We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.
The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6.
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.
In this video we demonstrate an in vivo electroporation protocol that results in labeling of single or small clusters of retinal neurons in postnatal mice with DNA constructs encoding fluorescent proteins. Small clusters of fluorescently labeled RGC projections to the dLGN and SC reproduced similar projection patterns as previous studies using RGC labeling with lipophilic dyes, indicating that electroporation did not interfere with normal RGC axon arbor refinement. We have utilized this protocol to analyze the r…
The authors have nothing to disclose.
The pCAG-gapEGFP plasmid was a gift from Dr. S. McConnell (Stanford, CA). pCAG-tdTomato plasmid was a gift from Dr. M. Feller (Berkeley, CA). We thank Dr. Edward Ruthazer for suggesting the use of a two-plasmid strategy for single cell labeling and Anne Schohl (Montreal, QC) for validating the two-plasmid Cre/loxP strategy in pilot studies and Crair lab members for technical support. Supported by R01 MH62639 (MC), NIH R01 EY015788 (MC) and NIH P30 EY000785 (MC).
Materials | Company | Catalog number |
---|---|---|
Dumont #5 Forceps | Fine Science Tools | 11252-20 |
Electrical Stimulator | Grass Instruments | Model S4 |
Oscilloscope | Agilent | Model 54621A |
Audio monitor | Grass Instruments | Model AM8B |
Puller | Sutter Instruments | Model P-97 |
Vannas Scissors a | World Precision Instruments | 14003 |
Micro Scissors b | Ted Pella | 1347 |
Dumont AA Forceps c | Fine Science Tools | 11210-20 |
Nanoinject II System | Drummond Scientific | 3-000-204 |
Glass Pipettes | Drummond Scientific | 3-000-203-G/X |
Foot pedal | Drummond Scientific | 3-000-026 |
Mineral Oil | Sigma-Aldrich | M3516 |
DiI | Invitrogen | D-383 |
N,N-Dimethylformamide | Sigma | D4551 |