Climbing fiber synapses rapidly and transiently inhibit neighboring Purkinje cells via ephaptic coupling

Abstract

Climbing fibers from the inferior olive make strong excitatory synapses onto cerebellar Purkinje cell (PC) dendrites and trigger distinctive responses known as complex spikes. We found that, in awake mice, a complex spike in one PC suppressed conventional simple spikes in neighboring PCs for several milliseconds. This involved a new ephaptic coupling, in which an excitatory synapse generated large negative extracellular signals that nonsynaptically inhibited neighboring PCs. The distance dependence of complex spike–simple spike ephaptic signaling, combined with the known CF divergence, allowed a single inferior olive neuron to influence the output of the cerebellum by synchronously suppressing the firing of potentially over 100 PCs. Optogenetic studies in vivo and dynamic clamp studies in slice indicated that such brief PC suppression, as a result of either ephaptic signaling or other mechanisms, could effectively promote firing in neurons in the deep cerebellar nuclei with remarkable speed and precision.

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Fig. 1: CSs in a PC rapidly and transiently suppress SS firing in neighboring PCs in awake mice.
Fig. 2: Extracellular potentials generated by SSs and CSs from PCs in vivo and in brain slice.
Fig. 3: CF stimulation evokes outward current in nearby PCs regardless of whether axons are intact.
Fig. 4: Reversing the CF EPSC by AMPAR activation in a PC reverses extracellular currents and currents in neighboring cells.
Fig. 5: Differential effects of extracellular dendritic and axonal stimulation.
Fig. 6: Mimicking extracellular signals that accompany a CS evokes outward currents and inhibits SSs.
Fig. 7: Dynamic clamp studies indicate that brief pauses in PC firing can effectively promote firing of neurons in the DCN.
Fig. 8: Brief suppression of PC SSs increases firing in the DCN and thalamus.

Data availability

The data that support the findings are available upon reasonable request from the corresponding author.

Code availability

Analyses used in this study are largely standard approaches for this type of data. The code that supports these findings is available upon request from the corresponding author.

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Acknowledgements

This work was supported by a National Institutes of Health grant to W.G.R. (R35NS097284), an NIH postdoctoral fellowship to C.H.C. (F32NS101889), the Stuart & Victoria Quan Fellowship in Neurobiology to C.G. and a National Science Foundation Graduate Research Fellowship under grant 1745303 to M.M.K. We thank the Neurobiology Imaging Facility for the consultation and instrument availability that supported this work. This facility is supported in part by the Neural Imaging Center as part of NINDS P30 Core Center grant NS072030. We thank T. Osorno for the slice two-photon imaging. We thank B. Bean, L. Witter and S. Rudolph for comments on the manuscript and M. Xu-Friedman for help with dynamic clamp studies.

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Contributions

K.-S.H., C.H.C. and W.G.R. conceived the experiments. C.H.C. conducted most of the experiments in Figs. 1 and 8, and Extended Data Figs. 1–4 and 6; M.M.K. performed the experiments in Fig. 7; and K.-S.H. conducted all other experiments. K.-S.H., C.H.C., M.M.K. and C.G. performed the analyses. K.-S.H., C.H.C. and W.G.R. wrote the manuscript.

Corresponding author

Correspondence to Wade G. Regehr.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks Chris I. De Zeeuw and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Subtraction of complex spikes from raw traces.

a, (top) Simple spikes and complex spikes recorded on a same cell (PC1 SS) are aligned to PC1 CS (red). (bottom) Complex spikes from PC1 were subtracted. b, (top) Simple spikes simultaneously recorded on a neighboring site (PC2 SS) are aligned to PC1 CS. Complex spikes from PC1 (PC1 CS, red) were detected in neighboring site (PC2). (bottom) Complex spikes from PC1 were subtracted.

Extended Data Fig. 2 Complex spikes in a Purkinje cell inhibit simple spikes in the same PCs in awake mice.

