Pseudorabies virus expressing enhanced green fluorescent protein: A tool for in vitro electrophysiological analysis of transsynaptically labeled neurons in identified central nervous system circuits

Physiological properties of central nervous system neurons infected with a pseudorabies virus were examined in vitro by using whole-cell patch-clamp t echniques. A strain of pseudorabies virus (PRV 152) isogenic with the Barth a strain of PRV was constructed to express an enhanced green fluorescent pr otein (EGFP) from the human cytomegalovirus immediate early promoter. Unila teral PRV 152 injections into the vitreous body of the hamster eye transsyn aptically infected a restricted set of retinorecipient neurons including ne urons in the hypothalamic suprachiasmatic nucleus (SCN) and the intergenicu late leaflet (IGL) of the thalamus. Retinorecipient SCN neurons were identi fied in tissue slices prepared for in vitro electrophysiological analysis b y their expression of EGFP. At longer postinjection times, retinal ganglion cells in the contralateral eye also expressed EGFP, becoming infected afte r transsynaptic uptake and retrograde transport from infected retinorecipie nt neurons. Retinal ganglion cells that expressed EGFP were easily identifi ed in retinal whole mounts viewed under epifluorescence. Whole-cell patch-c lamp recordings revealed that the physiological properties of PRV 152-infec ted SCN neurons were within the range of properties observed in noninfected SCN neurons. Physiological properties of retinal ganglion cells also appea red normal. The results suggest that PRV 152 is a powerful tool for the tra nssynaptic labeling of neurons in defined central nervous system circuits t hat allows neurons to be identified in vitro by their expression of EGFP, a nalyzed electrophysiologically, and described in morphological detail.