A Novel Method for Culturing Telencephalic Neurons in Axolotls

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.

Keywords: axolotl; primary neuron culture; regeneration; telencephalon.

FIGURE 1

Axolotl primary telencephalon culture procedure. (A) Dissection and removal of the brain. (B) Isolated telencephalon. (C) Brightfield images of cultured neurons during 2 weeks of incubation.

FIGURE 2

Viability and neurite extension in telencephalic cultures up to DIV14. (A–C) Normalized viability ratio of PI negative cells to total cell count in serum or serum‐free conditions in high (HC), medium (MC), and low confluency (LC) conditions, respectively. (D) Measurement of average maximum axon length over the course of 14 days in serum or serum‐free conditions. (E) Neurite extending cell count between DIV5 and DIV8 in serum (p value = 0.0414) and serum‐free (p value = 0.0707) conditions (n = 4).

FIGURE 3

Calcium imaging and axotomy injury. (A) Representative images of spontaneous calcium activity detected with Calcium Green‐1 (AM). (B) Representative fluorescence traces of calcium activity from 8 ROIs. (C) Time series representation of calcium activity in panel D, axotomy time point indicated with dashed line. (D) Representative images showing calcium imaging of an axotomized neuron, arrowhead points to the injury site.

FIGURE 4

Representative ICC images of axolotl primary telencephalon culture. (A) Cells expressing β‐III tubulin (red), synaptophysin (green), and DAPI (cyan), indicating a neuron identity (scale bar = 40 µm). (A’) Inlet image from selected rectangular area showing axons and filopodia region with positive immunoreactivity for β‐III tubulin (red) and synaptophysin (green). (B) Cells stained for β‐III tubulin (red), GFAP (green), and DAPI (cyan) (scale bar = 40 µm). (B’) Inlet image from selected rectangular area showing one cell with immunoreactivity for GFAP (green) in the cytoplasm and for β‐III tubulin (red) in both cytoplasm and the nucleus. (C) Secondary antibody control for panel B images.