摘要：  PURPOSE: Convergent evidence suggest that amacrine cells (ACs) might provide excitatory inputs to retinal ganglion cells (GCs), and such inputs might be non-glutamatergic. Here, we sought to optogenetically characterize these non-glutamatergic inputs in the VGAT-ChR2-EYFP mouse, in which channelrhodopsin-2 (ChR2) are expressed in inhibitory CNS neurons.
METHODS: Double-staining immunohistochemical assays were used to characterize the retinal expression profile of the transgene with various specific markers for retinal neurons. In a whole-mount preparation, when ACs were optogenetically activated, whole-cell recordings were applied on GCs and postsynaptic responses were examined. Excitatory and inhibitory inputs were separated by holding the membrane potential at -70 mV and 0 mV, respectively.
RESULTS: EYFP signals were detected in the vast majority of horizontal cells and ACs, but not in photoreceptors, bipolar cells and GCs, suggesting a confined ChR2 expression in retinal inhibitory interneurons. When glutamatergic transmission was blocked pharmacologically and ACs were stimulated optogenetically, an EPSC (named as non-glutamatergic light-evoked EPSC, or nG-L-EPSC) was elicited in most GCs (percentage: ON-type GCs, 82%; OFF-type GCs, 79%; ON-OFF-type GCs, 92%). Furthermore, in a population (~87%) of GCs, their nG-L-EPSCs survived superimposed cholinergic blockade (the residual responses further named as nGnC-L-EPSCs). The nGnC-L-EPSCs remained in the presence of inhibitory transmitter antagonists and MFA, a gap junction blocker, but was completely abolished by Co2+, indicating that it is not mediated by inhibitory signals, or by electrical coupling with neighboring ChR2-expressing ACs, but is indeed a postsynaptic response due to optogenetic activation of ACs. Finally, quantitative analysis showed that all three GC subclasses are driven by such nGnC excitatory inputs, but likely with moderate difference in input strength.
CONCLUSIONS: Most mouse GCs possess non-glutamatergic excitatory synaptic inputs, which are mediated by chemical synapses via acetylcholine and other unknown excitatory transmitters. ACs might play a more intricate role in inner retinal signal processing than ever thought.

关键词:  , retina, optogenetics, channelrhodopsin-2, amacrine cells, retinal ganglion cells

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