Using the visual cliff assay to assess binocular deficits in amblyopic mice

The visual cliff assay (VCA) has been used in multiple animal models including humans to assess stereopsis, the pinnacle percept of binocular function that is markedly disrupted in amblyopia. Amblyopia is a neurodevelopmental disorder of the visual system caused by refractive error, strabismus, or obscuration of the visual axis during infancy and early childhood. While the current standard of care can improve visual acuity in the amblyopic eye, gains are infrequently associated with improvements in stereoscopic depth perception and typically only achieved when treatment is initiated in early childhood. The mouse model of amblyopia induced by monocular deprivation (MD) is a powerful tool to study the mechanistic pathophysiology of this disorder and test novel therapeutics. However, to date, only one study has used the VCA to assess the effects of MD in mice in a limited capacity. Advantages of the VCA include no need for operant conditioning or training, completion in minutes, and minimal equipment. We comprehensively characterize and validate fundamental aspects of the VCA including test-retest reliability and contributions of binocularity using multiple monocular and binocular manipulations with clinically relevant paradigms. After long-term (3 weeks) MD, mice exhibit reduced predilection for the safe side, decreased latency to cross to the cliff side, and more crosses to the cliff side compared to sham littermate controls. Our data demonstrate that the VCA can detect binocular deficits in amblyopia but with important limitations that guide how the VCA should be applied to read out binocular function in both mechanistic and therapeutic studies in mice. ### Competing Interest Statement MFB: Luminopia, Inc. (scientific advisor, equity) EDG: Luminopia, Inc. (scientific advisor, equity, patent), Stoke Therapeutics Inc. (consultant)