Methods

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. We have described this technique in the following publications:

Haas, K, WC Sin, A Javaherian, Z Li and HT Cline. "Single-cell electroporation for gene transfer in vivo". Neuron. 29 (2001): 583 - 591.

Hewapathirane D.S., Haas K. "Single-cell electroporation in vivo". Journal Visualized Experiments, 17 (2008). 705.

Xue Feng Liu and Kurt Haas. “Single-cell Electroporation in Xenopus". In: Imaging in Developmental Biology: A Laboratory Manual. Eds. Rachel Wong and James Sharpe Cold Spring Harbor Laboratory Press. 2009.

Derek Dunfield and Kurt Haas. "Single-Cell Electroporation" . New Encyclopedia of Neuroscience. Eds.Larry Squire, Tom Albright, Floyd Bloom, Fred Gage and Nick Spitzer, Oxford University Press., 2009.

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. This technique has been used in the following study:

Xue Feng Liu, Parisa Karimi Tari, and Kurt Haas, "PKM zeta restricts dendritic arbor growth by filopodial and branch stabilization within the intact and awake developing brain" Journal of Neuroscience. 29 (2009)12229-35.

calcium imaging of network activity and plasticity

We employ bulk loading of calcium-sensitive dyes into Xenopus brains to image activity and plasticity within the intact and awake developing brain in response to natural visual stimuli. Rapid in vivo time-lapse imaging of a single optical plain through the optic tectum allows simultaneous sampling of networks of 100s of neurons, with single-cell resolution. Using this technique, we have demonstrated paradigms for inducing long lasting potentiation, depression and metaplasticity within the awake developing brain. See:

Derek Dunfield and Kurt Haas, "Metaplasticity governs natural experience-driven plasticity of nascent embryonic brain circuits", Neuron. 64 (2009) 240-50.