Abstract
Most of the excitatory synapses on principal neurons of the forebrain are located on specialized structures called dendritic spines. Their morphology, comprising a spine head connected to the dendritic branch via a thin neck, provides biochemical and electrical compartmentalization during signal transmission. Spine shape is defined and tightly controlled by the organization of the actin cytoskeleton. Alterations in synaptic strength correlate with changes in the morphological appearance of the spine head and neck. Therefore, it is important to get a better understanding of the nanoscale organization of the actin cytoskeleton in dendritic spines. A periodic organization of the actin/spectrin lattice was recently discovered in axons and a small fraction of dendrites using super-resolution microscopy. Here we use a small probe phalloidin-Atto647N, to label F-actin in mature hippocampal primary neurons and in living hippocampal slices. STED nanoscopy reveals that in contrast to β-II spectrin antibody labelling, phalloidin-Atto647N stains periodic actin structures in all dendrites and the neck of nearly all dendritic spines, including filopodia-like spines. These findings extend the current view on F-actin organization in dendritic spines and may provide new avenues for understanding the structural changes in the spine neck during induction of synaptic plasticity, active organelle transport or tethering.
Highlights
Most of the excitatory synapses on principal neurons of the forebrain are located on specialized structures called dendritic spines
We established a protocol that allows for triple fluorescence labelling: dendrites are visualized by an antibody directed against MAP2 coupled to AlexaFluor[488] or by overexpressed GFP as a cell fill, spectrin lattice or synapses by antibodies against β-II spectrin or the synaptic marker bassoon labelled with Aberrior Star 580, and actin filaments by phalloidin conjugated to Atto647N (A647N), a far-red high quantum yield fluorophore for STED imaging[27]
In a series of tests we found that following immunostaining of freshly fixed adult hippocampal primary neurons an overnight incubation with 0.165 nM phalloidin-A647N results in a strong A647N fluorescence signal in dendrites and dendritic spines (Supplementary Fig. S1)
Summary
Most of the excitatory synapses on principal neurons of the forebrain are located on specialized structures called dendritic spines Their morphology, comprising a spine head connected to the dendritic branch via a thin neck, provides biochemical and electrical compartmentalization during signal transmission. STED nanoscopy reveals that in contrast to β-II spectrin antibody labelling, phalloidin-Atto647N stains periodic actin structures in all dendrites and the neck of most dendritic spines, including filopodia-like spines. These findings extend the current view on F-actin organization in dendritic spines and may provide new avenues for understanding the structural changes in the spine neck during induction of synaptic plasticity, active organelle transport or tethering. In improving the accuracy of localization for proteins, have been made with the development of novel small probes and fluorescent labels[16,19,20,21]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
