Inhibition gates supralinear Ca2+ signaling in Purkinje cell dendrites during practiced movements
- Max Planck Florida Institute for Neuroscience, United States
- Gunma University Graduate School of Medicine, Japan
Figures
Figure 1 with 3 supplements
Climbing fiber-evoked Ca2+ signals in PCs during behavior.
(A) Head-fixed mice were trained to lick water from a port, cued by an audible tone, using the procedure shown. (B) Across-trial distribution of lick probability, aligned to the delivery of water, …
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Figure 1—source data 1
Source data for panels D and G.
- https://doi.org/10.7554/eLife.36246.008
Figure 1—figure supplement 1
Ca2+ activity measurements in parallel fibers.
(A) 2pLSM image from Crus II of an awake mouse previously injected with AAV containing GCaMP6f under control of the synapsin promoter. GCaMP6f expression is apparent in both parallel fibers (PFs) …
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Figure 1—figure supplement 1—source data 1
Source data for panels B and E.
- https://doi.org/10.7554/eLife.36246.004
Figure 1—figure supplement 2
Analysis of non-isolated PC dendritic Ca2+ events.
(A) Comparison of the fraction of responses composed of single isolated events or those containing overlapping events, grouped closely in time. Responses were categorized whether they occurred …
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Figure 1—figure supplement 2—source data 1
Source data for panels A-C.
- https://doi.org/10.7554/eLife.36246.006
Figure 1—figure supplement 3
PC Ca2+ event amplitudes do not co-vary with the level of MLI activity.
Examples from three mice plotting the amplitudes of all identified PC Ca2+ events (each point is an individual dendritic Ca2+ event) and the corresponding Ca2+ activity measurement in surrounding …
Figure 2 with 4 supplements
MLIs suppress climbing fiber-evoked dendritic Ca2+ signals in PCs during licking.
(A) Following an audible cue, head-fixed mice licked from a port during in vivo imaging with 2pLSM. In some sessions, CNO was administered by intraperitoneal injection prior to the start of the …
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Figure 2—source data 1
Source data for panels C-E and G.
- https://doi.org/10.7554/eLife.36246.016
Figure 2—figure supplement 1
MLI-mediated suppression of non-isolated PC Ca2+ events.
(A) Average PC dendritic Ca2+ responses composed of two climbing fiber-evoked events in close temporal apposition (within 150–200 ms). Peak amplitudes were significantly larger in CNO than in …
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Figure 2—figure supplement 1—source data 1
Source data for panels A and B.
- https://doi.org/10.7554/eLife.36246.011
Figure 2—figure supplement 2
Chemogenetic suppression of MLIs increases trial-averaged Ca2+ activity in PC dendrites.
(A) Trial-averaged Ca2+ activity in PCs dendrites during cued bouts of water consumption. Measurements were obtained in control or during chemogenetic suppression of MLI activity. (11 to 35 PCs each …
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Figure 2—figure supplement 2—source data 1
Source data for panels A and B.
- https://doi.org/10.7554/eLife.36246.013
Figure 2—figure supplement 3
Chemogenetic suppression of MLIs in Crus II does not affect licking rates.
(A) Average lick rate during cued bouts of licking in control sessions and those with chemogenetic disinhibition of the molecular layer (p=0.32; paired Student’s t-test; N = 7 mice). In all of our …
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Figure 2—figure supplement 3—source data 1
Source data for panels B and C.
- https://doi.org/10.7554/eLife.36246.015
Figure 2—video 1
Licking behavior in control and the disinhibited condition.
Left (hM4d alone) and right (hM4d + CNO) showing example licking behavior. Images were collected at 2000 Hz and slowed down 10-fold for replay. Total time is 1.5 s.
Figure 3
MLIs broadly influence climbing fiber-evoked Ca2+ signaling in PCs.
(A) The across-session change in trial-averaged PC Ca2+ activity, colored-coded based on the extent to which chemogenetic disinhibition affected responses during movement (MDM ratio; see Materials …
Figure 4
Disinhibition does not affect presynaptic climbing fiber activity.
