Inner limiting Membrane Peel ExtendsIn vivoCalcium Imaging of Retinal Ganglion Cell Activity Beyond the Fovea in Non-Human Primate
Authors: Baez Hector C. , LaPorta Jennifer M. , Walker Amber D. , Fischer William S. , Hollar Rachel , Patterson Sara , DiLoreto David A. , Gullapalli Vamsi , McGregor Juliette E. 
Published: June 2024

AbstractHigh resolution retinal imaging paired with intravitreal injection of a viral vector coding for the calcium indicator GCaMP has enabled visualization of activity dependent calcium changes in retinal ganglion cells (RGCs) at single cell resolution in the living eye. The inner limiting membrane (ILM) is a barrier for viral vectors, restricting transduction to a ring of RGCs serving the fovea in both humans and non-human primates (NHP). We evaluate peeling the ILM prior to intravitreal injection as a strategy to expand calcium imaging beyond the fovea in the NHP eye in vivo. Five Macaca fascicularis eyes (age 3-10y; n=3 individuals; 2M, 1F) underwent vitrectomy and 5 to 6-disc diameter ILM peel centered on the fovea prior to intravitreal delivery of 7m8:SNCG:GCaMP8s. Calcium responses from RGCs were recorded using a fluorescence adaptive optics scanning laser ophthalmoscope. In all eyes GCaMP was expressed throughout the peeled area, representing a mean 8-fold enlargement in area of expression relative to a control eye. Calcium recordings were obtained up to 11 degrees from the foveal center. RGC responses were comparable to the fellow control eye and showed no significant decrease over the 6 months post ILM peel, suggesting that RGC function was not compromised by the surgical procedure. In addition, we demonstrate that activity can be recorded directly from the retinal nerve fiber layer. This approach will be valuable for a range of applications in visual neuroscience including pre-clinical evaluation of retinal function, detecting vision loss, and assessing the impact of therapeutic interventions.Significance StatementThis research presents a groundbreaking advancement in visual neuroscience through the development of a novel technique involving the peeling of the inner limiting membrane (ILM) in conjunction with intravitreal injection to expand functional recording capabilities in the living primate eye. By utilizing high-resolution retinal imaging coupled with a viral vector-mediated expression of the calcium indicator GCaMP, the study achieves unprecedented visualization and assessment of retinal ganglion cell (RGC) activity at single-cell resolution. Importantly, the technique enables recording from regions of the retina previously inaccessible, significantly broadening the area for calcium imaging beyond the fovea. The results demonstrate stable RGC function post-procedure, suggesting minimal impact on retinal physiology. This innovative approach holds significant promise for diverse applications in visual neuroscience, including pre-clinical evaluation of retinal function, detection of vision loss, and evaluation of therapeutic interventions. Overall, the study represents a major step forward in understanding and potentially treating retinal degenerative disorders, offering new avenues for research and development in vision restoration.