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 with in vivo electroporation of plasmid DNA encoding eYFP ~24 hours before the first image was acquired. 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 every 10 minutes for 1 hour. After 60 minutes, the animal was placed in a 50 microMolar anisomycin solution for ~1.5 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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