Cre-Dependent Viral Tracing of Neuronal Circuits

Anterograde tracing was performed using adeno-associated virus (AAV), including Cre-activated (Flex) and Cre-silenced (Fas) vectors (“Cre-on/Cre-off” system). Viral stocks were prepared at the University of Pennsylvania Gene Therapy Program Vector Core (http://www.med.upenn.edu/gtp/vectorcore/). For Cre-dependent labeling of cell bodies and axons we used AAV pCAG.FLEX.tdTomato.WPRE (“FLEX-tdT,” Addgene plasmid #51503) or AAV pCAG.FLEX.EGFP.WPRE (Addgene plasmid #51502; Harris et al., 2012 ). Enhanced labeling of axon terminals was performed by Cre-dependent viral expression of a synaptophysin-EGFP fusion protein (sypGFP). The plasmid pCAG.Flex.sypEGFP.WPRE (“FLEX-sypGFP”) was constructed by replacing the EGFP moiety of pCAG-FLEX-EGFP-WPRE with the sypEGFP construct from phSyn1(S)-FLEX-tdTomato-T2A-SypEGFP-WPRE (Addgene #51509) by Julie Harris, Karla Hirokawa, and Hong Gu of the Allen Institute for Brain Science (gift of Julie Harris). In most experiments the tdTomato axonal tracer and the sypGFP synaptic tracer viruses were co-injected. Cre-inactivated expression was performed with pAAV-Ef1a-FAS-tdTomato-WPRE (“FAS-tdT,” Addgene #37092; Saunders et al., 2012 (link)). In most cases, FAS-tdT was co-injected with the FLEX-GFP or FLEX-sypGFP virus. Viral methods for optogenetic electrophysiological experiments are described below. All viruses used were AAV capsid strain 1.