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Ultra-rapid axon-axon ephaptic inhibition of cerebellar Purkinje cells by the pinceau

Nature Neuroscience volume 17, pages 289–295 (2014)Cite this article

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Abstract

Excitatory synaptic activity in the brain is shaped and balanced by inhibition. Because inhibition cannot propagate, it is often recruited with a synaptic delay by incoming excitation. Cerebellar Purkinje cells are driven by long-range excitatory parallel fiber inputs, which also recruit local inhibitory basket cells. The axon initial segment of each Purkinje cell is ensheathed by basket cell axons in a structure called the pinceau, which is largely devoid of chemical synapses. In mice, we found at the single-cell level that the pinceau mediates ephaptic inhibition of Purkinje cell firing at the site of spike initiation. The reduction of firing rate was synchronous with the presynaptic action potential, eliminating a synaptic delay and allowing granule cells to inhibit Purkinje cells without a preceding phase of excitation. Axon-axon ephaptic intercellular signaling can therefore mediate near-instantaneous feedforward and lateral inhibition.

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Figure 1: Nonsynaptic inhibition of the Purkinje cell.
Figure 2: Extracellular voltage field at the pinceau.
Figure 3: Two components of the pinceau field.
Figure 4: Pinceau model.
Figure 5: Extracellular voltage clamp.
Figure 6: Opposing direct excitation.

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Acknowledgements

We thank D. Attwell, M. Beato, N. Brunel, M. Casado, M. Diana, D. DiGregorio, N. Emptage, P. Faure, A. Feltz, V. Hakim, C. Léna, T. Margie, E. Schwartz, C. Sotelo, S. Supplisson and M. Wassef, as well as members of the Barbour laboratory and the IBENS Neuroscience Section, for helpful discussions and/or critical comments on the manuscript. This work was supported by the Agence Nationale de la Recherche (ANR-08-SYSC-005, ANR-08-BLAN-0023) and the Ecole Normale Supérieure (fellowship to A.B.).

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  1. Ecole Normale Supérieure, IBENS, Paris, France

    Antonin Blot & Boris Barbour

  2. CNRS, UMR 8197, Paris, France

    Antonin Blot & Boris Barbour

  3. INSERM, U1024, Paris, France

    Antonin Blot & Boris Barbour

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  1. Antonin Blot
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A.B. performed all of the experiments and analyses. B.B. designed and built the custom electronics. Both authors designed the experiments and analyses, interpreted the results, developed the model, and wrote the manuscript.

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Correspondence to Boris Barbour.

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Integrated supplementary information

Supplementary Figure 1 The pinceau.

(a), Diagram of Purkinje cells, basket cells and granule cells in the cerebellar cortex. (b), Organisation of the basket and pinceau. BC, basket cell; GC, granule cell; GCL, granule cell layer; ML, molecular layer; PF, parallel fibre; PC, Purkinje cell; PCL, Purkinje cell layer.

Supplementary Figure 2 Small effect of an action potential in the basket.

The Purkinje cell soma is surrounded by the axons of basket cells (diagram in Supplementary Fig. 1a). The propagation of the action potential of the interneurone in this perisomatic basket could itself affect the somatic voltage of Purkinje cell and thus its firing, though the control cells in Fig. 1i,k,m,o suggest this action is weak, if present. To gain insight into this possible mechanism, we modelled changes of extracellular voltage at the soma (black) by changing VSE and compared the result to the effects of the pinceau already modelled (orange) and of action potentials in both compartments (green). Spike propagation in the basket can be active, because an extracellular negativity could be observed there (Fig. 2c, traces 9 and 10). To estimate an upper limit on the contribution of an action potential in the basket we set VSE to twice the recorded trace with the maximal negativity (Fig. 2c trace 7), thus assuming a spatially uniform negativity of −70 μV around the soma. (both the amplitude and uniformity of this signal cause overestimation of its effect). This change of extracellular potential capacitivelyhyperpolarises the Purkinje cell soma (b) by 20 μV. The somatic transmembrane potential is depolarised (c). The intracellular hyperpolarisation propagates to the axon (d) with very little decrement. These changes do not affect the intra-pinceau potential (e), producing a net transmembranehyperpolarisation of the Purkinje cell axon (f). However, even in this overestimate, the hyperpolarisation caused in the Purkinje cell axon by the basket is small compared to the pinceau effect and the firing of the Purkinje cell is only weakly modulated (g). The basket-induced signals described above would render impossible the intracellular detection of the pinceau signal, which we showed in Fig. 4 would in any case be undetectably small. The only method able to demonstrate the pinceau effect on the Purkinje cell is therefore measurement of its effect on firing.

Supplementary Figure 3 Detection method.

The extracellular field induced by the spikes of basket cells was computed by averaging recording periods (triggered on basket cell action potentials) that were devoid of Purkinje cell spikes (a), (c) (blue). We subtracted from this first trace an average of periods with no spike in either the Purkinje cell or the Basket cell (b), (c) (green). The resulting field (c) (yellow) was subtracted from every trace (d) and Purkinje cell spikes were then detected using a simple threshold (d) (red) on these snippets (± 20 ms around each basket cell spike) after baseline subtraction by a 5 ms high-pass box filter.

Supplementary Figure 4 Average spike waveform of Purkinje cell.

(a) Average spike of the Purkinje cell in Supplementary Fig. 3. (b) Expanded view of the dashed box in a. Purkinje cell spikes are followed by a long-lasting overshoot. The repetitive firing of the Purkinje (at 70 Hz) explains the slope preceding the spike. Average of 137,672 spikes.

Supplementary Figure 5 Sensitivity analysis of the model.

Effects of varying the principal parameters of the model (Fig. 4) on the membrane potential of the Purkinje cell axon (VPA – VP). Changes of (a): RI, (b): CBA, (c): GK, (d): RL, (e): CPA, (f): RAS. Colour map: multiplicative change of the value indicated in each panel and used in the paper (green curves). Scale bar: 50 μV, 1 ms.

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Blot, A., Barbour, B. Ultra-rapid axon-axon ephaptic inhibition of cerebellar Purkinje cells by the pinceau. Nat Neurosci 17, 289–295 (2014). https://doi.org/10.1038/nn.3624

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