Immuno-Golgi as a Tool for Analyzing Neuronal 3D-Dendritic Structure in Phenotypically Characterized Neurons

Luísa Pinto,Mónica Morais,Nuno Sousa,João Miguel Bessa,António Mateus-Pinheiro,Anna Dunaevsky

doi:10.1371/journal.pone.0033114

Abstract

Characterization of neuronal dendritic structure in combination with the determination of specific neuronal phenotype or temporal generation is a challenging task. Here we present a novel method that combines bromodioxyuridine (BrdU) immunohistochemistry with Golgi-impregnation technique; with this simple non-invasive method, we are able to determine the tridimensional structure of dendritic arborization and spine shape of neurons born at a specific time in the hippocampus of adult animals. This analysis is relevant in physiological and pathological conditions in which altered neurogenesis is implicated, such as aging or emotional disorders.

Highlights

The unique phenomenon of adult neurogenesis adds a new dimension to neuroplasticity in the adult brain [1,2]
We studied the structure of newborn neurons in the dentate gyrus (DG) of control rats (Figure 1, 2) and compared to those of rats displaying depressivelike symptoms after exposure to chronic mild stress (CMS) and/or to the administration of MAM, an alkylating agent that arrests cellular proliferation
No significant differences in total dendritic length per neuron were found between the automatic AutoNeuron and manual reconstructions, showing that distinct reconstruction strategies can be used to study the structure of these neurons

Summary

Introduction

The unique phenomenon of adult neurogenesis adds a new dimension to neuroplasticity in the adult brain [1,2]. We describe a novel, non-invasive, method that combines BrdU immunohistochemistry with Golgi-impregnation; with this new method we can trace newborn neurons (BrdU labeling) and study their dendritic and spine structure, (3D morphometric analysis of Golgi-impregnated neurons) in distinct experimental conditions. For rats exposed to CMS, with and without MAM administration, 26 and 23 newborn neurons were reconstructed, respectively.

Results

Conclusion