/-, MPEP+LY, SCH and CPT for Figure 1h and l; Control and BAPTA for figure 2c, 2e and 2g; CNO-DREADDs, ACSF-DREADDs and CNO-Naive for Figure 3d; Control, SCH and AM 251 for figure 3f; Control, CPT and AM 251 for figure 3h.
11 a CeL-evoked IPSCs Condition Basal Post ND P value Control 0.39 ± ± 0.04 <0.001 AM ± ± GFAP-CB1R -/- mice 0.47 ± ± GFAP-CB1 WT mice 0.55 ± ± IP 3 R2 -/- mice 0.37 ± ± MPEP+LY 0.32 ± ± SCH 0.32 ± ± CPT 0.47 ± ± b BLA-evoked EPSCs Condition Basal Post ND P value Control 0.47 ± ± AM ± ± GFAP-CB1R -/- mice 0.39 ± ± GFAP-CB1 WT mice 0.5 ± ± IP 3 R2 -/- mice 0.63 ± ± MPEP+LY 0.39 ± ± SCH 0.5 ± ± CPT 0.37 ± ± Supplementary table 2. Absolute values of CeL-evoked IPSCs Pr and BLA-evoked EPSCs Pr. (a) Absolute probability of release values of CeL-evoked IPSCs in control conditions (n=22), in the presence of AM251 (n=11), in GFAP-CB1R WT (n=7), GFAP-CB1R -/- (n=7) and IP3R2 -/- (n=10) mice, in the presence of MPEP+LY (n=10), SCH (n=7) and CPT (n=13). P values are obtained comparing Basal and Post ND values with paired t-test. (b) Absolute probability of release values of BLA-evoked EPSCs in control conditions (n=24), in the presence of AM251 (n=12), and in GFAP-CB1R WT (n=11), GFAP-CB1R -/- (n=9) and IP3R2 -/- (n=10) mice, in the presence of MPEP+LY (n=13), SCH (n=12) and CPT (n=9). P values are obtained comparing Basal and Post ND values with paired Student s t-test.
12 Student s t-tests (main figures) Figure Comparison Condition Test t and p values 1d Basal vs Post ND Control (n=10) Two-tailed Student s paired t-test t 9 =6, p< d Basal vs Post ND AM251 (n=7) Two-tailed Student s paired t-test t 6 =0.04, p=0.96 1d Basal vs Post ND GFAP-CB1R-/- (n=10) Two-tailed Student s paired t-test t 9 =0.49, p=0.63 1d Basal vs Post ND GFAP-CB1WT (n=9) Two-tailed Student s paired t-test t 8 =-3.5, p= d Basal vs Post ND IP3R2-/- (n=8) Two-tailed Student s paired t-test t 7 =0.34, p=0.73 1h Basal vs Post ND control (n=22) Two-tailed Student s paired t-test t 21 =-4.48,p< h Basal vs Post ND AM251 (n=11) Two-tailed Student s paired t-test t 10 =1.89, p=0.74 1h Basal vs Post ND GFAP-CB1R-/- (n=7) Two-tailed Student s paired t-test t 6 =1.17, p=0.21 1h Basal vs Post ND GFAP-CB1WT (n=7) Two-tailed Student s paired t-test t 6 =-3.8, p= h Basal vs Post ND IP3R2-/- (n=10) Two-tailed Student s paired t-test t 9 =-0.01, p=0.3 1h Basal vs Post ND MPEP-LY (n=10) Two-tailed Student s paired t-test t 9 =0.84, p= h Basal vs Post ND SCH (n=7) Two-tailed Student s paired t-test t 6 =-0.9, h Basal vs Post ND CPT (n=13) Two-tailed Student s paired t-test t 12 =-3.3, p= l Basal vs Post ND Control (n=24) Two-tailed Student s paired t-test t 23 =3.14, p= l Basal vs Post ND AM251 (n=12) Two-tailed Student s paired t-test