Inner limiting membrane (ILM) peel extends in vivo calcium imaging of retinal ganglion cell (RGC) activity beyond the fovea in non-human primate

Abstract

Purpose : Fluorescence ophthalmoscopy paired with intravitreal injection of viral vectors coding for the calcium indicator GCaMP made it possible to record RGC activity in the living primate eye. Functional recording was previously limited to transduced RGCs surrounding the foveal pit. We test the hypothesis that surgically peeling the ILM will extend the area of transduced RGC thereby allowing in vivo functional recording well beyond the fovea.

Methods : The right eye of a male Macaca fascicularis underwent vitrectomy with induction of a posterior vitreous detachment followed by staining of the ILM with brilliant blue G (Sigma-Aldrich) and 21° by 16° diameter ILM peel centered on the fovea. 31 days later 7e¹⁴ vg of AAV2.7m8-SNCG-GCaMP8s was injected intravitreally. The fellow eye and an additional control eye were injected without ILM peel. The spatial extent of GCaMP8s expression was assessed using visible fluorescence cSLO. Functional responses were elicited with a 660nm 8uW/mm² flickering stimulus presented through a Maxwellian view system while GCaMP fluorescence (excitation 488nm, 90uW/mm², emission 535/20) was recorded using an adaptive optics scanning laser ophthalmoscope.

Results : In the ILM peeled eye GCaMP fluorescence was observed throughout the area of the ILM peel; a 13mm² region representing a > 4.5 fold spatial increase of GCaMP expression relative to the control eyes. 16 weeks post ILM peel, cellular scale RGC responses were recorded at the four meridians of the edge of the ILM peel region at 9.8° superior, 11.7° inferior, 7.3° nasal, and 8.1° temporal. We recorded 94 (40 ON, 54 OFF) RGCs responding 2σ above noise in the superior region. Responses at these eccentricities were not observed in the control eye. At 3.5° superior the averaged responses across a 2.5^o field of view were comparable between control and ILM peeled eyes (z-scores 0.34 and 0.48 respectively). Beyond the peeled region, in the nasal hemifield we recorded activity from the retinal nerve fiber layer.

Conclusions : ILM peel substantially extends the region of primate retina accessible for in vivo GCaMP recording beyond the foveal ring of RGCs. This strategy presents new opportunities for both physiological study of the retina and pre-clinical testing disease models. Furthermore, in vivo calcium imaging could be used to assess the long-term impact of ILM peel on RGC function.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.