Characteristics And Synaptic Mechanism Of Asynchrony Of ON/OFF System During Retinal Waves

Patterns of synaptic connections in the visual system are remarkably precise.The connectivity patterns of many early neuronal networks undergo extensive refinement process during development in which imprecise connections are weakened and eliminated by asynchronous activities and correctly targeted connections are strengthened and maintained by asynchronous activities.Much of this refinement is well underway or even completed before vision begins.Patterns of coordinated spontaneous activity have been proposed to guide circuit refinement in many parts of the developing nervous system.In retina,On vs.Off information is encoded in stage III waves and is relayed to central brain targets as differences in the temporal pattern of spiking and mean firing rate across these two RGC populations.It is unclear,however,whether the on-off asynchrony is present only in the level of output neurons,or it also exists among interneurons upstream of RGCs;what are the temporal dynamics of and spatial pattern of the glutamatergic drive for stage III retinal waves;what is the nature of the neurotransmitter interactions that shape this on-off asynchrony and what is the under-line synaptic mechanism dictate this on-off refinement.To answer those detailed and critical questions,we use calcium imaging and dual patch of ON and OFF starburst amacrine cells in whole mount retina and diverse double patch between on SAC and three different types of retinal ganglion cells(ON、OFF and ON-OFF)in transgenic mice to characterize the spatial and temporal patterns of ON、OFF cells during stage III waves;take the advantages of opto genetic technique which giving scientists an unprecedented ability to modulate neural circuits,combined with pharmacology method to reveal the synaptic mechanism of ON、OFF segregation in retinal glutamatergic spontaneous activities.Here we found that not only ganglion cells but also upstream cells(starburst amacrine cells)were dictated by the calcium dependent asynchrony which was caused by the dual-phase excitatory inputs from bipolar cells rather than direct postsynaptic inhibition of retinal ganglion cells or feedforward inhibition to starburst amacrine cells.Within a wave,cells undergo excitatory inputs in a fixed order:ON before OFF.This process was absent from stage II cholinergic retinal waves,which precede glutamate-dependent activity,in spite of the anatomical structural segregation of ON、OFF layer.The time difference between ON and OFF system was mediated by glycine,one of the major inhibitory transmitters in retina.Optogenetic modulation indicated a direct glycinergic cross-over inhibitory pathway from ON bipolar cell to OFF bipolar cell which drive the glutamate inputs to next level of neurons.A delayed rebound upon disinhibition of OFF bipolar cells at the end of ON bipolar cells effectively time-locking the ’ ON before OFF’ sequence.To conclude,spontaneous periodic activates in retina before vision starts provide a developmental sequence of distinct activity-encoded cues necessary to segregate ON、OFF system.Glycinergic cross-over inhibition between ON and OFF pathway mediate this ’ON leading OFF’ asynchrony in stage III glutamatergic waves.