Abstract

The axolotl (Ambystoma mexicanum), a neotenic salamander with remarkable regenerative capabilities, serves as a key model forstudying nervous system regeneration. Despite its potential, the cellular and molecular mechanisms underlying this regenerativecapacity remain poorly understood, partly due to the lack of reliable in vitro models for axolotl neural cells. In this study, wedeveloped a novel protocol for primary cultures of adult axolotl telencephalon/pallium, enabling the maintenance of viable andfunctionally active neural cells. Using calcium imaging and immunocytochemistry, we demonstrated the presence of neuronal andglial 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 robustneurite outgrowth and responsiveness to injury. This protocol addresses a critical gap in axolotl research by providing a controlledin 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 forneurodegenerative diseases and CNS injuries in humans.