t 11 =-0.48, p=0.66 1l Basal vs Post ND GFAP-CB1R-/- (n=11) Two-tailed Student s paired t-test t 10 =-1.18, p=0.25 1l Basal vs Post ND GFAP-CB1WT (n=9) Two-tailed Student s paired t-test t 8 =4.16, p= l Basal vs Post ND IP3R2-/- (n=10) Two-tailed Student s paired t-test t 9 =-1.4, p=0.17 1l Basal vs Post ND MPEP-LY (n=13) Two-tailed Student s paired t-test t 12 =2.87, p=0.01 1l Basal vs Post ND CPT (n=9) Two-tailed Student s paired t-test t 8 =1.61, p=0.14 1l Basal vs Post ND SCH (n=12) Two-tailed Student s paired t-test t 11 =2.29, p=0.04 2c Basal vs Post ND BAPTA (n=9) Two-tailed Student s paired t-test t 8 =1.5, p=0.16 2c Basal vs Post ND control (n=7) Two-tailed Student s paired t-test t 6 =-6.7, p< e Basal vs Post ND BAPTA (n=8) Two-tailed Student s paired t-test t 7 =1.54, p=0.16 2e Basal vs Post ND control (n=9) Two-tailed Student s paired t-test t 8 =-4.01, p= g Basal vs Post ND BAPTA (n=8) Two-tailed Student s paired t-test t 7 =0.49, p=0.6 2g Basal vs Post ND control (n=11) Two-tailed Student s paired t-test t 10 =2.48, p=0.03 3d Basal vs Post CNO CNO-DREADDs (n=7) Two-tailed Student s paired t-test t 6 =-5.3, p= d Basal vs Post ACSF ACSF-DREADDs (n=8) Two-tailed Student s paired t-test t 7 =1.51, p=0.17 3d Basal vs Post CNO CNO-naive (n=8) Two-tailed Student s paired t-test t 7 =0.21, p=0.83 3f Basal vs Post CNO Control (n=7) Two-tailed Student s paired t-test t 6 =-4.5, p= f Basal vs Post CNO SCH (n=7) Two-tailed Student s paired t-test t 6 =0.45, p=0.96 3f Basal vs Post CNO AM251 (n=8) Two-tailed Student s paired t-test t 7 =0.1, p= h Basal vs Post CNO Control (n=8) Two-tailed Student s paired t-test t 7 =2.8, p=0.02 3h Basal vs Post CNO CPT (n=7) Two-tailed Student s paired t-test t 6 =1.137, p=0.3 3h Basal vs Post CNO AM251 (n=6) Two-tailed Student s paired t-test t 5 =3.34, p=0.02 4c Basal vs Post saline Saline (n=23 neurons of 6 mice) Two-tailed Student s paired t-test t 22 =-0.43, p=0.6 4c Basal vs Post CNO CNO (n=28 neurons of 7 mice) Two-tailed Student s paired t-test t 27 =3.1, p= e 1 st minute of Test 1: Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =2.13, p= e 2 nd minute of Test 1: Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =3.65, p< e 3 rd minute of Test 1: Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =4.12, p< e 1 st minute of Test 2: Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =1.19, p=0.23 4e 2 nd minute of Test 2: Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =1.16, p=0.24 4e 3 rd minute of Test 2: Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =1.87, p= f Saline vs CNO CNO (n=33) and saline (n=30) Two-tailed Student s unpaired t-test t 61 =0.76, p=0.44 Supplementary table 3. Student s t-tests performed in main figures.
