Acquisition phase-specific contribution of climbing fiber transmission to cerebellum-dependent motor memory in mice
- Department of Physiology, Seoul National University College of Medicine, Republic of Korea
- Department of Biomedical Sciences, Seoul National University College of Medicine, Republic of Korea
- Memory Network Medical Research Center, Wide River Institute of Immunology, Seoul National University College of Medicine, Republic of Korea
- Neuroscience Research Institute, Seoul National University Medical Research Center, Republic of Korea
Figures
Figure 1
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). The error bars indicate ± SEM.
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Figure 1—source data 1
CF-EPSC.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig1-data1-v1.xlsx
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Figure 1—source data 2
Complex spike.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig1-data2-v1.xlsx
Figure 2
Optogenetic inhibition of climbing fiber transmission blocked Cs firing but not Ss firing in vivo.
(A) Schematic diagram of virus injection into IO and in vivo recording during 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 s traces without and with opto-stimulation, and the lower plots display a 1 s 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. The error bars indicate ± SEM.
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Figure 2—source data 1
IO_GFP.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig2-data1-v1.xlsx
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Figure 2—source data 2
IO_NpHR.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig2-data2-v1.xlsx
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Figure 2—source data 3
IO_GFP.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig2-data3-v1.xlsx
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Figure 2—source data 4
IO_NpHR.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig2-data4-v1.xlsx
Figure 3
Inhibition of climbing fiber transmission during memory acquisition of OKR.
(A) Illustration of the optokinetic reflex behavioral test (left) and experimental scheme (right). For training, visual stimulation was given by constant rotation of the screen, while the table, where a mouse’s head is fixed, remained stationary. For 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 s. (C) The learning curve from 0 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 58 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 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 to 50 min (right; Paired t-test, **** p < 0.0001), while the NpHR group showed no significant improvement of gain from 0 to 50 min (right; Paired t-test, p = 0.1046). The error bars indicate ± SEM.
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Figure 3—source data 1
OKR_Learning Curve_NpHR.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig3-data1-v1.xlsx
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Figure 3—source data 2
OKR_Learning Curve_GFP.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig3-data2-v1.xlsx
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Figure 3—source data 3
Change of Gain.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig3-data3-v1.xlsx
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Figure 3—source data 4
Pre vs Post with Opto.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig3-data4-v1.xlsx
Figure 4
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 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 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 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 s 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). The error bars indicate ± SEM.
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Figure 4—source data 1
OKR_Learning Curve_NpHR_0 min.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data1-v1.xlsx
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Figure 4—source data 2
OKR_Learning Curve_GFP_0 min.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data2-v1.xlsx
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Figure 4—source data 3
Consolidation Percentage_0 min.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data3-v1.xlsx
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Figure 4—source data 4
OKR_Learning Curve_NpHR_6 hr.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data4-v1.xlsx
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Figure 4—source data 5
OKR_Learning Curve_GFP_0 min.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data5-v1.xlsx
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Figure 4—source data 6
Consolidation Percentage_6 hr.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data6-v1.xlsx
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Figure 4—source data 7
Retrieval_24 hr_NpHR.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data7-v1.xlsx
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Figure 4—source data 8
Retrieval_24 hr_GFO.
- https://cdn.elifesciences.org/articles/95838/elife-95838-fig4-data8-v1.xlsx
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Other | AAV9.CaMKIIα.EGFP | Addgene | RRID:Addgene_50469 | Plasmid #50469 |
| Other | AAV1. CaMKIIα.eNpHR 3.0.EYFP | Addgene | RRID:Addgene_26971 | Plasmid #26971 |
| Chemical compound, drug | Zoletil | Virvac | ||
| Chemical compound, drug | Rompun | Bayer | ||
| Chemical compound, drug | Dexamethasone | Samyang Pharmaceutical | ||
| Chemical compound, drug | Meloxicam | Boehringer Ingelheim | ||
| Software, algorithm | ZEN | Zeiss | RRID:SCR_013672 | |
| Software, algorithm | Fiji | http://fiji.sc | RRID:SCR_002285 | |
| Software, algorithm | Patchmaster | HEKA | RRID:SCR_000034 | |
| Software, algorithm | Igor Pro | WaveMetrics | RRID:SCR_000325 | |
| Software, algorithm | GraphPad Prism | GraphPad | RRID:SCR_002798 |
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Acquisition phase-specific contribution of climbing fiber transmission to cerebellum-dependent motor memory in mice
eLife 13:RP95838.
https://doi.org/10.7554/eLife.95838.4
