Feb 16, 2026
Open-Field Locomotion Assay with Optogenetic Stimulation
- Cristian González-Cabrera1,
- Matthias Prigge1
- 1Leibniz Institute for Neurobiology, Magdeburg, Germany
Protocol Citation: Cristian González-Cabrera, Matthias Prigge 2026. Open-Field Locomotion Assay with Optogenetic Stimulation. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv55m35v1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 16, 2026
Last Modified: February 16, 2026
Protocol Integer ID: 243353
Keywords: locomotor responses during optogenetic manipulation, field locomotion assay with optogenetic stimulation, optogenetic stimulation, locomotor response, specific manipulation of vta neuron, optogenetic manipulation, optical stimulation, vta neuron, field locomotion assay, presynaptic terminal, pno pathway, vtapno projection, controlled illumination condition, directional movement parameter, offline for directional movement parameter, vta somata
Abstract
This protocol describes the open-field assay used to quantify locomotor responses during optogenetic manipulation of the VTA–PnO pathway. Experiments were conducted under controlled illumination conditions during the light phase and synchronized with optical stimulation delivered either to VTA somata (DAT-Cre experiments) or to presynaptic terminals (GAD2 experiments). The VTAPnO projection-defined subpopulation was selectively labeled using a retrograde Flp-dependent targeting strategy, enabling specific manipulation of VTA neurons projecting to the PnO. Locomotor behavior was recorded using overhead video acquisition and analyzed offline for directional movement parameters.
Guidelines
**Critical Steps**
- Maintain consistent illumination (50 lux) across sessions.
- Measure optical output at the fiber tip immediately before each session and adjust to 5 mW.
- Ensure stable patch cord positioning to avoid torque-induced artifacts.
- Maintain consistent stimulation timing parameters across animals.
Materials
**Arena**
- Square open-field arena: 50 cm × 50 cm
- Opaque walls
- Uniform illumination: 50 lux
- Experiments performed during the light phase
**Video Recording**
- Overhead camera
- Frame rate: 30 frames per second
- Continuous recording throughout session
- Stimulation synchronized via TTL or equivalent digital trigger
Troubleshooting
Before start
Immediately before each behavioral session, optical output was measured at the tip of the implanted fiber using a calibrated power meter. Output power was adjusted to 5 mW at the fiber tip before starting the experiment.
Procedure
Connect the optic patch cord to the implanted ferrule. Verify proper connection and ensure free cable movement. Place the mouse in the center of the open-field arena.
Begin video acquisition.
Deliver repeated stimulation cycles consisting of:
3 seconds stimulation
10 seconds pause
Continue cycles for a total duration of 5 minutes. Ensure synchronization between stimulation onset and video timestamps.
After 5 minutes, stop stimulation and recording. Disconnect the patch cord and return the animal to its home cage.
Data Acquisition and Synchronization
Stimulation onset timestamps were recorded.
Video frames were aligned to stimulation epochs for peri-stimulus analysis.
Directional locomotion metrics were extracted offline.