Nanostructure of the mammalian cone photoreceptor synapse in adult, developing and in vitro 3D retinas

Cone photoreceptor transplantation followed by the regeneration of mature functional connections with bipolar and horizontal cells remains an unsolved problem. We have developed a super-resolution microscopy approach that, in association with antibody labels, can reveal the protein organization of the cone synaptic terminal, synaptic cleft, and postsynaptic contacts at the scale of 10s of nanometers in 3D--at least 5-7 times better resolution than provided by standard light microscopy. We are currently using this approach to characterize the nanostructure of the ground squirrel cone photoreceptor to Off bipolar cell synapses in NEI-funded research. Ground squirrel retinas are cone-dominant (90% cones/10% rods). Our objective in this proposal is to extend the super-resolution studies to the ground squirrel cone to On bipolar cell synapses in the adult and On and Off bipolar cell synapses during development, mouse cone synapses with Off and On bipolar cells in the adult and during development, and to cone synapses in 3D retinas derived from mouse and ground squirrel intrinsic pluripotent stem cells (iPSCs). By identifying the proteins that participate in establishing and maintaining the connections at the cone synapse in the adult and during development, we will establish a rubric for evaluating cone photoreceptors in 3D retinal organoids with respect to the maturity and/or deficits in their synaptic connections and potentially hone in on ways to improve those connections (eg, by transfecting the genes for essential synaptic proteins). In this collaboration, members of the DeVries lab, including the PI, will train members of the Koike lab (and other participant labs in the Center for Systems Visual Science, CSVS, at Ritsumeikan University) in Chicago in the technology of obtaining 3D super-resolution STED images from the outer retina. Imaging will focus on the relationships between On bipolar cells and cones (a specialty of the Koike lab). In Japan, members of the Koike lab and CSVS will train members of the DeVries lab, including the PI, in techniques for growing 3D retinal organoids from iPSCs. Together the DeVries and Koike labs will work on producing 3D retinal organoids from the cone-dominant ground squirrel in Japan. Organoid technology will also be transferred to the DeVries lab in Chicago. This is an ideal cross-pollination of approaches where an expert in super-resolution microscopy and cone to Off bipolar cell synapses exchanges knowledge and technical know-how with experts on On bipolar cell transduction, retinal development, and in growing 3D retinoids from iPSCs. The career of the candidate will be enhanced through the incorporation of 3D retinal organoids into the physiological and ultrastructural studies in the lab. The career of the collaborator will be enhanced through learning the methodologies of 3D retinal nanoscopy. Both sets of investigators will benefit from joint studies on the structure of cone synapses. The DeVries and Koike labs have collaborated for six years beginning with the submission of an Audacious Goals Initiative grant. We have continued to work on aspects of the project since that time. However, the two labs have only rarely worked together in person.