Electric axon guidance in embryonic retina: Galvanotropism revisited

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Abstract

In addition to well-known mechanisms of chemical guidance, growing axons in the nervous system are directed by an extracellular electric field in a process known as galvanotropism. The galvanotropic behavior of nerve cells in vitro was first demonstrated as long ago as 1920. However, it remains unknown whether embryonic nerve tissues generate a similar electric field in order to guide growing axons. The present study reveals that an extracellular voltage gradient exists in the embryonic retina and that this gradient guides the axons of newborn retinal ganglion cells towards their targets. These findings indicate an important role for galvanotropism in the initial orientation of axons that extend over long distances, and provide insight into the mechanisms underlying the proper extension of developing axons in the embryonic brain.

Highlights

► An extracellular voltage gradient exists in the embryonic retina. ► The voltage gradient guides retinal ganglion cell axons towards their target. ► This study highlights galvanotropism in the initial orientation of axons.

Introduction

The prevailing belief is currently that growing axons are guided by attraction or repulsion in response to various chemical signals (chemotropism) during brain development. However, it has also been shown that cells may respond to an extracellular electric field by changing their morphology along the voltage gradient in a process termed ‘galvanotropism’ [1]. Galvanotropic behavior in nerve cells was demonstrated in cultured cells as early as 1920 [2]. The application of an extracellular electric field directs growing axons towards the cathode under a voltage gradient as low as 7 mV/mm [3]. However, since previous studies used culture systems to which exogenous electric fields were applied, it remains unknown whether electric gradients are normally generated in the embryonic nervous system, or whether developing axons use galvanotropism to find their targets in vivo. The present study reveals that an extracellular voltage gradient exists in the embryonic retina, and that axons of newborn retinal ganglion cells (RGCs) are directed towards their targets via a galvanotropic mechanism.

Section snippets

Preparation of retina

The optic cup was isolated from a chick embryo incubated for three days (E3) at 38 °C. The optic cup was positioned on the bottom of a recording chamber (volume, 0.2 mL) with the inner side up. The recording chamber was mounted on the fixed stage of an upright microscope (BX51WI, Olympus, Tokyo, Japan) under a water immersion objective (×100 or ×60), and was perfused at 2 mL/min with a normal bath solution (NBS) containing (mM); 137 NaCl, 5 KCl, 2.5 CaCl2, 1 MgCl2, 10 HEPES, 22 glucose, buffered

Results

Optic cups (precursors to the retina) were isolated from chick embryos incubated for three days (E3). Extracellular potentials were recorded from the dorsal (D), temporal (T), nasal (N), central (C), and ventral (V) parts of the optic cup, immediately inside the inner limiting membrane through which the axons of newborn RGCs travel. Upon penetration of the inner limiting membrane from the vitreous side with a microelectrode (Supplementary Fig. S1), a positive direct current (DC) potential was

Discussion

The molecular mechanisms involved in guiding RGC axons to the optic disc are not well understood [8]. Although netrin has been proposed to be an axon guidance molecule in the retina, RGC axons orient and extend towards the optic disc even in the absence of this molecule [9]. However, axons in these netrin-deficient retinas exhibit erroneous path-finding within the optic disc, and fail to exit via the optic nerve [9]. Thus, netrin-mediated signaling is essential for the local guidance of axons

Acknowledgments

This work was supported by the Japan Spina Bifida and Hydrocephalus Research Foundation (JSBHRF), Strategic Promotion System for Brain Science, Special Coordination Funds for Promoting Science and Technology (SCF), and Japan Society for the Promotion of Science (JSPS).

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