Abstract
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.
Highlights
Neocortical circuits are known to be established by genetic mechanisms and by environmental factors such as sensory-evoked neuronal activity
The results revealed that horizontally elongating axons pause in their growth after high-frequency stimulation, suggesting that developing firing activity contributes to circuit formation by regulating axonal growth
ChR2-EYFP was used for optogenetic stimulation (475 nm), while DsRed was used to observe axonal growth under a different excitation light (560 nm) without activating ChR2
Summary
Neocortical circuits are known to be established by genetic mechanisms and by environmental factors such as sensory-evoked neuronal activity. Intrinsic cortical circuits are thought to be established based on genetic and activitydependent mechanisms. Axon collaterals from layer 2/3 pyramidal neurons, called horizontal axons, connect cells within the same layer, and these connections provide a substrate for lateral interactions across cortical columns [2,3,4,5,6,7,8]. In the visual cortex of higher mammals, the horizontal connections primarily link cortical cells in columns having the same orientation [9,10,11,12,13,14,15]. Horizontal connections have been found in other cortical areas, and are the basis of information processing in the neocortex [16,17,18,19,20,21,22,23]
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