Axonal regeneration of cultured mouse hippocampal neurons studied by an optical nano-surgery system

During development, the axons of neurons in the mammalian central nervous system lose their ability to regenerate after injury. In order to study the regeneration process, we developed a system integrating an optical tweezers and a laser dissector to manipulate the sample. A sub-nanosecond pulsed UVA laser was used to inflict a partial damage to the axon of mouse hippocampal neurons at early days in vitro. Partial axonal transections were performed in a highly controlled and reproducible way without affecting the regeneration process. Force spectroscopy measurements, during and after the ablation of the axon, were performed by optical tweezers with a bead attached to the neuronal membrane. Thus, the release of tension in the neurite could be analyzed in order to quantify the inflicted damage. After dissection, we monitored the viscoelastic properties of the axonal membrane, the cytoskeleton reorganization, and the dynamics of the newly formed growth cones during regeneration. In order to follow cytoskeleton dynamics in a long time window by tracking a bead attached to the neuron, we developed a real-time control of the microscope stage position with submillisecond and nanometer resolution. Axonal regeneration was documented by long-term ( 24-48 hours) bright-field live imaging using an optical microscope equipped with a custom-built cell culture incubator.