Fig. 6: Transsynaptic tracing with ATLASsn.

a, Schematic of ATLASsn. (1) Synaptotagmin nanobody (SYTnb) fused to the transmembrane domain of VAMP2 (VAMP2Δ) targets ATLAS to synaptic vesicles. (2) AF-Cre is cleaved from VAMP2Δ. (3) The synaptic vesicle fuses with the plasma membrane at the presynaptic terminal. (4) AF-Cre is released into the synaptic cleft. (5) SYTnb-VAMP2Δ-At is recycled back into the terminal, labeling the presynaptic site. b, ATLAS tracing in dissociated cortical culture transfected with AAV8-ATLASsn_Cre (labeled with At, cyan) and AAV8-BACE-HA and infected with AAV8-DIO-mCherry. Scale bar, 10 μm. c, mPFC of the mouse injected with AAV8-ATLASsn_Cre (cyan) and incubated for 2 weeks. This experiment was performed in three mice. d, Str of the same mouse as in c, injected with AAV8-DIO-mCherry (red). e, Schematic showing projections from the M1 to Str and thalamus (Thal). This experiment was performed in three mice. f, M1 of a mouse coinjected with AAV8-BACE-GFP (green) and AAV8-ATLASsn_Cre. g, Str of the same mouse as in f injected with AAV8-DIO-mCherry showing mCherry-labeled neurons (red). This experiment was performed in three mice. h, Thalamus of the same mouse as in f and g injected with AAV8-DIO-mCherry showing mCherry-labeled neurons (red). i, Schematic showing pathway from the ventral CA1 to the nucleus accumbens (ACB) and then to the LHA in the rat. This experiment was performed in three rats. j, Ventral CA1 region of a rat injected with AAV8-ATLASsn_Cre showing cell labeled with At (cyan). k, ACB of the same rat as in j injected with AAV8-DIO-tdTomato, showing tdTomato-labeled neurons (red): aco refers to the anterior commissure. l, LHA of the same rat as in j and k showing nerve terminals labeled with tdTomato (red). Scale bars 100 μm in c,d,f,g,h, 500 μm in j; 200 μm in k,l.