# Analyzing engram reactivation and long-range connectivity

## TL;DR

The paper introduces a protocol to study engram cell reactivation and long-range brain connections using genetic tools and tissue-clearing techniques.

## Contribution

A novel protocol for labeling and tracing engram cells in mice using transgenic methods and modified CLARITY.

## Key findings

- A method to tag and measure reactivation of two engram cell populations in the same mouse.
- A modified CLARITY technique enables tracing of axonal pathways in cleared brain tissue.
- The protocol supports investigations into long-range connectivity and overlapping cell populations.

## Abstract

Here, we present a protocol for marking engram cells to efficiently measure reactivation levels and their projection pathways. We describe steps for genetic manipulation utilizing transgenic mice and viral infections, labeling engram cells, and a modified version of CLARITY, a tissue-clearing technique. This protocol can be adapted to various research inquiries that involve assessing the overlap of cell populations and uncovering novel long-range connectivity pathways.

For complete details on the use and execution of this protocol, please refer to Refaeli et al. (2023).1

•Tagging two populations of engram cells in the same mouse•Measuring the level of engram reactivation using transgenic mice and viral vectors•Tracing axonal paths of engram cells in cleared brains•Using a modified CLARITY tissue-clearing technique

Tagging two populations of engram cells in the same mouse

Measuring the level of engram reactivation using transgenic mice and viral vectors

Tracing axonal paths of engram cells in cleared brains

Using a modified CLARITY tissue-clearing technique

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

Here, we present a protocol for marking engram cells to efficiently measure reactivation levels and their projection pathways. We describe steps for genetic manipulation utilizing transgenic mice and viral infections, labeling engram cells, and a modified version of CLARITY, a tissue-clearing technique. This protocol can be adapted to various research inquiries that involve assessing the overlap of cell populations and uncovering novel long-range connectivity pathways.