Active neuronal tracing in postmortem large mammalian brains
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Genetics
First Advisor
Sestan, Nenad
Abstract
Systematic mapping of the brain's connectome, particularly focusing on long-range axonal projections, is essential for decoding the complexities of the nervous system and understanding its functional mechanisms. Comprehensive maps of neural circuits have been generated in experimental animals using various techniques, including active neuronal tracing using non-viral chemical and viral vectors. However, these methods often involve invasive procedures such as craniectomy and survival surgery, posing significant challenges when attempting to apply them to larger animals and entirely unfeasible for human subjects. These limitations impede multimodal interrogation of brain mapping through active neuronal tracing and overall understanding of mesoscale connectome in large mammalian brains, including human brain. To fill this gap and access the viable brain in large mammals without the need for survival surgery, we previously developed the BrainEx technology, a pulsatile perfusion system and synthetic, cytoprotective perfusate, that restored brain microcirculation and cell viability. In an ex vivo porcine model, with 6h of BrainEx perfusion for up to 4 hours after death, we successfully restored brain metabolism and certain cellular functions, without eliciting global electrical activity in a porcine model. Importantly, the perfused brains demonstrated preserved anatomic architecture and cellular functions, which provided the potential to deliver and trace active vectors that are required to understand the global brain connectivity at mesoscale. The work presented here describes my graduate studies in Dr. Nenad Sestan’s lab, focused on providing approaches to mapping of large mammalian brains at cellular resolution. The work encompassed four distinct facets: 1) optimizing the BrainEx technology to extend the viability of brain cells in postmortem large mammalian brain tissue, 2) implementing active neuronal tracing at a cellular resolution in postmortem brains, and 3) exploring the potential of BrainEx technology for application not only in isolated brain but also to other organs of the body in vivo. In the first chapter, I provide an extensive review about the historical view of our understanding of fiber pathways as well as state-of-the-art brain mapping approaches utilized in both experimental animals and in humans, across varying levels of granularity. Following this, I introduce the present challenges that impede our understanding of the human connectome at a cellular level, and discuss how these gaps can potentially be bridged using postmortem brain tissue. In the second chapter, I describe the optimization of the previously established BrainEx technology to extend the viability of cells within intact porcine brains under ex vivo conditions. Furthermore, I report robust cellular uptake of chemical neuroanatomic tracers and their active intracellular transport within major brain regions through 24 hours of normothermic BrainEx perfusion. Of particular significance, this work provides the first evidence that viruses can infect neurons in isolated postmortem brains and express viral genetic material. Furthermore, I explore the application of MAPseq for deciphering projection patterns. This work serves as a proof-of-principle for actively tracing neurons in isolated pig brains postmortem, and it paves the way for a novel approach to map the connectome of large mammalian brains at the mesoscale level. In the third chapter, I present the extension of BrainEx technology to an in vivo whole body perfusion setting (OrganEx). We report that after 1 hour of warm ischaemia, the application of OrganEx restored global circulation, preserved tissue integrity and restored certain molecular and cellular processes across multiple vital organs. Taken together, this work highlights the largely unexplored potential of postmortem large mammalian brains as invaluable assets for achieving brain mapping at cellular resolution. By ensuring proper preservation of cell viability, these brains offer a unique opportunity to enhance our understanding of brain connectivity and cellular functions. Moreover, this proof-of-concept study presents a translatable approach, holding promise for applicability connectivity mapping pursuits in multiple other large species.
Recommended Citation
Zhang, Shupei, "Active neuronal tracing in postmortem large mammalian brains" (2023). Yale Graduate School of Arts and Sciences Dissertations. 1236.
https://elischolar.library.yale.edu/gsas_dissertations/1236
