Objective Direction-selective retinal ganglion cell (dsRGC) has characteristic responses to light stimuli: When light stimulus moving in its preferred direction, dsRGC will response with a large number of action potentials; when the light stimulus moving in its null direction, dsRGC could not response with action potentials, but only excitatory postsynaptic potential (EPSP) can be recorded.The different responses caused by the asymmetric inhibitory connections exist between dsRGC and starburst amacrine cells (SACs): dsRGC has inhibitory connections with the SACs located in its null side significantly stronger than with the SACs located in its preferred side.Our study focused on when the asymmetric inhibitory connections between dsRGC and SACs formed.Methods We first puffed glutamate (10 mM) around adult mouse retinal dsRGC soma in different orientations and recorded dsRGCs responses.We found puff glutamate could be used to detect the asymmetric inhibitory connections between dsRGC and SACs: When glutamate was puffed at the preferred side the response intensity was significantly higher than when glutamate was puffed at the null side.Results and Conclusion We puffed glutamate around adult and P8-11 mouse retinal dsRGCs, used this method we ultimately found in mouse retina the asymmetric inhibitory connections between dsRGC and SACs started to be formed on the 10th day after mouse was born.