Neuronal growth cones respond to laser-induced axonal damage

Abstract

Although it is well known that damage to neurons results in release of substances that inhibit axonal growth, release of chemical signals from damaged axons that attract axon growth cones has not been observed. In this study, a 532 nm 12 ns laser was focused to a diffraction-limited spot to produce site-specific damage to single goldfish axons in vitro. The axons underwent a localized decrease in thickness ('thinning') within seconds. Analysis by fluorescence and transmission electron microscopy indicated that there was no gross rupture of the cell membrane. Mitochondrial transport along the axonal cytoskeleton immediately stopped at the damage site, but recovered over several minutes. Within seconds of damage nearby growth cones extended filopodia towards the injury and were often observed to contact the damaged site. Turning of the growth cone towards the injured axon also was observed. Repair of the laser-induced damage was evidenced by recovery of the axon thickness as well as restoration of mitochondrial movement. We describe a new process of growth cone response to damaged axons. This has been possible through the interface of optics (laser subcellular surgery), fluorescence and electron microscopy, and a goldfish retinal ganglion cell culture model.

Nanotechnology platforms in Parkinson’s Disease

“…This resulted in the vaporization of femtolitres of axon volumes and subsequent cutting of the axon processes [122]. Besides, the technique was robust allowing the use of confocal fluorescence imaging and laser scanning brightfield for real-time imaging from the first incision to the end [122].The surgical technique has improved understanding of a number of processes in nerve cell injury [123] and has also paved way for an all new concept of nanomanipulation.…”

Directing growth cones of optic axons growing with laser scissors and laser tweezers

“…1. For the laser scissors system 1,6 , the second harmonic (532 nm, 12 ns) of a Nd:YOV 4 laser was coupled to a Zeiss microscope through the epi-fluorescence port by scanning of a Fast Steering Mirror (FSM) and expanded to fill the back aperture of a 63X objective (NA=1.4). The number of the pulses irradiated on the axons is controlled by a mechanical shutter.…”

“…Goldfish retinal ganglion cells are used as samples in these studies 1,7 . The optic nerve of an adult goldfish was firstly crushed and the goldfish was then put back into the tank for the damaged axon to regenerate.…”

“…In addition, laser tweezers generates localized microfluidic flow 2 , which can guide the axonal growth. By generating controlled sub-axotomy laser microbeam injury and localized microfluidic flow on retinal ganglion axons derived from goldfish retina explants 1,7 , we demonstrate the response of near-by axonal growth cones to the damage, in which filopodia growing towards and touching the localized damage induced by laser pulses, and to the microfluidic flow, in which the axon growth cones turn in response to the optically driven microfluidic flow. …”

“…By exploiting laser micromanipulation technologies, such as laser scissors and laser tweezers, the whole neuronal cells or intracellular organelles can be studied with high spatial precision 1,2 . The ability to study the guided growth of growth cones responding to focused damage, and localized microfluidic flow applied on a single axon is novel and opens up opportunities not only for basic research on individual axons but also for studies on repair and regeneration that will impact our understanding of traumatic brain, spinal cord injury and retinal degeneration.…”