ELECTROPHYSIOLOGICAL ANALYSIS OF DORSAL-ROOT GANGLION NEURONS - PRE-COEXPRESSION AND POST-COEXPRESSION OF GREEN FLUORESCENT PROTEIN AND FUNCTIONAL 5-HT3-RECEPTOR

Aequorea green fluorescent protein (GFP) is an excellent marker to exa mine genetically altered live cells in whole animals or culture. Its p otential use in identifying genetically modified neurons, however, has not been investigated extensively. To examine the usefulness, toxicit y, and potential electrophyiological effects of GFP expression in neur ons, we generated adenovirus containing the mGFP4 cDNA. One week after virus transfection of dorsal root ganglion neurons (DRG), 10% of post natal DRG neurons appeared brightly fluorescent, labelling the soma an d neurites. Temporal examination of these neurons demonstrated no toxi city to DRG neurons even after several weeks in culture with repeated daily epifluorescent exposure. Electrophysiological analysis and compa rison of control and viral exposed (GFP- and GFP+) DRG neurons did not demonstrate any differences in whole cell resistance, resting potenti al, action potential (AP) threshold, AP duration, AP amplitude, or who le cell capacitance. To investigate the usefulness of GFP as a marker for identifying neurons genetically altered to express a novel neurotr ansmitter receptor, a second adenovirus construct was generated contai ning both GFP and serotonin type 3 (5-HT3) receptor cDNAs. Transfectio n of DRG neurons with this virus produced an inward current in the pre sence of serotonin only in DRG neurons that were GFP-positive. It is c oncluded that adenoviral transfection of neurons with GFP, for cellula r labeling, and coexpression of GFP-neurotransmitter constructs are sa fe, nontoxic, methods for electrophysiologically investigating neurons over several weeks. The uniqueness of the vector used in these experi ments is that it was constructed to express GFP in a second cassette s o that it would label the transduced cells, but have no potential for interfering with the function of the foreign 5-HT3 receptor.