Role of electrical activity in horizontal axon growth in the developing cortex: a time-lapse study using optogenetic stimulation

PLoS One. 2013 Dec 23;8(12):e82954. doi: 10.1371/journal.pone.0082954. eCollection 2013.


During development, layer 2/3 neurons in the neocortex extend their axons horizontally, within the same layers, and stop growing at appropriate locations to form branches and synaptic connections. Firing and synaptic activity are thought to be involved in this process, but how neuronal activity regulates axonal growth is not clear. Here, we studied axonal growth of layer 2/3 neurons by exciting cell bodies or axonal processes in organotypic slice cultures of the rat cortex. For neuronal stimulation and morphological observation, plasmids encoding channelrhodopsin-2 (ChR2) and DsRed were coelectroporated into a small number of layer 2/3 cells. Firing activity induced by photostimulation (475 nm) was confirmed by whole-cell patch recording. Axonal growth was observed by time-lapse confocal microscopy, using a different excitation wavelength (560 nm), at 10-20-min intervals for several hours. During the first week in vitro, when spontaneous neuronal activity is low, DsRed- and ChR2-expressing axons grew at a constant rate. When high-frequency photostimulation (4 or 10 Hz) for 1 min was applied to the soma or axon, most axons paused in their growth. In contrast, lower-frequency stimulation did not elicit this pause behavior. Moreover, in the presence of tetrodotoxin, even high-frequency stimulation did not cause axonal growth to pause. These results indicate that increasing firing activity during development suppresses axon growth, suggesting the importance of neuronal activity for the formation of horizontal connections.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials / drug effects
  • Action Potentials / physiology*
  • Animals
  • Animals, Newborn
  • Axons / drug effects
  • Axons / metabolism
  • Axons / ultrastructure*
  • Channelrhodopsins
  • Electroporation
  • Gene Expression
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Optogenetics
  • Patch-Clamp Techniques
  • Photic Stimulation
  • Plasmids
  • Rats
  • Tetrodotoxin / pharmacology
  • Time-Lapse Imaging
  • Tissue Culture Techniques
  • Visual Cortex / cytology
  • Visual Cortex / drug effects
  • Visual Cortex / growth & development*
  • Visual Cortex / metabolism


  • Channelrhodopsins
  • Luminescent Proteins
  • fluorescent protein 583
  • Tetrodotoxin

Grant support

This study was supported by Grants-in-Aid for Scientific Research on Innovative Areas “Mesoscopic Neurocircuitry” (number 23115102) of the Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT) to N.Y., and for Scientific Research (B) (numbers 20300110 and 23300118) of the Japan Society for the Promotion of Science to NY. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.