13 Student s t-tests (supplementary figures) Figure Comparison Condition Test t and p values Supp. 2b Basal vs Post ND Control (n=22) Two-tailed Student s paired t-test t 21 =0.14, p=0.88 Supp. 2b Basal vs Post ND AM251 (n=11) Two-tailed Student s paired t-test t 10 =1.89, p=0.087 Supp. 2b Basal vs Post ND GFAP-CB1R-/- (n=7) Two-tailed Student s paired t-test t 6 =0.6, p=0.56 Supp. 2b Basal vs Post ND GFAP-CB1WT (n=7) Two-tailed Student s paired t-test t 6 =1.8, p=0.12 Supp. 2b Basal vs Post ND IP3R2-/- (n=10) Two-tailed Student s paired t-test t 9 =-0.09, p=0.92 Supp. 2b Basal vs Post ND MPEP-LY (n=10) Two-tailed Student s paired t-test t 9 =0.8, p=0.42 Supp. 2b Basal vs Post ND SCH (n=7) Two-tailed Student s paired t-test t 6 =-0.9, p=0.37 Supp. 2b Basal vs Post ND CPT (n=11) Two-tailed Student s paired t-test t 10 =0.22, p=0.82 Supp. 2d Basal vs Post ND Control (n=24) Two-tailed Student s paired t-test t 23 =-1.3, p=0.2 Supp. 2d Basal vs Post ND AM251 (n=9) Two-tailed Student s paired t-test t 8 =-1.93, p=0.089 Supp. 2d Basal vs Post ND GFAP-CB1R-/- (n=10) Two-tailed Student s paired t-test t 9 =-1.24, p=0.24 Supp. 2d Basal vs Post ND GFAP-CB1WT (n=8) Two-tailed Student s paired t-test t 7 =1.5, p=0.16 Supp. 2d Basal vs Post ND IP3R2-/- (n=10) Two-tailed Student s paired t-test t 9 =1.34, p=0.21 Supp. 2d Basal vs Post ND MPEP-LY (n=13) Two-tailed Student s paired t-test t 12 =-1.59, p=0.13 Supp. 2d Basal vs Post ND CPT (n=9) Two-tailed Student s paired t-test t 8 =0.3, p=0.75 Supp. 2d Basal vs Post ND SCH (n=12) Two-tailed Student s paired t-test t 11 =0.58, p=0.56 Supp. 4c Basal vs Post ND IPSCs (n=6) Two-tailed Student s paired t-test t 5 =-3.31, p=0.02 Supp. 4c Basal vs Post ND EPSCs (n=6) Two-tailed Student s paired t-test t 5 =2.6, p=0.04 Supp. 4d Basal vs Post ND IPSCs (n=6) Two-tailed Student s paired t-test t 5 =1.9, p=0.1 Supp. 4d Basal vs Post ND EPSCs (n=6) Two-tailed Student s paired t-test t 5 =-0.04, p=0.96 Supp. 5b Basal vs Post CNO IPSCs Control (n=6) Two-tailed Student s paired t-test t 5 =0.84, p=0.93 Supp. 5b Basal vs Post CNO IPSCs SCH (n=7) Two-tailed Student s paired t-test t 6 =0.45, p=0.3 Supp. 5b Basal vs Post CNO IPSCs AM251 (n=8) Two-tailed Student s paired t-test t 7 =0.1, p=0.87 Supp. 5b Basal vs Post CNO EPSCs control (n=8) Two-tailed Student s paired t-test t 7 =0.4, p=0.48 Supp. 5b Basal vs Post CNO EPSCs CPT (n=7) Two-tailed Student s paired t-test t 6 =-1.7, p=0.12 Supp. 5b Basal vs Post CNO EPSCs AM251 (n=6) Two-tailed Student s paired t-test t 5 =0.29, p=0.77 Supp. 5d Basal vs post CNO IPSCs CNO (n=8) Two-tailed Student s paired t-test t 7 =-1.17, p=0.3 Supp. 5d Basal vs Post SCH IPSCs SCH (n=3) Two-tailed Student s paired t-test t 2 =-0.12, p=0.91 Supp. 5d Basal vs post CNO EPSCs CNO (n=6) Two-tailed Student s paired t-test t 5 =0.56, p=0.59 Supp. 5d Basal vs Post CPT EPSCs CPT (n=4) Two-tailed Student s paired t-test t 3 =1.44, p=0.24 Supp. 6b Basal vs Post CNO Control (n=6) Two-tailed Student s paired t-test t 5 =-4.07, p=0.009 Supp. 6d Basal vs post CNO IPSCs CNO (n=9) Two-tailed Student s paired t-test t 8 =-4.32, p=0.002 Supp. 6d Basal vs Post SCH IPSCs SCH (n=3) Two-tailed Student s paired t-test t 2 =0.16, p=0.88 Supp. 6d Basal vs post CNO EPSCs CNO (n=6) Two-tailed Student s paired t-test t 5 =16.5, p= Supp. 6d Basal vs Post CPT EPSCs CPT (n=4) Two-tailed Student s paired t-test t 3 =1.47, p=0.23 Supp. 7a 1 st minute of Test 1: Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 18 =0.14, p=0.88 Supp. 7a 2 nd minute of Test 1: Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 18 =0.86, p=0.39 Supp. 7a 3 rd minute of Test 1: Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 18 =-1.19, p=0.24 Supp. 7a 1 st minute of Test 2: Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 18 =0.12, p=0.99 Supp. 7a 2 nd minute of Test 2: Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 18 =0.23, p=0.81 Supp. 7a 3 rd minute of Test 2: Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 18 =0.38, p=0.70 Supp. 7b Saline vs CNO CNO (n=10) and saline (n=10) Two-tailed Student s unpaired t-test t 17 =-1.2, p=0.24 Supp. 9b Basal vs Post ND CB1WT (n=6) Two-tailed Student s paired t-test t 5 =3.59, p=0.015 Supp. 9b Basal vs Post ND CB1R-/- (n=6) Two-tailed Student s paired t-test t 10 =3.67, p=0.014 Supplementary table 4. Student s t-tests performed in supplementary figures.
