Rapid 3D enhanced resolution microscopy reveals the dynamics of cortical dendritic spinules

Abstract

Dendritic spinules are thin, membranous protrusions formed by neuronal dendritic spines that are not adequately resolved by diffraction-limited light microscopy. Hence, our understanding of spinules is inferred predominantly from fixed-tissue electron microscopy (EM).Super-resolution modalities have enabled live-cell nanoscopic imaging, but their utility for fast, time-lapse, volumetric imaging has been restricted. Herein, we utilized rapid structured illumination microscopy (SIM) and 'enhanced resolution' confocal microscopy to study spatiotemporal spinule dynamics in live cultured cortical pyramidal neurons. Spinules on mushroom spines typically recurred at the same topographical locations and most were short-lived, originating near simple post-synaptic densities (PSDs), while a subset was long-lived and elongated, emerging from complex PSDs. Ca 2+ puncta within spinules synchronized with spine head transients and Ca 2+ depletion drastically decreased spinule number. Moreover, we uncovered evidence of differential Ca 2+ -mediated regulation of short-lived and long-lived spinules. Thus, we identified unique spinule classes divergent in lifespan, dynamics, morphology, relationship to the