Optimizing Optogenetic Activation of Purkinje Cell Axons to Investigate the Purkinje Cell - DCN Synapse.

Kim M Gruver,Alanna J Watt

doi:10.3389/fnsyn.2019.00031

Abstract

Optogenetics is a state-of-the-art tool for interrogating neural circuits. In the cerebellum, Purkinje cells serve as the sole output of the cerebellar cortex where they synapse on neurons in the deep cerebellar nuclei (DCN). To investigate the properties of this synaptic connection, we sought to elicit time-locked single action potentials from Purkinje cell axons. Using optical stimulation of channelrhodopsin-2 (ChR2)-expressing Purkinje cells combined with patch-clamp recordings of Purkinje cells and DCN neurons in acute cerebellar slices, we determine the photostimulation parameters required to elicit single time-locked action potentials from Purkinje cell axons. We show that axons require longer light pulses than somata do to elicit single action potentials and that Purkinje cell axons are also more susceptible to light perturbations. We then demonstrate that these empirically determined photostimulation parameters elicit time-locked synaptic currents from postsynaptic cells in the DCN. Our results highlight the importance of optimizing optogenetic stimulation conditions to interrogate synaptic connections.

Highlights

Optogenetics is a powerful tool that has transformed the investigation of neural circuits
We find that focal illumination of Purkinje cell axons requires longer light pulses than somata, and that axons are more susceptible to perturbations from ambient light
We show that axons require longer pulse durations than somata to elicit the same number of action potentials, and that axonal photostimulation causes longer latencies to spike than somatic photostimulation

Summary

Introduction

Optogenetics is a powerful tool that has transformed the investigation of neural circuits. Targeting axons with focal optical stimulation can be an effective means by which to probe connectivity, especially in acute slices where presynaptic axons are preserved even if their soma is lesioned This approach raises the question of whether focal stimulation of a neuron’s axon requires different conditions than focal stimulation of its soma. This is important to address given that there are several recent reports showing that focal axonal stimulation with inhibitory optogenetic tools paradoxically produces excitation rather than inhibition (Mahn et al, 2016; Messier et al, 2018). These studies highlight the importance of empirically testing conditions for optogenetic experiments

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