The axolotl (Ambystoma mexicanum) has extraordinary regenerative abilities鈥攖he salamander can regrow lost limbs and repair complex organs, including the retina and the brain鈥攎aking the axolotl an ideal model for studying both how neural circuits form and how they regenerate after injury.

So far, brain regeneration in the axolotl has been studied with classical methods, such as by employing tracers and antibodies. However, researchers have lacked the tools for capturing the dynamics of circuit regeneration, interrogating functional recovery and manipulating neuronal function in the axolotl brain.

Now, Katharina Lust and Elly Tanaka at the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences present an efficient method for delivering genes into axolotl neurons using viruses, enabling researchers to dynamically visualize neurons and transfer novel genes into neurons. Their findings were in the Proceedings of the National Academy of Sciences on March 5.

Lighting up axolotl neurons

Genes can be introduced into cells using different methodologies, such as by hijacking harmless viruses to deliver genes. So far, virus-mediated gene delivery into axolotl neurons had not been achieved.

In the newly published study, Lust and Tanaka demonstrated for the first time that adeno-associated viral vectors (AAVs) can efficiently deliver transgenes into axolotl neurons. By testing different AAV serotypes鈥攙ariants that target different cell types鈥攖he scientists identified the most effective serotype for delivering transgenes into axolotl neurons.

Using this gene delivery method, the scientists introduced the marker GFP into the neurons of a live axolotl. In this way, the scientists were able to fluorescently label different neuronal types and to visualize the projections connecting neurons.

From eye to brain鈥攁nd back

Visualizing neural connections allows scientists to map the circuits linking different brain areas. Using viral gene delivery in the axolotl's retina, the scientists mapped the connections that allow retinal neurons to transmit visual information to different brain regions. The scientists also identified neuronal projections traveling in the opposite direction鈥攆rom the brain to the retina鈥攕uggesting that the brain influences and fine-tunes retinal function.

"This technology provides a window into visualizing neuronal activity in vivo in the brain and tracing how brain circuits regenerate after injury," explains first author Lust, postdoctoral researcher in Tanaka's lab.

Expanding the limits of axolotl brain research

In addition to opening up new ways for dynamically visualizing neurons in the axolotl brain, this study also establishes viral vectors as powerful tools for introducing new genes into axolotl neurons and interrogating neuronal organization.

"Viral vectors could be used to manipulate neural circuits or to probe the roles of specific genes in axolotl brain repair," says corresponding author Tanaka, Scientific Director of IMBA. "This tool will unlock experimental opportunities previously unattainable in the axolotl.

"This work establishes the axolotl as a key vertebrate representative in the world of molecular neuroscience, helping us understand the essential features of the vertebrate brain."