A platform for stereological quantitative analysis of the brain-wide distribution of type-specific neurons

Chen Zhang,Anan Li,Jing Yuan,Hui Gong,Cheng Yan,Miao Ren,Tingwei Quan

doi:10.1038/s41598-017-14699-w

Abstract

Quantifying the distribution of specific neurons throughout the whole brain is crucial for understanding physiological actions, pathological alterations and pharmacological treatments. However, the precise cell number and density of specific neurons in the entire brain remain unknown because of a lack of suitable research tools. Here, we propose a pipeline to automatically acquire and analyse the brain-wide distribution of type-specific neurons in a mouse brain. We employed a Brain-wide Positioning System to collect high-throughput anatomical information with the co-localized cytoarchitecture of the whole brain at subcellular resolution and utilized the NeuroGPS algorithm to locate and count cells in the whole brain. We evaluated the data continuity of the 3D dataset and the accuracy of stereological cell counting in 3D. To apply this pipeline, we acquired and quantified the brain-wide distributions and somatic morphology of somatostatin-expressing neurons in transgenic mouse brains. The results indicated that this whole-brain cell counting pipeline has the potential to become a routine tool for cell type neuroscience studies.

Highlights

The brain is an important and extremely complex organ
We performed immunostaining using an antibody against SOM in sections from the primary motor area (MOp), the hippocampal formation (HPF) and the caudoputamen (CP) and collected images using a Zeiss 710 confocal microscope with a 20× objective (Fig. 1b)
The overlapping images of fluorescently labelled somas and immunostained molecules showed that the majority of the enhanced yellow fluorescent protein (EYFP)-labelled neurons were SOM-positive (SOM+), consistent with previous studies[32,39]

Summary

Introduction

The brain is an important and extremely complex organ. In 1888, Santiago Ramón y Cajal revolutionarily modified Golgi’s method to stain neurons[1]; since the neuroscience field has developed for more than a century. Brain-wide Positioning System (BPS)[27] immersed the sample in a propidium iodide (PI) solution during data acquisition and employed the interval between sectioning and imaging to counterstain co-located cytoarchitecture for fluorescence-labeled neurons in real time This method provides an accurate anatomical reference at single-neuron resolution for studying the pattern characteristics of neural structures in the brain. We propose a platform to analyse the brain-wide distribution of type-specific neurons from a 3D high-resolution fluorescence MOST dataset This platform combined whole-brain optical imaging using the BPS and stereological localization using NeuroGPS to generate an accurate stereological cell count in the whole mouse brain. To demonstrate the power of this platform, we obtained high-resolution brain-wide datasets for SOM-IRES-Cre:Ai3-EYFP mouse brains and quantitatively analysed the distribution patterns of somatostatin (referred to as SOM in this manuscript and referred to as SST) expression and characterized the morphological features of SOM neurons in the whole brain

Methods

Results

Conclusion