An Optimized and Detailed Step-by-Step Protocol for the Analysis of Neuronal Morphology in Golgi-Stained Fetal Sheep Brain

Abstract

Antenatal brain development during the final trimester of human pregnancy is a time when mature neurons become increasingly complex in morphology, through axonal and dendritic outgrowth, dendritic branching, and synaptogenesis, together with myelin production. Characterizing neuronal morphological development over time is of interest to developmental neuroscience and provides the framework to measure grey matter pathology in pregnancy compromise. Neuronal microstructure can be assessed with Golgi staining, which selectively stains a small percentage (1-3%) of neurons and their entire dendritic arbor. Advanced imaging processing and analysis tools can then be employed to quantitate neuronal cytoarchitecture. Traditional Golgi staining protocols have been optimized and commercial kits are readily available offering improved speed and sensitivity of Golgi staining to produce consistent results. Golgi stained tissue is then visualized under light microscopy and image analysis may be completed with several software programs for morphological analysis of neurons, including freeware and commercial products. Each program requires optimization, whether semi-automated or automated, requiring different levels of investigator intervention and interpretation, which is a critical consideration for unbiased analysis. Detailed protocols for fetal ovine brain tissue are lacking and therefore, we provide a step-by-step workflow of computer software analysis for morphometric quantification of Golgi-stained neurons. Here, we utilized the commonly applied FD Rapid GolgiStain kit (FD NeuroTechnologies) on ovine fetal brains collected at 127 days (0.85) gestational age for the analysis of CA1 pyramidal neurons in the hippocampus. We describe the step-by-step protocol to retrieve neuronal morphometrics using Imaris imaging software to provide quantification of apical and basal dendrites for measures of dendrite length (μm), branch number, branch order and Sholl analysis (intersections over radius). We also detail software add-ons for data retrieval of dendritic spines including the number of spines, spine density and spine classification, which are critical indicators of synaptic function. The assessment of neuronal morphology in the developing brain using Rapid-Golgi and Imaris software is labour-intensive, particularly during the optimization period. The methodology described in this step-by-step description is novel, detailed, and aims to provide a reproducible, working protocol to quantify neuronal cytoarchitecture with simple descriptions that will save time for the next users of these commonly used techniques.

Progressive inflammation reduces high-frequency EEG activity and cortical dendritic arborisation in late gestation fetal sheep

“…Coded region-matched Golgi-stained tissue sections (10 serial sections per subject) were used to assess basal dendrites from pyramidal neurons in layers 3 and 5 of the lateral cortex as described previously [ 33 , 34 ]. The Golgi staining tended to produce less complete filling of the apical dendrites relative to the basal dendrites.…”

Progressive inflammation reduces high frequency EEG activity and cortical dendritic arborisation in late gestation fetal sheep

“…Coded region-matched Golgi-stained tissue sections (10 serial sections per subject) were used to assess basal dendrites from pyramidal neurons in layers 3 and 5 of the lateral cortex using methods described previously [30,31]. The Golgi staining tended to produce less complete lling of the apical dendrites relative to the basal dendrites.…”

Role of Chemokine Cxcl12a in Mediating the Stimulatory Effects of Ethanol on Embryonic Development of Subpopulations of Hypocretin/Orexin Neurons and Their Projections

“…The number of Hcrt neurons in the aAH and pAH and also further anterior in the POA was quantified manually using the "Spots" function in Imaris. We then created 3D reconstructions of neuronal projections from Hcrt neurons located in the aAH, POA and pAH using the "Filaments" function of Imaris [35]. Spot sizes for the start point and seeding points of the filament were 7 µm and 0.9 µm with a threshold of 1350 and 350, respectively, followed by manual correction.…”