Optogenetic inhibition of climbing fiber transmission derived from the inferior olive.

A. Schematic diagram showing virus injection for optogenetic manipulation of CF transmission.

B. Bi. A virus is expressed specifically only in the IO region (IO, inferior olive; py, pyramidal tract; ml, medial lemniscus). Scale bar, 100 μm. Bii. Virus expression along CF in the cerebellar flocculus region. (GCL, granule cell layer; PCL, Purkinje cell layer; ML, molecular layer; FL, flocculus). Scale bar, 100 μm.

C. A scheme of optogenetic suppression using a yellow laser (593 nm, 3–5 mW) while recording CF EPSCs under voltage clamp mode.

D. Left, representative traces of CF EPSCs of the GFP (top) and NpHR group (bottom) with (dark line) and without (light line) opto-stimulation. Right, Quantitative analysis of suppression of CF EPSCs (GFP: n = 10 cells/2 mice, NpHR: n = 9 cells/5 mice, *** p < 0.001, Unpaired t-test).

E. A scheme of optogenetic suppression while recording complex spikes under current clamp mode.

F. Representative traces of complex spike of the GFP (top) and NpHR group (bottom) with (dark line) and without (light line) opto-stimulation. Quantitative analysis of number of complex spike spikelets (GFP: n = 8 cells/1 mouse, NpHR: n = 8 cells/5 mice, ** p = 0.002; Paired t-test).

Optogenetic inhibition of climbing fiber transmission bl ked Cs firing but not Ss firing in vivo

A. Schematic diagram of virus injection into IO and in vivo recordin ring optogenetic manipulation of CF transmission. Arrow heads indicate NpHR-expressing CF terminals in the molecular layer of the cerebellar cortex. Scale bar, 50 μm

B. Representative electrophysiological recording traces of PC firing from the GFP and NpHR groups, without (OFF) and with (ON) yellow (593 nm) opto-stimulation. The upper plots show 10-second traces without and with opto-stimulation, and the lower plots display a 1-second segment from the respective upper traces. Asterisks denote Cs

C. Comparison of Ss firing rates during optogenetic inhibition. For the GFP group, firing rates with laser off (n = 11 cells from 4 mice) versus on (n = 11 cells from 4 mice) were not significantly different (Mann-Whitney test, P = 0.8594). Similarly, for the NpHR group, no significant difference was observed between OFF (n = 10 cells from 6 mice) and ON (n = 10 cells from 6 mice) conditions (Mann-Whitney test, p > 0.9999).

D. Comparison of Cs firing rates during optogenetic inhibition. In the GFP group, no significant difference was observed between OFF (n = 8 cells from 4 mice) and ON (n = 8 cells from 4 mice) conditions (Mann-Whitney test, p = 0.4563). In the NpHR group, Cs firing rates were significantly reduced during opto-stimulation (OFF, n = 6 cells from 6 mice; ON, n = 6 cells from 6 mice; Mann-Whitney test, ** p = 0.0022). Of note, 6 out of 8 recorded cells were responsive to optogenetic suppression.

Inhibition of climbing fiber transmission during memory acquisition of OKR.

A. Illustration of the optokinetic reflex behavioral test (left) an experimental scheme (right). For training, visual stimulation was given by constant rotat n of the screen, while the table, where a mouse’s head is fixed, remained stationary. r day 1, a mouse was trained for a total of 50 minutes, and on day 2, an amount of sustained memory was validated via gain check. Scale bar, 100 μm

B. Representative traces of the screen (black sinusoidal curve) and eye movements (grey sinusoidal curve) divided into the GFP (i) and NpHR (ii) groups. Top traces (pre) were acquired before learning, and bottom traces (post) were obtained after 50 min of learning. The vertical scale bar represents 10 degrees per second and the horizontal scale bar represents 0.5 seconds.

C. The learning curve from 0 min to 50 min, and +24 hr period. Change of gain is indicated at each time point (+10 min increment). Yellow boxes indicate opto-stimulation (12.5 mW for 10 min/session, total of 5 sessions). The left graph represents the GFP (n = 8) and the right graph indicates the NpHR (n = 9) group.

D. Comparison of gain changes between the GFP and NpHR groups (GFP, n = 8 mice; NpHR, n = 9 mice). Percentages of gain increment from 0 min to 50 min were calculated. The change of gain of the GFP group was significantly larger than that of the NpHR group (left; Unpaired t-test, p = 0.0007). The gain of the GFP group was significantly increased from 0 min to 50 min (right; Paired t-test, **** p < 0.0001), while the NpHR group showed no significant improvement of gain from 0 min to 50 min (right; Paired t-test, p = 0.1046).

Inhibition of climbing fiber transmission during memory consolidation and retrieval phase.

A. Illustration of the experimental scheme. 10 min of optokinetic reflex training was repeated 5 times, and then opto-stimulation (12.5 mW) was given at 0 min post-learning for 30 min.

B. The learning curve from 0 min to 50 min, and +24 hr period. Change of gain is indicated at each time point (+10 min increment). A yellow box indicates opto-stimulation at 0 min post-learning. The left graph represents the GFP and the right graph indicates the NpHR group.

C. Comparison of consolidation percentage between the GFP and NpHR groups (GFP group, n = 7 mice; NpHR group, n = 9 mice). The percentage of sustained memory after 24 hr was calculated. No difference was observed between the groups (p = 0.3173; Unpaired t-test).

D. Illustration of the experimental scheme. 10 min of optokinetic reflex training was repeated 5 times, and then opto-stimulation (12.5 mW) was given at 0 min post-learning for 6 hr.

E. The learning curve from 0 min to 50 min, and +24 hr period. Change of gain is indicated at each time point (+10 min increment). A yellow box indicates opto-stimulation at 0 min post-learning. The left graph represents the GFP group, and the right graph indicates the NpHR group.

F. Comparison of gain changes between GFP and NpHR groups (GFP group, n = 6 mice; NpHR group, n = 6 mice). Percentages of gain increment from 0 min to 50 min were calculated. No difference was observed between the groups (Unpaired t-test, p = 0.6405).

G. Illustration of the experimental scheme. Following proper motor learning on day 1, the remaining gain value was checked on day 2, after testing 1st gain retrieval without opto-stimulation, 2nd gain retrieval was tested with opto-stimulation to validate the effect of CF inhibition on gain retrieval (opto-stimulation was given 3 times for 24 seconds each at 12.5 mW).

H. Gain retrievals before and during opto-stimulation were compared. The left graph represents the GFP group, and the right shows the NpHR group (GFP group, n = 6 mice; NpHR group, n = 8 mice). There was no significant variation between the groups (NpHR group, p = 0.2439; GFP group, p = 0.3180; Paired t-test).