This is IMAGE #4 (day 3) of a time lapse series contained with in this image group. It shows a neuron from the optic tectum of an albino Xenopus laevis tadpole CNS, stage 47. The images were acquired daily over 3 days and show dendritic arbor development and morphological plasticity of the dendritic arbor with branch additions and retractions. The superficial layers of the z-stack show the autofluorescence of a pigment cell. This cell was transfected ~24 hours before the first image 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.
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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