Reduced transmission at PC-DCN synapses by GPR55 in slice and culture

(A) Image of patch-clamp recording from a DCN neuron in slice. A magnified image of the recorded neuron is shown as an inset (a patch pipette is indicated in blue). A pipette for electrical stimulation (magenta) was placed at the white matter. (B, C) Individual (gray) and averaged (black) traces (B), and time courses of eIPSC amplitude (C, left) and CV (C, right) before and after the AM251 application (at time 0), n = 6. (D) Image of dual whole-cell recordings from a presynaptic EGFP-labeled PC soma and its postsynaptic target neuron in culture. (E-H) Representative traces of eIPSCs before and 5 minutes after the application of AM251 (E), AM281 (F), AM251 in the presence of CID (G), or LPI (H). In E, presynaptic Na+ and the following K+ currents (INa+, IK+) at a presynaptic PC soma are also shown. (I) Time courses of eIPSC amplitude and CV. n = 6 (AM251), 5 (AM281), 5 (CID + AM251) and 5 cells (LPI). CID was applied from the beginning of the recording. The internal solution contained 0.5 mM EGTA. Data are shown as mean ± SEM.

Localization of GPR55 at PC axonal boutons

(A) Representative confocal fluorescent images of GFP and T1117 labeling in control and transfected cells with GPR55-targeting CRISPR/Cas9 vectors or Cas9-alone. White dotted lines in the T1117 images indicate the outlines of GFP-labeled axons. (B) Spatial profiles of T1117 signals in GFP(+) or GFP(-) boutons. Averaged data are shown as mean ± SEM. Fluorescence peak of GFP (for GFP(+) boutons) or T1117 (for GFP(-) boutons) was aligned to the position 0. (C) FT1117 signal density (averaged fluorescence per pixel) in GFP(+) boutons (control, 455; GPR55-KD, 161; Cas9-alone, 270 boutons) or that in GFP(-) boutons near GFP(+) ones (control, 124; GPR55-KD, 116; Cas9-alone, 104 boutons). Data for individual boutons (circles) and mean ± SD (squares) are shown. ***, p < 0.001; n.s., not significant.

Reduced vesicle exocytosis by GPR55 without changing presynaptic Ca2+ influx

(A) Image of direct patch-clamp recording from an EGFP-labeled PC axon terminal in culture. (B-E) Representative traces (B, D) and amplitude of presynaptic Ca2+ currents during 5 ms depolarization (ICa2+) and the resultant Cm increase (ΔCm, C, E) before and after the AM251 application in wild-type (n = 6) (B, C) or transfected cells with Cas9-alone (n = 5), or CRISPR/Cas9 for GPR55 (n = 6) (D, E). Both ICa2+ and ΔCm were normalized by the size of presynaptic Cm under the voltage-clamp. The internal solution contained 0.5 mM EGTA. Data are mean ± SEM. * p < 0.05; *** p < 0.001; n.s., not significant.

GPR55 has no effect on APs or postsynaptic responsiveness

(A) Image of direct recording from an EGFP-labeled PC axon terminal in culture. (B, C) Representative traces (B), amplitude and half-width (C) of APs recorded from PC terminals before and 5 minutes after the AM251 application (n = 5). (D) Image of mIPSC recordings from a neuron innervated by lots of EGFP-positive PC boutons in culture. (E) Representative traces of mIPSCs before and 5 minutes after the AM251 application. (F) Time courses of mIPSC amplitude and frequency (n = 4). At 0 min, AM251 was applied. The internal solution contained 0.5 mM EGTA. Data are shown as mean ± SEM. n.s., not significant.

Decrease in RRP vesicles by GPR55

(A) Representative traces of presynaptic ICa2+ (middle) and the resultant Cm increase (bottom) recorded with presynaptic 0.5 mM EGTA upon 1, 2, 5, 10, 20, or 50 ms of depolarization pulses (to 0 mV) (top) without (left) or with (right) AM251. Experiments were performed with a presynaptic patch pipette containing 0.5 mM or 5 mM EGTA. (B, C) Amplitude and time constant for activation of ICa2+ (B), and Cm change (C) upon depolarization pulses recorded without (black, 0.5 mM EGTA, n = 6; 5 mM EGTA, n = 6) or with AM251 (red, 0.5 mM EGTA, n = 7; 5 mM EGTA, n = 8). Single exponential fits for each are shown as dotted lines. Both ICa2+ and Cm change were normalized by the size of presynaptic Cm under the voltage-clamp. In B, data for individual cells (circles) and mean ± SEM (squares) are shown. (D) Ratio of Cm increase (Cm increase with AM251 divided by that in control) recorded with 0.5 or 5 mM presynaptic EGTA is plotted against the logarithm of pulse duration.

Halved velocity of exocytosis shown by pre- and postsynaptic paired recordings

(A) Image of paired recordings from a presynaptic EGFP-labeled PC bouton and its postsynaptic neuron (upper left), and representative traces of the presynaptic ICa2+ (upper right), IPSC (lower left), and velocity of vesicular fusion (bottom right; calculated by the deconvolution of IPSC trace) upon 2 ms depolarization before and after the AM251 application. (B, C) Amplitude of ICa2+ (B, left, calibrated by the presynaptic Cm under the voltage-clamp), IPSC (B, right), maximal release velocity (C, left) and synaptic delay (C, right) before and after application of AM251. The internal solution contained 0.5 mM EGTA. Data are mean ± SEM. * p < 0.05; n.s., not significant. n = 6 pairs.