(a, a) Average complex spike recorded on a single site (PC1 CS). (a, b) Simple spikes simultaneously recorded on the same cell (PC1 SS) are aligned to PC1 CS. (a, c) Raster plot of simple spikes from (a, b). (a, d) Histogram summarizing the data in (a, b). (a, e) Average of firing rate of simple spikes from the same PCs (PC1 SS) after complex spikes from PC1 (PC1 CS). Shaded gray is SEM. (b, a-d) Four example pairs of nearest neighbor cells showing histograms of PC1 and PC2 SSs relative to CSs in PC1. Data are mean ± s.e.m.

Extended Data Fig. 3 Optogenetic silencing of MLIs does not disrupt CS suppression of SSs in neighboring cells.

CKit cre mice54 were injected with 250 nl of AAV9-Ef1a-DIO eNpHR 3.0-EYFP nine sites in the cerebellum. Experiments were conducted 2–3 weeks later (see Methods). Slices were cut in order to evaluate expression and or ability to suppress MLI firing a, b. a, Image of eNpHR 3.0-EYFP labeling showing an expression pattern that is characteristic of membrane labelling of MLIs, including the pinceaux associated with basket cells. 3 times reproduced. b, c, On cell recordings were used to assess the effect of light on MLI firing, and we found that firing was eliminated in all MLIs. After waiting for 2–3 weeks, in vivo recordings proceeded similarly to experiments shown in Fig. 1. Once a pair was located, 5 s illumination was alternated with 5 s of no light, and this continued for at least 30 minutes in order to record sufficient complex spikes for each condition. d, Light increased PC firing of a pair of closely spaced (25 µm) cells. e, CS induced decreases in SS firing rate are shown for control condition (no light, left) and when MLI firing was suppressed with light (right). f, The effects of light on PC firing is shown for another pair of cells (50 µm). g, CS induced decreases in SS firing rate are shown for control condition (no light, left) and when MLI firing was suppressed with light (right).

Extended Data Fig. 4 Simple spikes promote synchrony whereas complex spikes suppress firing for neighboring cells.

The CC firing of the PC pairs of Fig. 1 were analyzed as described previously20. a, Average firing rate of simple spikes from neighboring PCs (PC2 SS) after simple spikes from PC1 (PC1 SS). Recording sites were separated by 25 µm (top), 50 µm (middle), and more than 75 µm (bottom). b, Summary of normalized firing rates of PC2 SS after PC1 SS as a function of distance between recording sites. c, Summary of inhibition of PC2 SS by PC1 CS (from Fig. 1) as a function of synchrony between PC1 SS and PC2 SS. Box plots indicate median and interquartile range with the whiskers indicating the range.

Extended Data Fig. 5 Subtracted extracellular voltage responses by complex spikes.

(top) Complex spikes by CF stimulation. A threshold stimulus intensity was used that stochastically evoked successes (left) or failures (middle) (individual trials: gray and average: black). Average success – average failure is shown (right) (bottom) Extracellular signals near proximal dendrite by successful stimulation of the CF input (left), but no extracellular signals were observed when stimulation failed to evoke complex spikes (middle). Average success – average failure is shown (right).

Extended Data Fig. 6 Example light-evoked responses in PCs (a), DCN neurons (b), and in the motor thalamus (c).

The bottom row shows example cells in each area that did not respond significantly to stimulation.

Extended Data Fig. 7 Schematic of PCs affected by CS inputs.

Model showing how PCs are affected by a single inferior olive neuron. Climbing fibers typically have 7 different branches and each contacts a single PC. Based on PC packing density55,56,57, a hexagonal packing pattern, each individual branch will ephaptically inhibit about 18 neighboring PCs while directly exciting 7 PCs.

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Han, K., Chen, C.H., Khan, M.M. et al. Climbing fiber synapses rapidly and transiently inhibit neighboring Purkinje cells via ephaptic coupling. Nat Neurosci (2020). https://doi.org/10.1038/s41593-020-0701-z

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