3D neural circuit visualization by neural tracing and tissue clearing for dementia study

Abstract

Background: To date, different techniques were developed for mapping neural connections, including diffusion tensor imaging (DTI) and neural tracers. DTI allows tracing of connections in macroscale but with low resolution. While neural tracers can allow visualization at single cell level, their signals are usually visualized in 2D sections. This results in missing information from the damages of brain tissues during tissue sectioning and inaccuracy of computation reconstruction. Therefore, we decided to trace neural circuits by using neural tracers in 3D intact tissues instead of 2D sections. In this study, we aim to establish 3D visualization protocol by combining neural tracing and tissue clearing techniques. We tested the potential of this protocol on tracing the neural circuit involved in Parkinson’s disease dementia (PDD) by using both chemical-based and viral-based neural tracers. Medial forebrain bundle (MFB) connects striatum, substantia nigra and frontal cortex. We at first focused on dopaminergic pathway projecting from MFB. Method: Cholera toxin subunit B (CTB) conjugated with Alexa Fluor-647 (CTB-647) or adeno-associated virus (AAV) expressing nerve growth factor and green fluorescent protein (AAV-NGF-GFP) was injected into MFB by stereotaxic injection. The rats were then euthanized by perfusion at different day points (10 days for CTB-647; 5 weeks for AAV-NGF-GFP) after the injection. The brains were treated with 4% sodium dodecyl sulfate (SDS) plus Optical Properties-adjusting Tissue Clearing agent (OPTIClear) solution for 5 days and then incubated into OPTClear solution for 2 days. The fluorescent signals were visualized by confocal microscopy. Result: CTB-647 and AAV-NGF-GFP signals were detected in striatum. It showed that the CTB-647 can travel from MFB to striatum through nigrostriatal pathway, while AAV-NGF-GFP can be absorbed by the neurons projecting from MFB to striatum to express green fluorescent proteins. It is feasible to trace neural circuits by neural tracing with tissue clearing. Conclusion: Taken together, nigrostriatal pathway, connecting substantia nigra and striatum, can be traced by using neural tracers together with tissue clearing. With this protocol, the neural circuits related to the dementia processes in PD can be further studied.