Case study | Silver wire for grounding in optophysiology and Neuropixels recordings in vivo

Optogenetic stimulation lets researchers probe how brain circuits function in living animals by delivering light to a target region and recording neuronal responses.

This STAR Protocols paper addresses a known complication. Optical stimulation in mice can unintentionally activate endogenous retinal opsins, producing off-target responses that propagate to the cortex and risk confounding experimental interpretation.

The authors describe two main ways these unwanted effects can arise. The first is direct light leakage from the implant site and exposed skull. The second is light propagation through brain tissue to the retina, which can be detected as light exiting the eye and can drive activity in visual pathways.

To minimise these effects, the paper presents a step-by-step workflow for optical device implantation that reduces light leakage from the implant and skull, paired with in vivo electrophysiology techniques that test whether off-target neuronal activity is still present. The protocol covers both acute and chronic approaches, using Neuropixels recordings paired with optical stimulation at 473 nm, 594 nm, and 637 nm.

The work was produced by researchers at the Sainsbury Wellcome Centre, part of University College London, with the Neurobiological Research Facility at the same centre also listed on the paper.

Study overview (STAR Protocols paper)

A central practical element of the protocol is reducing light emission from the implant and dorsal skull. The authors describe applying layers of black dental cement over the skull and implant area, and they include measurements showing how these layers attenuate wavelength-dependent light leakage.

The second element is verification. Rather than assuming that light control measures have worked, the protocol sets out how to assay off-target stimulation by recording neuronal activity during laser pulses in darkness and under controlled ambient light conditions. The paper explains how retinal light adaptation can be used in this assessment workflow and reports the conditions under which off-target activity is expected to be abolished for different wavelengths and intensities.

Finally, the protocol includes post-hoc validation of the optical fibre and recording probe location, including registration and segmentation workflows to reconstruct tracks in the brain.

The Science Made Simple

Researchers shine light into the mouse brain to stimulate a target region. The risk is that some of that light causes brain activity for the wrong reason.

Light can leak out around the implant and skull. Light can also reach the retina from inside the head. If the retina is activated, it can drive responses through visual pathways that show up in brain recordings, even when the brain itself does not contain any engineered light-sensitive proteins.

This protocol shows how to reduce those effects during implantation, then how to test for off-target activity directly in the brain signals recorded during optical stimulation.

Advent Research Materials in use | Silver wire

The protocol uses Advent's silver wire as part of the grounding and reference setup for electrophysiology, including steps that describe preparing a grounding pin with a short piece of silver wire and securing it during chronic Neuropixels implantation.

Protocols like this matter because they help labs improve experimental control and reduce confounding signals when combining optical stimulation with high-density electrophysiology.

For researchers building similar optophysiology setups, this paper provides a clear workflow for identifying and reducing off-target stimulation effects, alongside a detailed list of practical components used in the method.

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Full citation

Weiler, S., Vélez-Fort, M., O’Hara, L., Lenzi, S. C., Amaniti, E. M., & Margrie, T. W. (2026). Protocol for minimizing off-target neuronal activation during optical stimulation in vivo. STAR Protocols, 7, 104342. https://doi.org/10.1016/j.xpro.2025.104342