Abstract
Efficient methods for visualizing cell morphology in the intact animal are of great benefit to the study of structural development in the nervous system. Quantitative analysis of the complex arborization patterns of brain cells informs cell-type classification, dissection of neuronal circuit wiring, and the elucidation of growth and plasticity mechanisms. Time-lapse single-cell morphological analysis requires labeling and imaging of single cells in situ without contamination from the ramified processes of other nearby cells. Here, using the Xenopus laevis optic tectum as a model system, we describe CRE-Mediated Single-Cell Labeling by Electroporation (CREMSCLE), a technique we developed based on bulk co-electroporation of Cre-dependent inducible expression vectors, together with very low concentrations of plasmid encoding Cre recombinase. This method offers efficient, sparse labeling in any brain area where bulk electroporation is possible. Unlike juxtacellular single-cell electroporation methods, CREMSCLE relies exclusively on the bulk electroporation technique, circumventing the need to precisely position a micropipette next to the target cell. Compared with viral transduction methods, it is fast and safe, generating high levels of expression within 24 h of introducing non-infectious plasmid DNA. In addition to increased efficiency of single-cell labeling, we confirm that CREMSCLE also allows for efficient co-expression of multiple gene products in the same cell. Furthermore, we demonstrate that this method is particularly well-suited for labeling immature neurons to follow their maturation over time. This approach therefore lends itself well to time-lapse morphological studies, particularly in the context of early neuronal development and under conditions that prevent more difficult visualized juxtacellular electroporation.
Highlights
The nature of the chaotic complexity of intermingled fibers and cellular structures in the nervous system constituted one of the great mysteries of anatomy, until Camillo Golgi’s serendipitous discovery of his “black reaction” in 1873 (Golgi, 1873)
We have presented a quantitative characterization of the CREMSCLE technique for sparse but bright labeling of single cells through the co-electroporation of extremely low concentrations of plasmid encoding Cre recombinase together with an enhanced green fluorescent protein (EGFP) or other expression vector into which a stop cassette has been inserted upstream of the open reading frame to inhibit protein translation in cells lacking Cre expression
This method, which benefits from the versatility and ease of bulk electroporation, provides bright labeling of sparsely distributed cells, which is an ideal condition for in vivo imaging of cellular morphology and time lapse imaging of axonal and dendritic branch remodeling
Summary
The nature of the chaotic complexity of intermingled fibers and cellular structures in the nervous system constituted one of the great mysteries of anatomy, until Camillo Golgi’s serendipitous discovery of his “black reaction” in 1873 (Golgi, 1873). Golgi’s stain, which miraculously produced sparse, but highly contrasted labeling of cells in nervous tissue, prompted the brilliant Spanish neuroanatomist Santiago Ramon y Cajal to embrace the “neuron doctrine,” which states that individual brain cells, or neurons, are the fundamental units from which nervous tissue is formed (Jones, 2010). Our efforts to dissect and decipher the nervous system continue to rely heavily on techniques that permit the labeling, observation and quantitative analysis of single cells within the richly complex network of brain cells and their elaborate processes from which neural circuits are constructed. Single-cell electroporation (SCE) is a juxtacellular labeling technique that has been used successfully to transfect and visualize individual cells with Golgi-like contrast in intact brain tissue and in organotypic slice cultures (Haas et al, 2001; Uesaka et al, 2005). By locally disrupting the plasma membrane of the cell while iontophoretically expelling the charged contents of the micropipette, the contents of the pipette are efficiently delivered into the cell, which promptly reseals its plasma membrane and traps the material within
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