14 Post-hoc Holm- tests Figure Comparison Condition Test t and p values 1d Control Post ND vs other conditions Post ND Control (n=10), AM251 (n=7), GFAP-CB1R-/- (n=10), GFAP-CB1WT (n=9) and IP3R2-/- (n=8) Control vs AM251 (t=3.3, p=0.004), GFAP-CB1R-/- (t=3.26, p=0.003), GFAP-CB1WT (t=0.78, p=0.42) and IP3R2-/- (t=5.5, p<0.001) 1h Control Post ND vs other conditions Post ND control (n=22), AM251 (n=11), GFAP-CB1R-/- (n=7), GFAP-CB1WT (n=7), IP3R2-/- (n=10), MPEP-LY (n=10), SCH (n=7) and CPT (n=13) Control vs AM251 (t=4.71, p<0.001), GFAP-CB1R-/- (t=4.82, p<0.001), GFAP-CB1WT (t=1.86, p=0.18), IP3R2-/- (t=5.1, p<0.001), MPEP-LY (t=0.92, p=0.35), SCH (t=4.23, p<0.001) and CPT (t=1.13, p=0.45) 1l Control Post ND vs other conditions Post ND Control (n=24), AM251 (n=12), GFAP-CB1R-/- (n=11), GFAP-CB1WT (n=9), IP3R2-/- (n=10), MPEP-LY (n=13), CPT (n=9) and SCH (n=12) Control vs AM251 (t=3.8, p<0.001), GFAP-CB1R-/- (t=4.6, p<0.001), GFAP-CB1WT (t=0.17, p=0.98), IP3R2-/- (t=3.47, p=0.002), MPEP-LY (t=1.32, p=0.46), SCH (t=0.05, p=0.96) and CPT (t=4.45, p<0.001) 2c Control condition vs BAPTA conditions BAPTA (n=9) and control (n=7) BAPTA vs Control before ND (t=2.55, p=0.016) and after ND (t=7.47, p<0.001) 2e Control Post ND vs BAPTA conditions Post ND BAPTA (n=8) and control (n=9) BAPTA vs Control after ND (t=3.43, p=0.002) 2g Control Post ND vs BAPTA conditions Post ND BAPTA (n=8) and control (n=11) BAPTA vs Control after ND (t=2.84, p=0.007) 3d CNO-DREADDs post application vs other conditions post application CNO-DREADDs (n=7), ACSF-DREADDs (n=8) and CNO-naive (n=8) Post CNO puff in CNO- DREADDs vs ACSF-DREADDs (t=7.43, p<0.001) and CNO-naive (t=7.13, p<0.001) 3f Control Post CNO vs other conditions post CNO Control (n=7), SCH (n=7) and AM251 (n=8) Control vs SCH (t=4.9, p<0.001) and AM251 (t=1.56, p=0.12) 3h Control Post CNO vs other conditions post CNO Control (n=8), CPT (n=7) and AM251 (n=6) Control vs CPT (t=3.89, p<0.001) and AM251 (t=1.62, p=0.1) Supplementary table 5. Post-hoc Holm- tests.
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