The formation of actin waves during regeneration after axonal lesion is enhanced by BDNF

During development, axons of neurons in the mammalian central nervous system lose their ability to regenerate. To study the regeneration process, axons of mouse hippocampal neurons were partially damaged by an UVA laser dissector system. The possibility to deliver very low average power to the sample reduced the collateral thermal damage and allowed studying axonal regeneration of mouse neurons during early days in vitro. Force spectroscopy measurements were performed during and after axon ablation with a bead attached to the axonal membrane and held in an optical trap. With this approach, we quantified the adhesion of the axon to the substrate and the viscoelastic properties of the membrane during regeneration. The reorganization and regeneration of the axon was documented by long-term live imaging. Here we demonstrate that BDNF regulates neuronal adhesion and favors the formation of actin waves during regeneration after axonal lesion

Long-Distance Signals Are Required for Morphogenesis of the Regenerating Xenopus Tadpole Tail, as Shown by Femtosecond-Laser Ablation

tadpoles has recently emerged as an important model for these studies; we explored the role of the spinal cord during tadpole tail regeneration.Using ultrafast lasers to ablate cells, and Geometric Morphometrics to quantitatively analyze regenerate morphology, we explored the influence of different cell populations. For at least twenty-four hours after amputation (hpa), laser-induced damage to the dorsal midline affected the morphology of the regenerated tail; damage induced 48 hpa or later did not. Targeting different positions along the anterior-posterior (AP) axis caused different shape changes in the regenerate. Interestingly, damaging two positions affected regenerate morphology in a qualitatively different way than did damaging either position alone. Quantitative comparison of regenerate shapes provided strong evidence against a gradient and for the existence of position-specific morphogenetic information along the entire AP axis.We infer that there is a conduit of morphology-influencing information that requires a continuous dorsal midline, particularly an undamaged spinal cord. Contrary to expectation, this information is not in a gradient and it is not localized to the regeneration bud. We present a model of morphogenetic information flow from tissue undamaged by amputation and conclude that studies of information coming from far outside the amputation plane and regeneration bud will be critical for understanding regeneration and for translating fundamental understanding into biomedical approaches

Exploring the ultrashort pulse laser parameter space for membrane permeabilisation in mammalian cells.

The use of ultrashort femtosecond pulsed lasers to effect membrane permeabilisation and initiate both optoinjection and transfection of cells has recently seen immense interest. We investigate femtosecond laser-induced membrane permeabilisation in mammalian cells as a function of pulse duration, pulse energy and number of pulses, by quantifying the efficiency of optoinjection for these parameters. Depending on pulse duration and pulse energy we identify two distinct membrane permeabilisation regimes. In the first regime a nonlinear dependence of order 3.4-9.6 is exhibited below a threshold peak power of at least 6 kW. Above this threshold peak power, the nonlinear dependence is saturated resulting in linear behaviour. This indicates that the membrane permeabilisation mechanism requires efficient multiphoton absorption to produce free electrons but once this process saturates, linear absorption dominates. Our experimental findings support a previously proposed theoretical model and provide a step towards the optimisation of laser-mediated gene delivery into mammalian cells.Publisher PDFPeer reviewe

Laser Fusion of Mouse Embryonic Cells and Intra-Embryonic Fusion of Blastomeres without Affecting the Embryo Integrity

Manipulation with early mammalian embryos is the one of the most important approach to study preimplantation development. Artificial cell fusion is a research tool for various biotechnological experiments. However, the existing methods have various disadvantages, first of them impossibility to fuse selected cells within multicellular structures like mammalian preimplantation embryos. In our experiments we have successfully used high repetition rate picosecond near infrared laser beam for fusion of pairs of oocytes and oocytes with blastomeres. Fused cells looked morphologically normal and keep their ability for further divisions in vitro. We also fused two or three blastomeres inside four-cell mouse embryos. The presence of one, two or three nuclei in different blastomeres of the same early preimplantation mouse embryo was confirmed under UV-light after staining of DNA with the vital dye Hoechst-33342. The most of established embryos demonstrated high viability and developed in vitro to the blastocyst stage. We demonstrated for the first time the use of laser beam for the fusion of various embryonic cells of different size and of two or three blastomeres inside of four-cell mouse embryos without affecting the embryo’s integrity and viability. These embryos with blastomeres of various ploidy maybe unique model for numerous purposes. Thus, we propose laser optical manipulation as a new tool for investigation of fundamental mechanisms of mammalian development

Femtosecond optical transfection of individual mammalian cells

Laser-mediated gene transfection into mammalian cells has recently emerged as a powerful alternative to more traditional transfection techniques. In particular, the use of a femtosecond-pulsed laser operating in the near-infrared (NIR) region has been proven to provide single-cell selectivity, localized delivery, low toxicity and consistent performance. This approach can easily be integrated with advanced multimodal live-cell microscopy and micromanipulation techniques. The efficiency of this technique depends on an understanding by the user of both biology and physics. Therefore, in this protocol we discuss the subtleties that apply to both fields, including sample preparation, alignment and calibration of laser optics and their integration into a microscopy platform. The entire protocol takes similar to 5 d to complete, from the initial setup of the femtosecond optical transfection system to the final stage of fluorescence imaging to assay for successful expression of the gene of interest