DECODING RETINAL CIRCUIT FUNCTION WITH TWO-PHOTON CALCIUM IMAGING ACROSS HEALTH AND DISEASE

Retinal degenerative diseases are a leading cause of irreversible visual impairment worldwide. Age-related macular degeneration (AMD), the most common cause of vision loss in older adults, highlights the profound impact of retinal degeneration on quality of life. Although structural changes during degeneration are well characterized, how these alterations affect retinal function remains poorly understood. A major challenge in addressing this gap is the lack of robust experimental and analytical platforms to monitor neuronal activity in degenerating retinas. Here, we establish and validate a functional imaging pipeline to investigate retinal circuit dynamics using two-photon calcium imaging. We employ Thy1-GCaMP6s transgenic mice to monitor neuronal activity in the ganglion cell layer. Retinal explants are prepared under infrared light and maintained in oxygenated medium to preserve viability and light responsiveness. Using a custom-built two-photon microscope, we perform stable, long-duration recordings of calcium activity. Visual stimulation is delivered with a 380 nm UV LED using chirp stimuli, while calcium signals are acquired at 5 Hz over a 300 × 300 µm² field of view. In healthy retinas, this approach reliably captures robust, light-evoked responses across populations of ganglion cells. An automated analysis pipeline—segmentation, signal extraction, filtering, and clustering—identifies distinct functional response groups, demonstrating that our platform can resolve the functional diversity of retinal output pathways. Applying this framework to a chemically induced degeneration model reproducing key features of AMD will allow us to define how disease progression alters retinal activity and functional organization, providing mechanistic insight to guide strategies for preserving visual function.