This research effort will focus on gene delivery of fluorescent proteins to neurons in the retina using HSV vectors in order to elucidate details of synaptic circuitry.
In Aim 1, targeted gene delivery to specific neuronal subtypes will be undertaken by selective infection and by placing transgene expression under a cell specific promoter. The major heparin sulfate binding domain of the virion will be replaced with BDNF to increase infection of retinal ganglion cells that express high levels of the TrkB receptor on their cell surface. Transcriptional targeting will be undertaken by using the Thy-1 promoter, specifically expressed in this same neuronal population, either to drive expression of a reporter gene in HSV amplicon vectors, or by blocking expression of a reporter under a strong promoter in the vector through insertion of a IoxP-flanked stop codon between these sequences with subsequent infection of Thy-1-Cre transgenic mice to activate transgene expression.
In Aim 2, we will attempt to label specific synaptic pathways by vector-mediated delivery of informative fluorescent fusion proteins. A GFP-tetanus toxin heavy chain fusion protein will be used to selectively label presynaptic contacts of the infected neurons. A BDNF-GFP fusion protein will be used to label synaptic partners of the infected cell which are enriched for TrkB-BDNF receptors.
Aim 3 will undertake labeling of synaptically connected neurons by expression of reporter genes carried in a replicating vector system intended to have minimal-to-no toxicity for the host neurons. Vector propagation will be controlled by placing an essential viral gene for ICP4 under a tight tetracycline regulated (tet-on) promoter in an HSV amplicon vector and coupling infection with a mutant helper virus deleted for this gene. Both vector and helper virus will express reporter genes, and co-infection with both will allow drug-dependent regulation of the levels of virions produced and passed onto synaptically connected neurons. The proposed targeting and labeling methods should be adaptable to other types of receptors and synaptic connections throughout the nervous system and should be very useful in dissecting and monitoring synaptic circuitry in living and fixed preparations.
