Unveiling Glial Heterogeneity in the Inner Retina: Non-Visual Photic Spectrum Responses and Calcium Dynamics in Müller Glial Cells vs. Neurons

Traditional calcium (Ca2+) imaging analysis often overlooks the non-stationary temporal complexity inherent in retinal circuits, reducing dynamic signaling traces to static summary descriptors. To resolve these dynamic signatures, we implemented a reproducible analytical framework combining Continuous Wavelet Transform (CWT) time-frequency decomposition with Sinc baseline filtering and Ward’s agglomerative hierarchical clustering, applied directly to standardized high-dimensional feature spaces, reserving Uniform Manifold Approximation and Projection (UMAP) strictly for low-dimensional visualization. This pipeline was deployed across three independent experimental models under blue light stimulation: mixed primary embryonic chicken retinal neurons, primary chicken Müller glial cells (MGCs), and the human MIO-M1 Müller cell line. Functional clustering resolved retinal neurons into three distinct kinetic subpopulations (k = 3), characterized by a kinetically stable core maintaining mid-frequency oscillations (Cluster 0), a cohort undergoing sharp post-stimulus period contraction with late amplitude surges (Cluster 1), and a low-frequency cohort with persistent period elongation (Cluster 2). Chicken MGCs partitioned into two operational clusters (k = 2), contrasting a period-stable, amplitude-decaying subpopulation (Cluster 0) with a dynamic subpopulation exhibiting light-evoked kinetic acceleration (Cluster 1). Similarly, human MIO-M1 glia separated into two major kinetic clusters (k = 2), distinguishing a low-amplitude, longer-period cohort from a high-amplitude, sustained subpopulation operating within an 8–80 s dynamic period band. Restricting phase-coupling and circular statistical analyses strictly to simultaneous Field-of-View (FOV) recordings revealed distinct stimulus-entrained network behaviors: primary avian MGCs maintain a decentralized, weakly coupled regime (“mosaic of autonomy”) that processes light across independent temporal windows, whereas human MIO-M1 glia and embryonic neuronal assemblies exhibit significant stimulus-evoked phase realignment and relative response latency sequencing. These findings demonstrate that inner retinal functional identity is fundamentally rhythmic and cluster-dependent, establishing a quantitative benchmark for decoding multi-scale Ca2+ signaling in healthy retinal preparations.