Haas Lab Methods

Xenopus Tadpoles as a Model System

In our laboratory, we use electroporation to examine the function of genes in the morphological and electro-physiological development of neurons in the albino Xenopus laevis tadpole brain. By adapting techniques worked out in the chick embryo and by devising new delivery methods, we have been able to control electroporative transfection in the Xenopus brain to small or large brain regions, or to individual cells. In general, we employ two distinct transfection strategies: one to maximize the numbers of neurons transfected in a specific brain region or throughout the brain, and an opposing strategy designed to restrict transfection to a single neuron. Albino Xenopus is ideal for these applications as the optic tectum is directly visible through the skin, permitting direct visualization of transfected neurons.

Single Cell Electroporation

Single-cell electroporation (SCE) is a technique developed by Dr. Kurt Haas for the selective delivery of macromolecules to individual neurons within the intact brain. SCE can be used to deliver plasmid DNA for neuronal transfection, to load cells with dye, or to fill cells with synthetic oligonucleotides morpholinos. SCE is particularly useful for labeling individual neurons in vivo by GFP expression or filling with fluorescent dye for imaging neuronal morphology and time-lapse imaging of neuronal growth. Setting up equipment and implementing SCE is simple and straightforward.

Dynamic Morphometrics

We have developed a new method for accurately analyzing brain neuron dendritic arbor growth involving rapid in vivo two-photon time-lapse imaging and post-imaging identification, tracking and measuring of all dendritic filopodia and branches within growing arbors. Our data allows comprehensive and sensitive analysis of rapid dynamic dendritic growth behaviors and how this growth relates to long-term persistent changes in neuronal morphology.