Functional Assay of ARNO and ARF6 in Neurite Elongation 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).
References (28)
- et al.
Netrin‐1 and slit‐2 regulate and direct neurite growth of ventral midbrain dopaminergic neurons
Mol. Cell. Neurosci.
(2005) - et al.
Essential role of type I(alpha) phosphatidylinositol 4‐phosphate 5‐kinase in neurite remodeling
Curr. Biol.
(2002) - et al.
Regulation of cortical dendrite development by Slit‐Robo interactions
Neuron
(2002) - et al.
Phosphatidylinositol 4‐phosphate 5‐kinase is essential for ROCK‐ mediated neurite remodeling
J. Biol. Chem.
(2002) - et al.
Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones
J. Neurochem.
(2004) - et al.
ADP‐ribosylation factor 6 and a functional PIX/p95‐APP1 complex are required for Rac1B‐mediated neurite outgrowth
Mol. Biol. Cell
(2003) - et al.
Culturing Nerve Cells
(1998) Trophic interactions between astroglial cells and hippocampal neurons in culture
Science
(1980)- et al.
Rat hippocampal neurons in dispersed cell culture
Brain Res.
(1977) - et al.
Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures
Neurobiology
(1972)
ADP‐ribosylation factor 6 regulates actin cytoskeleton remodeling in coordination with Rac1 and RhoA
Mol. Cell Biol.
The establishment of polarity by hippocampal neurons in culture
J. Neurosci.
Differential effects of NgCAM and N‐cadherin on the development of axons and dendrites by cultured hippocampal neurons
J. Neurocytol.
Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP‐ribosylation factor nucleotide exchange factor ARNO
Mol. Biol. Cell
Cited by (2)
Exclusion of integrins from CNS axons is regulated by Arf6 activation and the AIS
2015, Journal of NeuroscienceARF6 directs axon transport and traffic of integrins and regulates axon growth in adult DRG neurons
2012, Journal of Neuroscience