Implementation of tissue clearing and 3‐dimensional volumetric imaging approaches for visualization of anatomical structures in autonomic nuclei
Authors: Arestakesyan Hovhannes , Young Colin N. 
Published: May 2022

Alterations within central nervous system (CNS) autonomic nuclei contribute to a wide array of diseases. In this context, significant advancements have been made into disease‐related molecular mechanisms in autonomic brain regions. While helpful, it is important to consider that information processing within the CNS occurs through dynamic micro‐architectural structural interactions, such astrocyte‐neuron synaptic connections. However the traditional method for evaluating CNS morphology and synaptic contacts between cells is performed with immunohistochemistry on thin brain sections. An inherent limitation of this approach is not only the narrow 2‐dimensional field of view, but also the lack of optical transparency that minimizes high‐resolution imaging of anatomical structures and cell‐cell interactions. To overcome these limitations, here we sought to optimize tissue clearing approaches combined with 3D volumetric imaging for autonomic CNS nuclei. We focused our initial efforts on the forebrain subfornical organ (SFO), a region lacking a blood‐brain‐barrier, given: 1 Alterations in the SFO have been described to contribute to numerous disease conditions; and 2 The SFO represents a unique technical challenge for 3D imaging due to the complexity of cellular processes and surrounding lateral ventricle space. As a first step, we used a “passive” CLARITY tissue approach in 8‐week‐old C57Bl/6J male mice. Mice were transcardially perfused with an acrylamide‐based hydrogel in standard fixative. Subsequently, a brain section encompassing the entire SFO (~1mm) was obtained, and a series of thermal diffusion steps was performed to place existing protein structures in a “scaffold” and remove the lipid contents ‐ rendering the tissue transparent. Whole‐mount immunohistochemistry for astrocytes was then conducted using GFAP and DAPI as a nuclei marker, followed by 3D volumetric multiphoton imaging to acquire high‐resolution visualization of the entire SFO in three planes (Figure top). In brief, this approach proved to be a significant advancement beyond standard 2D histology, and 3D visualization of intricate astrocyte processes throughout the whole SFO was possible. However, a major limitation was the extensive amount of time required to process a single sample; 2 weeks for tissue clearing and 2 weeks for primary and secondary antibody incubation. Based on this, we adapted our protocol to an “active” CLARITY approach and employed electrophoretic‐based clearing of large brain sections (n=5), which resulted in a reduction in clearing time from 2 weeks to 4 hours. It was also readily apparent that this approach rendered the tissue more transparent than passive clearing, allowing for greater visualization of astrocyte processes (Figure middle). Preliminary analysis with IMARIS software to analyze astrocyte surface area and dendritic processing (Figure bottom) revealed highly complex SFO astrocytic structures with some dendrites extending from soma at the lateral ventricle throughout and surrounding nearly the entire SFO; these processes are not visualized with standard histology. Collectively, these approaches highlight the utility of tissue‐based clearing for 3D visualization of anatomical structures within autonomic nuclei and provide a framework to understand structure‐function relationships within these brain regions in the regulation of physiological and pathophysiological conditions.