Halved vesicular releases by GPR55 imaged with pHluorin

(A) Schematic illustration for imaging of vesicle exocytosis with synapto-pHluorin. Upon vesicular fusion at the AP arrival, pHluorin becomes fluorescent because of exposure to the neutral pH. NH4+ application forcefully neutralizes vesicle lumen. (B) Representative images of synapto-pHluorin fluorescence and the color-coded fluorescence increase upon 400 APs (ΔF400APs) in PC terminals before and after the AM251 application. The right-most panel shows the ratio of fluorescence increase after the AM251 application relative to that before, represented in pseudo-color. (C) Time courses of 400 APs-triggered pHluorin fluorescence changes before and after the AM251 application. (D) Plot of ΔF400APs along a PC axon (indicated as a magenta line in B) before and after the AM251 application. (E) Ratio of ΔF400APs after the AM251 application relative to that before is plotted against the ΔF400Aps before the AM251 application. Data from different cells are shown as different symbols. n = 8 cells. Dashed line represents data fitting with a function: y = 1.02e-0.0334x + 0.355, R2 = 0.506. (F) Images of pHluorin fluorescence and the color-coded fluorescence increases in a PC axon upon 400 APs and the following NH4Cl application (50 mM). (G) Time courses of synapto-pHluorin fluorescence changes upon 400 APs and the following NH4Cl application without or with AM251. Inset shows enlarged traces. (H) ΔF400APs is plotted against the 2/3rd of ΔF caused by NH4Cl (ΔFNH4Cl) for individual boutons without or with AM251. The effect of AM251 was predicted (blue) by conversion of dataset for control (black) based on the relationship shown in E, showing similar distribution to the actual data obtained with AM251 (red). Fitted line for control: y = 1.03x – 4.05, R2 = 0.704; for AM251, y = 0.307x + 10.9, R2 = 0.441. Data are mean ± SEM. *** p < 0.001; n.s., not significant.

Increase in reluctant vesicles insensitive to APs by GPR55

(A, B) Schematic illustration (top) and time courses of synapto-pHluorin fluorescence changes at PC axon varicosities upon the repetitive 400 APs trains (20 Hz) before and after the application of bafilomycin and KCl (finally 50 mM) (A), or ionomycin (B), followed by the NH4Cl application, in the presence or absence of AM251. Data are mean ± SEM. *** p < 0.001; n.s., not significant. (C) Fluorescent images of GCaMP7f expressed in PC axon terminals (left) and color-coded relative fluorescence increase (ΔF/F) upon 400 APs at 20 Hz (middle) or the following ionomycin application (right). (D, E) Distribution patterns along an axonal segment (D; indicated as the magenta line in C) or representative time courses (E) of ΔF/F caused by 400 APs or ionomycin.

No effects of GPR55 on IPSCs time courses and somatic Na+ currents

(A) 20-80% risetime of eIPSCs before and after application of AM251 or LPI. Data for individual cells are also shown (open circles). (B, C) Representative traces (B) and amplitudes (C) of Na+ current before and after application of AM251 or LPI. AM251, n = 6; LPI, n = 5. Data are mean ± SEM. n.s., not significant.

No effects of GPR55 on time courses of mIPSCs

20-80% risetime or half-width of mIPSCs before and after application of AM251 (n = 5). Data for individual cells are also shown (open circles). Data are mean ± SEM. n.s., not significant.

AM251 and LPI occlude the suppressive effects on vesicle release each other

(A, B) Amplitudes and time constants for activation of presynaptic ICa2+ (A), and Cm changes (B) upon depolarization pulses recorded with LPI only (orange, n = 6) or with LPI and AM251 (purple, n = 6). For comparison, control and AM251 data (shown in Figure 5) are also presented. Single exponential fits for each are shown as dotted or solid lines. Both ICa2+ and Cm change were normalized by the size of presynaptic Cm under the voltage-clamp. In A, data for individual cells (open circles) and mean ± SEM (squares) are shown. The presynaptic internal solution contained 0.5 mM EGTA.

GPR55-mediated suppression of release in PC boutons with 5 mM EGTA

Representative traces of presynaptic ICa2+ (middle) upon 1, 2, 5, 10, 20 and 50 ms of depolarization (to 0 mV, depicted on the top) of a PC bouton, and the resultant Cm increase (bottom) in the presence or absence of AM251. Recording was performed with a patch pipette containing 5 mM EGTA.

No effects of GPR55 on somatic Na+ currents upon repetitive stimulation at 20 Hz

(A) Representative traces of Na+ and K+ currents before and after application of AM251. (B) Amplitudes of Na+ current upon repetitive stimulation at 20 Hz before and after application of AM251 (n = 7). Data for individual cells are also shown (open circles). Data are mean ± SEM. n.s., not significant.

Ca2+ influx induced by AP trains, external high K+ or ionomycin

(A) Color-coded relative fluorescence increase of GCaMP7f (ΔF/F) upon 400 APs at 20 Hz (at the 1st round or the 8th) or the following KCl application (finally 50 mM). (B) Time course of GCaMP7f ΔF/F upon the repetitive 400 APs trains and KCl before and after application of bafilomycin. (C) GCaMP7f ΔF/F at boutons on axons upon 400 APs, application of KCl or of ionomycin (iono). n = 35 (400APs), 10 (KCl) and 20 (ionomycin) boutons, or 41 (400 APs), 15 (KCl) and 26 (ionomycin) sites in axon. Data are mean ± SEM.