Slit2/Robo1 signaling constrains image stabilization responses to preserve ethologically favorable directional asymmetry.

Visual sensation relies on retinal circuitry to receive environmental inputs and convey relevant information for behavioral outputs. Many species depend on gaze stabilization behaviors, such as the optokinetic reflex (OKR), to perceive and correct for global motion. OKR calculation begins in the retina, where ON direction-selective ganglion cells (oDSGCs) respond to slow visual motion and deliver information to the accessory optic system (AOS) to inform oculomotor outputs. Here, we find that the guidance receptor roundabout-1 (Robo1) and its ligand Slit2 are selectively expressed in mammalian retinal AOS components and refine oDSGC circuitry to constrain OKR gain for appropriate image stabilization responses. Robo1 or Slit2 loss-of-function (LOF) mutants exhibit an increase in OKR gain, reducing directional asymmetry by elevating naturally weaker OKR responses. These behavioral enhancements largely arise from alterations in the retina, and loss of Slit2 in starburst amacrine cells (SACs) enhances oDSGC firing properties, with increased spike responses, excitatory synaptic puncta, and downstream AOS activation. These findings reveal that the Slit2/Robo1 signaling tunes oDSGC circuitry to maintain regulated image stabilization behavior.