# CHLOK: a chemigenetic multicolor labeling system to visualize neuronal birthdate and circuit integration

## TL;DR

CHLOK is a new method using fluorescent dyes to track how neurons develop and integrate into brain circuits in zebrafish larvae.

## Contribution

CHLOK introduces a non-invasive, multicolor labeling system for studying neuronal birthdate and circuit integration with high resolution.

## Key findings

- CHLOK enables precise multicolor labeling of neurons based on their maturation stage.
- The method reveals distinct functional roles of early- and late-born neurons in zebrafish development.
- CHLOK is compatible with calcium imaging, optogenetics, and voltage imaging for functional analysis.

## Abstract

Understanding how neurons integrate into developing circuits and contribute to functional activity is essential for decoding brain development and plasticity. However, current methods to study neuronal integration often suffer from low throughput, limited spatiotemporal resolution, or invasive procedures that hinder in vivo functional analysis. To overcome these challenges, we present a birthdate-labeling strategy, named CHLOK, based on HaloTag technology and a broad palette of fluorescent synthetic dyes. This approach enables precise multicolor labeling of neurons according to their maturation stage and allows flexible integration into functional assays through compatibility with calcium imaging and optogenetics. We validated CHLOK by mapping birthdate-resolved neuronal activity in the developing visual and motor systems of zebrafish larvae. Our results reveal distinct functional contributions of early- versus late-born neurons, providing new insights into the temporal dynamics of circuit formation. Furthermore, we demonstrate the versatility of this approach, showcasing age-specific multicolor calcium and voltage imaging as well as optogenetic manipulation. By overcoming key limitations of existing techniques, CHLOK offers a powerful, versatile and non-invasive tool for studying neural integration, circuit development and function in vivo.