# Protocol for minimizing off-target neuronal activation during optical stimulation in vivo

## TL;DR

This paper introduces a protocol to reduce unintended neuronal activation during optogenetic experiments in mice by minimizing light leakage and monitoring off-target effects.

## Contribution

A novel protocol for implanting optical devices and reducing off-target activation during in vivo optogenetics.

## Key findings

- The protocol minimizes light leakage from optical implants and the skull.
- Electrophysiological techniques are described to assess and reduce off-target neuronal responses.
- Post hoc validation ensures correct placement of optical and recording devices.

## Abstract

In vivo optogenetic stimulation is a powerful approach for dissecting neuronal circuit function, yet it can inadvertently lead to activation of endogenous retinal opsins, producing off-target responses that propagate to the cortex and therefore confound experimental interpretation. Here, we present a protocol for optical device implantation in the mouse brain that minimizes light leakage from the implant and skull. We also describe in vivo electrophysiological techniques to assay off-target neuronal activity and ensure its amelioration.

For complete details on the use and execution of this protocol, please refer to Weiler et al.1

•Procedures for implanting optical devices and acute or chronic recording probes•Instruction for reducing light emission from optical implants and the dorsal skull•Steps to assess and minimize stimulation light-induced off-target neuronal responses•Post hoc validation of the location of optical fiber and recording probe

Procedures for implanting optical devices and acute or chronic recording probes

Instruction for reducing light emission from optical implants and the dorsal skull

Steps to assess and minimize stimulation light-induced off-target neuronal responses

Post hoc validation of the location of optical fiber and recording probe

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

In vivo optogenetic stimulation is a powerful approach for dissecting neuronal circuit function, yet it can inadvertently lead to activation of endogenous retinal opsins, producing off-target responses that propagate to the cortex and therefore confound experimental interpretation. Here, we present a protocol for optical device implantation in the mouse brain that minimizes light leakage from the implant and skull. We also describe in vivo electrophysiological techniques to assay off-target neuronal activity and ensure its amelioration.