Genetic tools have revealed the localization and functions of specific neuronal populations expressing distinctive neurotransmitters such as dopamine, serotonin, and noradrenaline. However, these genetically identified neurons sometimes contain subpopulation which has independent projections and independent physiological functions. Therefore, it is important to reveal the projection pattern and manipulate them selectively to elucidate the functional unit of neuronal circuits. To achieve this goal, we developed Cre recombinase fused with tetanus toxin fragment C (Cre-TTC) which can be expressed by adeno-associated virus (AAV) vectors. TTC is used for projection tracing, taking advantage of its retrograde trans-synaptic transport. To test the function of Cre-TTC, we selected dopamine neurons in the ventral tegmental area (VTA) and the substantia nigra pars compacta (SNc). It is known that dopamine neurons in the VTA project to the nucleus accumbens (NAc), while dopamine neurons in the SNc project to the caudate putamen (CPu). When we injected Cre-TTC vector in the CPu and FLEX-GFP vector in the VTA-SNc region, we detected GFP only in the SNc not in the VTA. On the other hand, when we injected Cre-TTC vector in the NAc and FLEX-GFP vector in the VTA-SNc region, we observed GFP only in the VTA not in the SNc. These results show that Cre-TTC is trans-synaptically and retrogradely transported from projected neurons and induce Cre-dependent gene expression in upstream projecting neurons. Our new expression system using AAV vectors can be used to visualize and manipulate the activity of the upstream neurons of specific neuronal populations.