Periodic Reporting for period 1 - Imagine (Imaging Functional Integration Of Newborn Neurons Into Neural Circuits Of The Axolotl Brain)
Reporting period: 2022-03-01 to 2024-02-29
In this project we have analyzed neurogenesis in the axolotl salamander brain during homeostasis and regeneration and developed methodology to analyse neuronal projections and connections. Neurogenesis, the generation of new neurons from stem or progenitor cells, occurs universally during embryonic development across all animals. Following embryonic development, certain species maintain ongoing neurogenesis into adulthood to varying degrees. In humans, regions like the hippocampus exhibit continuous adult neurogenesis, producing approximately 700 new neurons daily. Conversely, species like teleost fish and amphibians experience lifelong growth, necessitating constant cell generation in multiple brain areas. In zebrafish and axolotl, continual neurogenesis is observed in various brain regions, including the telencephalon, mesencephalon, and olfactory bulb. Notably, these species possess the unique ability to regenerate lost neurons after injury, a feature distinct from mammals, where neuronal death typically results in glial cell hypertrophy and scar formation, impeding neuronal regeneration. Both adult neurogenesis and neural regeneration face a crucial challenge: integrating newborn cells into existing neural networks. Maintaining and restoring neural connectivity and circuit function is vital for controlling animal behavior and regulating physiology. Thus, newborn cells must be appropriately integrated and connected to target cells to ensure the functionality of the neural network is preserved or restored. The objectives of this Marie Skłodowska Curie Action (MSCA) have been to (a) Develop in vivo imaging of neurogenesis during homeostasis and regeneration of the axolotl brain, to (b) set up in vivo imaging of functional neural circuits, their growth and regeneration in the axolotl and (c) develop methods to visualize functional connections of neurons. We have been successful in developing a variety of methods visualize functional connections of neurons which will in the future be useful to determine neuronal circuit recovery after brain regeneration in the axolotl.
To understand neurogenesis and dissect the cellular basis for continuous neurogenesis and regeneration in the axolotl telencephalon, we have performed Cre-loxP-mediated lineage tracing of stem cells. We established conditions for single cell conversion, which allowed to trace individual stem cells and understand their contribution to growth of the telencephalon. Using this approach, we were able to understand the clonal dynamics, clone patterns and migration patterns of neurons. To understand neuronal function, we have established an imaging and staining pipeline to map neuronal response to sensory stimulation. To understand neuronal circuitry before and after regeneration we have established different methods to label neurons and understand their projection and connection patterns in the axolotl. For a relatively fast overview of projection patterns, we used Neurobiotin injections (retrograde and anterograde tracer). To determine projection betters in a more defined way we have established adeno-associated viruses (AAVs) in the axolotl. To determine synaptically connected neurons, we have been working on the establishment of GFP reconstitution across synaptic partners (GRASP), Synapse protein fusions as well as viral monosynaptic retrograde tracing. To establish AAVs in the axolotl we have defined the effective serotypes which can be used to transduce cells in larval and juvenile axolotl brains and the retina. We determined the cellular tropism finding that both excitatory and inhibitory neurons are preferentially expressing virally delivered transgenes. We then analyzed both retrograde and anterograde spreading capacities of different serotypes and use them explore neuronal projections in the axolotl visual pathway. By this we confirmed canonically known projections from the retina to the brain and in addition to that describe, for the first time in axolotls, the retinopetal projection from the brain to the retina. The results of this work have been made available under: “Adeno-associated viruses for efficient gene expression in the axolotl nervous system”
Katharina Lust, Elly M. Tanaka, bioRxiv 2024.02.15.580426; doi: https://doi.org/10.1101/2024.02.15.580426(opens in new window)
Katharina Lust, Elly M. Tanaka, bioRxiv 2024.02.15.580426; doi: https://doi.org/10.1101/2024.02.15.580426(opens in new window)
Amphibians occupy a central position in the phylogeny of tetrapods and are now an exciting system to integrate behavior and the cell and circuit biology of the nervous system. The salamander axolotl, in addition, represents a powerful genetic model to understand the regeneration of neural circuitry. The study of amphibian nervous systems was, until now, lacking viral tools to transduce gene expression in post-mitotic neurons. We have described for the first time extensive characterization and successful use of Adeno-associated viruses to induce gene expression, and perform projection mapping in the axolotl brain and retina (Lust and Tanaka, 2024). We hope that these contributions are of broad impact as it brings onboard key vertebrate representatives to the world of molecular neuroscience to help us understand the essential features of vertebrate brain regeneration.