Spine neck plasticity regulates compartmentalization of synapses

Dendritic spines have been proposed to transform synaptic signals through chemical and electrical compartmentalization. However, the quantitative contribution of spine morphology to synapse compartmentalization and its dynamic regulation are still poorly understood. We used time-lapse super-resolution stimulated emission depletion (STED) imaging in combination with fluorescence recovery after photobleaching (FRAP) measurements, two-photon glutamate uncaging, electrophysiology and simulations to investigate the dynamic link between nanoscale anatomy and compartmentalization in live spines of CA1 neurons in mouse brain slices. We report a diversity of spine morphologies that argues against common categorization schemes and establish a close link between compartmentalization and spine morphology, wherein spine neck width is the most critical morphological parameter. We demonstrate that spine necks are plastic structures that become wider and shorter after long-term potentiation. These morphological changes are predicted to lead to a substantial drop in spine head excitatory postsynaptic potential (EPSP) while preserving overall biochemical compartmentalization

A comprehensive framework for fluorescence cross-correlation spectroscopy

Dual-colour fluorescence cross-correlation spectroscopy is a powerful method of studying binding between labelled biomolecules in vitro as well as in vivo. However, numerous artefacts and experimental complexities complicate quantitative measurements. Here, we show that a combination of dual-colour fluorescence correlation spectroscopy (FCS) with dual-focus FCS avoids artefacts due to chromatic aberrations or saturation and circumvents the calibration of the detection volumes. In addition, we present a comprehensive mathematical framework that allows us to accurately analyse correlation curves even in the presence of spectral cross-talk, incomplete or stochastic labelling, multiple binding sites, a fluorescent background and depletion due to photobleaching. We demonstrate the merits of this approach using dual-colour dual-focus scanning FCS, which allows binding measurements on membranes not affected by membrane movements