The nigrostriatal dopaminergic pathway is implicated in Parkinson disease, although the precise mechanisms by which dopaminergic axons innervate striatal neurons are still unclear. We have established a method to evaluate dopaminergic innervation of striatal neurons by using primary dissociated cells. Mecencephalic and striatal cells derived from rat embryos were plated inside and outside an isolation wall, respectively. When mecencephalic cells were adjacently paired-cultured with striatal cells for 10 days after removal of the isolation wall, dopaminergic neurons in the mesencephalic cell region extended their axons to the striatal cell region. We have previously showed that pharmacological inhibition of integrin α5β1, one of cell adhesion molecules, attenuated the dopaminergic innervation of the striatal neurons. In this study, we investigated whether integrin α5β1 which is expressed in dopaminergic neurons is involved in the innervation. We prepared a lentivirus expressing shRNA targeted to integrin α5 and the fluorescent reporter protein Venus. Local application of the lentivirus vector inside the isolation wall induced preferential infection of mesencephalic cells, and transfection with integrin α5 shRNA led to the knockdown of target gene expression in mecencepahalic cells without affecting the expression in striatal cells. The application of the lentivirus vector achieved a tranfection efficiency of about 70-80% in dopaminergic neurons. Quantitative analysis of Venus-positive dopaminergic neurite lengths revealed that selective transfection of mecencepahalic cells with integrin α5 shRNA siginificantly suppressed the neurite outgrowth toward the striatal cell region. These results suggest that integrin α5 expressed in mesencephalic dopaminergic neurons is important for their axons to innervate striatal neurons.