(A) AAVs containing genetically encoded activity reporters and effectors were injected in the inferior olive and lobule Crus II of Kit::Cre mice, respectively. Image from fixed tissue showing …
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Figure 4—source data 1
Source data for panel C.
- https://doi.org/10.7554/eLife.36246.020
Figure 5 with 2 supplements
Feed-forward inhibition attenuates supralinear Ca2+ signaling in PC dendrites.
(A) In acute slices, parallel fibers were stimulated in conjunction with climbing fibers during whole-cell patch recording from PCs in lobule Crus II. Ca2+ imaging was performed using 2pLSM in PC …
Figure 5—figure supplement 1
Optogenetic elimination of MLI-mediated feed-forward inhibition.
(A) Confocal image from the cerebellum of a Kit::Cre mouse injected with AAV containing Cre-dependent GtACR2-YFP showing transduction in MLIs (DAPI counterstain in blue). During a cell-attached …
Figure 5—figure supplement 2
Temporal dynamics of conjunctive parallel fiber-climbing fiber Ca2+ signaling in PC dendrites.
(A) The timing between the parallel fiber tetanus and the climbing fiber stimulus was systematically varied. Supralinear enhancement of climbing fiber-evoked Ca2+ transients by preceding parallel …
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Figure 5—figure supplement 2—source data 1
Source data for panel B.
- https://doi.org/10.7554/eLife.36246.024
Figure 6 with 1 supplement
Activity-dependent recovery of supralinear climbing fiber Ca2+ signaling is sensitive to MLI inhibition.
(A) Acute slice recording configuration. In a subset of experiments, bReaChES was expressed in MLIs by Cre-dependent AAV in Kit::Cre mice. (B) Comparison of average climbing fiber-evoked Ca2+ …
Figure 6—figure supplement 1
Influence of optogentic MLI activation on PC dendritic and somatic excitation.
(A) Left: diagram of recording configuration. Right: average Ca2+ activity responses in a PC dendrite (corresponding to the same site as Figure 6E) from trials with a parallel fiber tetanus (PF; …
Tables
Key resources table
| Reagent type or resource | Designation | Source or reference | Additional information |
|---|---|---|---|
| strain, (Mus Musculus) | Kit::Cre | Amat et al., 2017 | on C57Bl/6 background |
| transfected construct | AAV1-Pcp2.4-GCaMP6f | University of North Carolina | custom |
| transfected construct | AAV1-Pcp2.4-FLPo | University of North Carolina | custom |
| transfected construct | AAV1-CAG-Flex(FRT) rev-RCaMP2 | University of North Carolina | custom |
| transfected construct | AAV1-CAG-Flex(loxP) rev-RCaMP2 | University of Pennsylvania | custom |
| transfected construct | AAV1-CAG-Flex(FRT) rev-GCaMP6f | University of North Carolina | custom |
| transfected construct | AAV1-CAG-Flex(loxP) rev-ChR2.HA-2a-hM4d | ViGene | custom |
| transfected construct | AAV1-Syn-GCaMP6f | University of Pennsylvania | AV-1-PV2822 |
| transfected construct | AAV1-CaMKIIα-GCaMP6f | University of Pennsylvania | AV-1-PV2822 |
| transfected construct | AAV1-EF1α-Flex(loxP) rev-GtACR2.eYFP | ViGene | custom |
| transfected construct | AAV5-EF1α-Flex(loxP) rev-bReachES-TS-YFP | University of North Carolina | shelf |
| antibody | anti HA | Abcam | #ab9110 |
| software, algorithm | Prism | GraphPad | Statistical analysis |
| software, algorithm | Matlab | Mathworks | Image analysis |
| software, algorithm | ImageJ | NIH | Image analysis |
| software, algorithm | bControl | Carlos Brody, Princeton | Behavior control |
| software, algorithm | ScanImage | Vidrio Technologies | Microscope control |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36246.027
