Amacrine cells, which are pivotal interneurons in the retina, influence stimulus processing by modulating synapses between bipolar and ganglion cells, which form the retinal output. However, understanding their specific roles is challenging because of the diversity of amacrine cell types (60 in total), making analysis difficult using conventional methods. This study aims to investigate the role of two novel amacrine cells in retinal processing using an innovative approach. By expressing optogenetics with specific promoters (ProB13 and ProD20), we use single-cell 2-photon holographic stimulation to target these amacrine cells, alongside large-scale recordings to record ganglion cell activity. These two novel amacrine cells are named ProB13 AC and ProD20 AC, respectively. Confocal imaging of flat-mount retinas revealed that ProB13 ACs are located predominantly in the inner plexiform and ganglion cell layers, suggesting that correspond to displaced ACs. ProD20 exhibits strong expression in a specific type of narrow amacrine cell within the inner nuclear layer. Interestingly, the activation of ProB13 ACs individually by 2-photon holography inhibits the spontaneous activity of OFF αRGCs, where the strength of this inhibition depends on the distance between the Prob13 AC-OFF αRGCs and is mainly a GABA-mediated inhibition. Conversely, ProD20 ACs suppress ON RGC activity independent of distance.Our data provide initial insights into the role of these novel inhibitory amacrine cell types. Future investigations will explore their roles in processing natural scenes using a combination of optogenetics, 2-photon holography, and large-scale recordings, a question that could not be addressed with classical tools.