Optimization of neural tracing protocol by using adeno-associated viruses and tissue clearing technique

Abstract

Studying neuron connections has been an important task in the field of neuroscience. Elucidation of different neural circuits can help understand brain functions and disease-progressing mechanisms. However, the visualization was limited by the complexity of the neuronal network and the resolution of the imaging technique. Hence, the neural circuit visualization was performed by non-invasive macroscale methods with poor resolution in the past. To increase the imaging resolution, neural tracing was carried out in the mesoscale, in which the connections of a particular group of neurons in a specific brain region were mapped.

With the use of confocal microscopy, imaging on intact tissues under cellular resolution became possible. This leads to the development of tissue clearing, which can render 3D tissue transparent. In addition, specific labeling of the neural circuits becomes possible by the manipulation of different viruses as neural tracers. Hence, the tissue clearing method and viral tracing system were optimized for 3D mesoscale tracing in this thesis.

In the first part, the tissue clearing condition was modified from the Accu-OptiClearing method, which was developed by our former lab members. Two permeabilizing agents, urea and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), were added to the clearing solutions of Accu-OptiClearing. The transparency, tissue size change and protein loss were assessed to screen out the optimal condition as the urea additive. Optimal urea concentration was assessed by the same tests in short-term and long-term clearing conditions. Together with the staining by antibodies and PM-ML myelin probes, 2 M urea + 10% 1,2 hexanediol (HxD) in Optical Properties-adjusting Tissue Clearing agent (OPTIClear) was found to be the optimal clearing solution. Hence, this new clearing solution was applied in neural tracing in the following parts.

In the second part, a combinatorial adeno-associated virus (AAV) tracing system was established from the modification of the dual virus system used in other studies. The new system enables specific labeling of neural circuits by the flip-excision (FLEx) switch carried in the recombinant AAVs (rAAVs). The tracing system was validated by mapping the known nigrostriatal pathway. By the nigrostriatal tracing, the expression timepoint of the tracing system was optimized as 3 weeks. In addition to the motor regulating nigrostriatal pathway, the tracing system was used to successfully trace the mesocortical pathway together with the circuits connecting the ventral tegmental area (VTA) to CA1, CA2 and CA3 regions. These mapped circuits link the midbrain to executive and cognitive functions.

In the third part, the degeneration of the traced circuits was visualized in the 6-hydroxydopamine (6-OHDA) Parkinson’s disease model. It was found that the axons in the terminal regions of different circuits degenerated to different extents. This indicated different degeneration disorders in different brain regions. It showed that the nigrostriatal pathway degenerated first, followed by the mesocortical pathway and the VTA-CA2 circuit.

From the results, it was confirmed that the optimized 2 M urea + 10% HxD-OPTIClear solution and the combinatorial AAV tracing system can be applied to trace neural circuits in physiological and pathological conditions.