# A Novel Method for Culturing Telencephalic Neurons in Axolotls

## TL;DR

A new method for growing and studying axolotl brain neurons in the lab could help understand regeneration and improve treatments for human brain injuries.

## Contribution

A novel in vitro protocol for culturing axolotl telencephalic neurons with functional integrity is introduced.

## Key findings

- Cultured axolotl neurons show synaptic connections and spontaneous calcium activity.
- Neurons maintain viability and neurite outgrowth in both serum and serum-free conditions.
- Cells respond to injury, indicating functional excitable properties.

## Abstract

The axolotl (Ambystoma mexicanum), a neotenic salamander with remarkable regenerative capabilities, serves as a key model for studying nervous system regeneration. Despite its potential, the cellular and molecular mechanisms underlying this regenerative capacity remain poorly understood, partly due to the lack of reliable in vitro models for axolotl neural cells. In this study, we developed a novel protocol for primary cultures of adult axolotl telencephalon/pallium, enabling the maintenance of viable and functionally active neural cells. Using calcium imaging and immunocytochemistry, we demonstrated the presence of neuronal and glial markers, synaptic connections, and spontaneous calcium activity, highlighting the functional integrity of the cultured cells. Our findings reveal that these cultures can be maintained in both serum and serum‐free conditions, with neurons exhibiting robust neurite outgrowth and responsiveness to injury. This protocol addresses a critical gap in axolotl research by providing a controlled in vitro system to study neurogenesis and regeneration. By offering insights into the regenerative mechanisms of axolotl neurons, this work lays the foundation for comparative studies with mammalian systems, potentially informing therapeutic strategies for neurodegenerative diseases and CNS injuries in humans.

A protocol for primary neuron culture from axolotl telencephalon was developed. Spontaneous and axotomy‐induced calcium transients showed excitable cells and neuronal and glial characterization was performed with immunocytochemistry.