Electrophysiological analysis of dorsal root ganglion neurons pre- and post-coexpression of green fluorescent protein and functional 5-HT3 receptor.
Aequorea green fluorescent protein (GFP) is an excellent marker to examine genetically altered live cells in whole animals or culture. Its potential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicity, and potential electrophyiological effects of GFP expression in neurons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of postnatal DRG neurons appeared brightly fluorescent, labelling the soma and neurites. Temporal examination of these neurons demonstrated no toxicity to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and comparison of control and viral exposed (GFP- and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potential, action potential (AP) threshold, AP duration, AP amplitude, or whole cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotransmitter receptor, a second adenovirus construct was generated containing both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfection of DRG neurons with this virus produced an inward current in the presence of serotonin only in DRG neurons that were GFP-positive. It is concluded that adenoviral transfection of neurons with GFP, for cellular labeling, and coexpression of GFP-neurotransmitter constructs are safe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experiments is that it was constructed to express GFP in a second cassette so that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3 receptor.
Smith
G M
GM
The Eugene McDermott Center for Pain Management, Department of Anesthesiology and Pain Management, University of Texas Southwestern Medical Center, Dallas 75235-9068, USA.
Berry
R L
RL
Yang
J
J
Tanelian
D
D
D000818:Animals; D000831:Animals, Newborn; D002478:Cells, Cultured; D005727:Ganglia, Spinal / Q000166:cytology / Q000502:physiology*; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics*; D008564:Membrane Potentials / Q000235:genetics / Q000502:physiology; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000502:physiology / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D011985:Receptors, Serotonin / Q000502:physiology*; D044406:Receptors, Serotonin, 5-HT3; D009435:Synaptic Transmission / Q000502:physiology*; D014162:Transfection / Q000235:genetics*
D008164:Luminescent Proteins; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D049452:Green Fluorescent Proteins