Elsevier

Methods in Enzymology

Volume 404, 2005, Pages 242-252
Methods in Enzymology

Functional Assay of ARNO and ARF6 in Neurite Elongation and Branching

https://doi.org/10.1016/S0076-6879(05)04023-1Get rights and content

Abstract

During development of the nervous system, neurite outgrowth is necessary for the formation of connections between nerve cells. Neurons are highly polarized cells that send out distinct processes, axons, and dendrites; however, the molecular regulation of the differential growth of these processes remains incompletely understood. Primary cultures of rat hippocampal neurons have been used to study many aspects of neuronal cell biology, including neurite extension, establishment of polarity, biogenesis of synapses, and membrane trafficking. After attachment to the substrate, hippocampal neurons begin sending out multiple processes by approximately 12 h after plating. The axonal process is derived from one of these processes, and is evident after 48 h in culture. Complete polarity of axons and dendrites is established after 7 days in culture. The establishment of these cultures and the ability to transfect them with potential regulatory genes allows the researcher to dissect out the pathways relevant to neurite extension. To study the role of small GTPases in neurite extension and branching, we describe methods for culture of hippocampal neurons, for transfection of these cells, and assessment of neurite extension and branching.

Introduction

Neurons have axonal and dendritic processes that define their unique morphology and function. These processes contain specialized sites to receive and send out information, which are critical for proper nervous system wiring. Controlled neurite elongation and branching are developmental events crucial for neurons to acquire their mature functional morphology. These events must be tightly regulated, and the molecular machinery involved in these processes is starting to be unraveled. An array of molecules has been shown to regulate neurite extension and branching, including cell surface receptors and ligands (Lin 2005, Whitford 2002), Rho GTPases and their effectors (Govek 2005, Luo 2000), and lipid modifying enzymes (Hernandez‐Deviez 2004, van Horck 2002, Yamazaki 2002). In addition, the Arf family of small GTPases has been implicated in membrane trafficking (Palacios 2001, Radhakrishna 1997, Radhakrishna 1999) as well as reorganization of the cytoskeleton in non‐neuronal cells (Boshans 2000, Frank 1998, Radhakrishna 1996), and we have shown that ARF6 and its exchange factor ARNO regulate dendrite branching and axonal elongation and branching in cultured rat hippocampal neurons (Hernandez‐Deviez 2002, Hernandez‐Deviez 2004).

The hippocampus is a source of a homogeneous population of pyramidal neurons. The sequence of developmental events that cultured hippocampal neurons follow to acquire their mature morphology has been previously described (Dotti et al., 1988), and Banker and Goslin (1991) have described in detail the preparation of primary culture of rat hippocampal neurons. Here we present a description of the methods we employed to study the role of the small GTPase ARF6 and its exchange factor ARNO during neurite elongation and branching.

In our studies of neurite elongation and branching in vitro, hippocampi from embryonic day 17–18 rats are used as the source of neurons. These dissociated hippocampal cells were plated on coated coverslips, transfected with the plasmids of interest, and the effects upon neurite extension and branching quantified.

Section snippets

Culture of Rat Hippocampal Neurons

Successful culture of hippocampal neurons for transfection requires that the coverslip substrates be properly prepared to promote adhesion as well as for survival during the transfection process.

Morphological Analysis of Hippocampal Neurons Expressing ARNO, ARF6, and Mutant Forms of ARNO and ARF6

Because many small GTPases have been shown to affect different aspects of neuronal development, including axonal growth and dendritic complexity (Albertinazzi 2003, Li 2000, Luo 2000, Nakayama 2000, Ng 2002), it is important when studying the effects of ARNO and ARF6 expression on neurite development to analyze the morphology of the transfected cells through immunofluorescent labeling and microscopy and to then quantify the differences upon neurite length and branching.

Acknowledgments

This work was supported by NIH DK43329 (JMW) and Consejo Nacional de Investigaciones Científicas y Tecnológicas, Venezuela and University of Los Andes, Mérida‐Venezuela (DJH).

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