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The aim is to provide a comprehensive protocol for using imaging and optogenetics to study memory mechanisms in mice.

July 4, 2026STAR Protocols0 citationsOpen Access

Protocol for longitudinal two-photon calcium imaging and holographic optogenetic manipulation to investigate memory in mice

Key Points

The aim is to provide a comprehensive protocol for using imaging and optogenetics to study memory mechanisms in mice.
Conduct longitudinal two-photon calcium imaging during behavior analysis.
Implant chronic cranial windows and perform stereotaxic viral delivery for targeted neuron manipulation.
Implement optical stimulation alongside imaging for direct observation of neuronal activity in real time.
Allows for simultaneous recording of neuronal activity and behavioral responses.
Facilitates causal interrogation of specific neuronal populations during memory tasks.

Abstract

Here, we present a protocol for longitudinal two-photon calcium imaging and holographic optogenetic manipulation to investigate memory in mice. We describe chronic cranial window implantation, stereotaxic viral delivery, and head-fixed contextual fear conditioning synchronized with imaging acquisition and optical stimulation of identified neurons. We further detail procedures for image data processing pipelines for cross-session registration and longitudinal tracking of the same neuronal population. This protocol enables simultaneous neuronal activity recording, behavioral monitoring, and causal interrogation of neuronal ensembles. For complete details on the use and execution of this protocol, please refer to Wang et al. 1

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