We sought to investigate the types of bipolar cells and amacrine cells that are connected to ON DS cells. We injected the retrograde tracer AAVs or herpes viruses expressing TVA receptor and rabies-G protein into the medial terminal nucleus. As a result, ON DS cells expressed the TVA receptor and the rabies-G protein (Figure 2A). We then injected GCaMP3-expressing, EnvA-coated G-deleted rabies viruses into the eye. Since EnvA specifically binds to TVA, rabies virus infected only ON DS cells. Due to the presence see more of rabies-G expressed from AAV/herpes viruses in ON DS cells, the G-deleted
rabies viruses were complemented with rabies-G and crossed one synapse retrogradely to mark the monosynaptically connected cells (Wickersham et al., 2007b and Yonehara et al., 2011) (Figure 2A). The transsynaptic spread of rabies virus has been observed to be specific to synaptically connected neurons and not to adjoining neurons that are either not connected or gap-junction connected (Ugolini, 2011). Injection of EnvA-coated rabies virus into the eye without supplying the TVA receptor did not result buy INCB024360 in any labeling of retinal cells in 15 independent eye injections (Figure S2). Immunohistochemistry, together with three-dimensional (3D) confocal image reconstruction, showed that bipolar and amacrine cells were labeled together with ON DS cells. Most of the labeled amacrine cells were ChAT-positive
starburst amacrine cells located Thymidine kinase in the ganglion cell layer (Figure 2B), confirming previous results that starburst cells are presynaptic to ON DS cells (Yonehara et al., 2011). Based on the confocal image stacks, we identified a morphological type of bipolar cell as presynaptic to ON DS cells. The axon terminals of all (21/21)
bipolar cells were positioned slightly above the proximal ChAT-labeled layer and were therefore categorized as type-5 bipolar cells (Ghosh et al., 2004) (Figures 2C, 2D, and S2). We did not find any labeled type-6 or type-7 bipolar cells, even though the axon terminals of these bipolar cells are physically close to the dendrites of ON DS cells at the proximal ChAT-labeled layer (Ghosh et al., 2004) and have therefore had opportunities to contact ON DS cell dendrites. We used the same combination of rabies and AAV/herpes viruses that we had used for circuit labeling to record calcium responses via GCaMP3 from the axon terminals of labeled bipolar cells. We stimulated retinas with a positive contrast spot moving in eight different directions. We first imaged the cell body of an ON DS cell and made sure that it was direction selective. Next, we imaged calcium responses in the axon terminal endings of connected bipolar cells. Each axon terminal button of a connected bipolar cell was visible under the two-photon microscope (Figure 3A). Based on their appearance (large buttons), we could differentiate them from ON DS dendrites and starburst processes.