Reduced transmission at PC-DCN synapses by GPR55

(A) Image of patch-clamp recordings from a DCN neuron in slice. A magnified image of the recorded neuron is shown as inset (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), amplitudes (C, left, open and filled squares) and CV (C, right) of eIPSCs before and after application of AM251 in slices (n = 6). Open circles connected by lines represent individual cells. (D) Image of dual whole-cell recordings from a presynaptic EGFP-labeled PC soma and its postsynaptic target neuron in cerebellar culture. (E-J) Representative traces (E, G, I), amplitude and CV (F, H, J) of eIPSCs at a postsynaptic cell before and after application of AM251 (E, F, n = 6), LPI (G, H, n = 5), or AM251 in the presence of CID (I, J, n = 5). In E, presynaptic Na+ and the following K+ currents (INa+, IK+) at a presynaptic PC soma are also shown. (K) Image of mIPSC recordings from a neuron innervated by lots of EGFP-positive PC boutons in culture. (L, M) Representative traces (L), amplitude and frequency (M) of mIPSCs before and after the AM251 application. n = 5 cells. Data are mean ± SEM. * p < 0.05; 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, C) Representative traces (B), amplitude and half-height width (C) of APs recorded from PC terminals before and after the AM251 application. n = 5 boutons. (D, E) Representative traces (D) and amplitude (E) of presynaptic Ca2+ currents (ICa2+) and Cm increase upon 5 ms depolarization before and after the AM251 application. n = 6 boutons. Both ICa2+ and Cm increase were normalized by the size of presynaptic Cm under the voltage-clamp. Data are mean ± SEM. * p < 0.05; n.s., not significant.

Decrease in RRP vesicles by GPR55

(A) Representative traces of presynaptic ICa2+ (middle) and the resultant Cm increase (bottom) upon 1, 2, 5, 10, 20, or 50 ms of depolarization pulses (to 0 mV) (top) without (left) or with (right) AM251. Recordings were performed by a presynaptic patch pipette containing 0.5 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 (open 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 duration of log depolarization pulse.

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+ (right middle), IPSC (lower left), and velocity of vesicular fusion (calculated by the deconvolution of IPSC trace) upon 2 ms depolarization before and after the AM251 application (right bottom). (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 (connected by lines) application of AM251. 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 of 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 AM251 application. (D) Plot of ΔF400APs along a PC axon (indicated as the 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 upon 400 APs and the following NH4Cl application (50 mM) in a PC axon. (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 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 = 0.307 + 10.9, R2 = 0.441. Data are mean ± SEM. *** p < 0.001; n.s., not significant.

Increase of reluctant vesicle insensitive to APs by GPR55

(A, B) Schematic illustration (top) and time courses of synapto-pHluorin fluorescence changes upon the repetitive 400 APs trains (20 Hz) before and after 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 pattern in an axonal segment (D, along a magenta line indicated in C) and representative time course (E) at the middle bouton of ΔF/F caused by 400 APs or ionomycin. (F) ΔF/F of GCaMP7f at PC boutons (n = 20 boutons from 4 PCs) or axonal segments (n = 26 segments) upon 400 APs or application of ionomycin.

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

(A) 20-80 % risetime of eIPSCs before and after application of AM251 (left, n = 6) or LPI (right, n = 5). 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 mIPSCs time courses

Representative averaged traces (from 20 events for each, A) and 20-80 % risetime or half-width (B) 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.

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.

Repetitive somatic Na+ currents unaffected by GPR55 activation

Representative traces (A) and amplitudes (B) of somatic Na+ currents at 20Hz before and after application of AM251 (n = 7). In B, data for individual cells are also shown (open circles). Averaged data represent mean ± SEM. n.s., not significant.