Mapping neuronal circuits using a novel transsynaptic labelling approach

ABSTRACT

Understanding how genetically defined neural circuits drive specific behaviors is crucial for neuroscience and the development of targeted therapies for neurological disorders. While existing neuronal circuit tracers are effective, no approach allows anterogradely labeling of distinct postsynaptic populations that receive inputs from genetically defined presynaptic populations across all cell types. The goal of this thesis is to develop a novel transsynaptic labeling tool named Transsynaptically-Induced Recombinase-Activated Neuron Tracing (TIRANT). To achieve transsynaptic labeling, TIRANT employs a synthetic ligand expressed in presynaptic neurons and a synthetic Notch receptor fused to a recombinase in postsynaptic neurons. Ligand-receptor binding triggers cleavage of the intracellular domain, releasing the functional recombinase and enabling genetic labeling of connected neurons. We cloned and tested over 30 TIRANT receptor constructs, assessing their functionality, including ligand-independent activation and ligand-dependent activation in vivo. This screening identified two top-performing receptors. We then packaged these receptors into Adeno-associated virus (AAVs) and demonstrated ligand-dependent activation in vivo using AAV-mediated delivery. Additionally, we generated Synaptophysin-GFP, a presynaptic ligand for which we assessed the efficacy for tracing neuronal circuits in vivo, including both local innervation and dopaminergic pathways. The results revealed approximately 10% ligand-independent activation in our top-performing receptors, and inconclusive results in terms of circuit tracing. While we are unable to conclusively demonstrate successful circuit tracing with our transsynaptic labeling approach, these findings offer valuable insights for the development of more precise and versatile strategies for genetically specific neural circuit mapping and the progress of circuit-tracing methodologies