This is IMAGE #3 (20 minutes) of a time lapse series contained with in this image group. It shows neurons from the optic tectum of an albino Xenopus laevis tadpole CNS, stage 47. The images were acquired with ~10 minute intervals and show morphological plasticity of the dendritic arbor with branch additions and retractions. These cells were transfected ~24 hours earlier with in vivo electroporation of plasmid DNA encoding eYFP. The intact, living tadpole was anesthetized, positioned under a glass coverslip and this image was taken directly through its transparent skin using a custom built two-photon microscope. Images were acquired a 0, 10, 20, 30, and 50 minutes. After 60 minutes, the animal was placed in a 50 microMolar anisomycin solution for ~4 hours. A second time lapse series (10 minute interval for 60 minutes) was then acquired.
Specifications of the custom built two-photon microscope, including the laser sources, signal amplification, PMT specifications, and YFP filter sets are described in: Ruthazer ES, Li J, Cline HT. 2006. Stabilization of axon branch dynamics by synaptic maturation. J Neurosci 26(13):3594-3603. The methods of cell transfection with electroporation can be found here: Bestman JE, Ewald RC, Chiu S-L, Cline HT. 2006. In vivo single-cell electroporation for transfer of DNA and macromolecules. Nat Protocols 1(3):1267-1272. Further explanation can be found here: Bestman JE, Cline HT. 2008. The RNA binding protein CPEB regulates dendrite morphogenesis and neuronal circuit assembly in vivo. Proc Natl Acad Sci U S A 105(51):20494-20499. Bestman JE, Cline HT. 2009. The Relationship between Dendritic Branch Dynamics and CPEB-Labeled RNP Granules Captured in Vivo. Front Neural Circuits 3:10.
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