Laser microbeam surgery: ultrastructural changes associated with neurite transection in culture

The exposure of neuronal and glial cell processes to a large number (up to 300) of 12-nsec laser pulses at a wavelength of 337 nm and energy densities below the threshold for nonlinear absorption results in a gradual, gentle process transection in the laser focus. Within 10 to 20 sec after cessation of firing, the process pinches in the target area. During this time, mitochondria become swollen and bleached, the plasma membrane develops an obvious tautness, microtubules disappear, and organelles accumulate to either side of the process constriction. Depending on the irradiation parameters, a local pinching may proceed to a transection in about 30 sec or it may reverse to yield a normal-appearing process in approximately 5 min. Severe process pinching is accompanied by a sudden depolarization that may last for 2 to 5 min and is usually followed by a repolarization to the original resting potential even if the process has transected. Spiral retraction of cut processes and cytoplasmic spillage observed after mechanical transections are not seen with this laser method. Process stretching is minimized or eliminated. Extensive vacuolization often associated with mechanical transections does not develop unless substrate involvement in the form of shock waves is apparent. For the performance of cell surgery in culture, this method appears to offer a reliable approach to morphological alteration of single cells and to the tailoring of two-dimensional neuronal networks. It should also allow more quantitative and better-controlled studies of axonotmesis, degeneration, and regeneration on the single cell level, and it may be used as a probe for the investigation of cytoskeletal dynamics. A mechanism describing the cytoskeletal changes associated with laser-induced cell process transection is proposed.