Journal: bioRxiv
Article Title: Microglia monitor and protect neuronal function via specialized somatic purinergic junctions
doi: 10.1101/606079
Figure Lengend Snippet: a) Schematic illustration and result of CX3CR1+/GFP mouse in utero electroporation with pCAG-IRES-tdTomato. Upper right shows the outspread of tdTomato-positive neurons in red. Lower panels show the non-overlapping staining of electroporated neurons (red) and microglia (green). Cell nuclei are visible in blue. b) CLSM images showing three microglial markers. Upper row shows complete overlap of immunolabelings against CX3CR1 (yellow), Iba1 (magenta) and P2Y12R (cyan) in CX3CR1+/GFP mice. Lower row shows the presence of overlapping CX3CR1 and Iba1 signals with the complete absence of P2Y12R labeling in P2Y12R-/- animals. c) Electron microscopic images showing microglial processes (m) establishing direct contact with neuronal cell body (left, n), dendritic shaft (d) and presynaptic bouton (b) in mice. Microglia are visualized by immunoperoxidase reaction against Iba1. Pseudocoloring shows microglia in yellow, neuronal cell body and dendritic processes in magenta, presynaptic bouton in cyan and nucleus (nucl.) in purple. Arrow points at a dendritic spine receiving asymmetric synaptic contact. d) Microglial processes contacting cortical inhibitory and excitatory synapses. Presynaptic terminals are visible by stainings against vGAT (red) and vGluT1 (cyan), while postsynaptic side is characterized by stainings against gephyrin (blue) and Homer1 (magenta). White arrowheads point at colocalization of pre- and postsynaptic markers. Microglial contacts in white circles are enlarged in the panels right, with the previous and following Z-planes from the stack (Z-step = 250 nm). Empty arrowheads point at the presynaptic marker, white arrows point at the postsynaptic side. e) Heatmap shows Kv2.1 clusters of a human cortical neuron. f) CLSM image shows that a P2Y12R-labeled microglial process contacts a human cortical neuron exactly at the spot where a strong Kv2.1 cluster is located. g) Heatmap shows that Kv2.1-clustering of mouse cortical neurons is not affected by microglia depletion. i) P2Y12R clustering differs depending on location. Microglial processes were classified depending on neuronal contacts established: contact processes are pale green, non-contact processes are pale magenta on bottom panel. j) Analysis of P2Y12R cluster and LP density on different microglial processes contacting distinct cell populations. Microglial contact processes on CamK2a (red) and PVA-positive cells (orange) possess significantly higher cluster density, than non-contact segments, while there is a similar trend observable in the case of GAD65-positive cells (yellow, for detailed results see .). Contact processes had significantly higer number of P2Y12R LP-s in all populations. Anti-PV and vGluT3 labelings were used to confirm cell specificity. k) Superresolution imaging of microglial Iba1 shows no clustering at somatic junctions. CLSM images show neuronal Kv2.1 (green) and a contacting microglial process made visible by Iba1-labeling (red). Iba1 (yellow) STORM-signal is overlaid and shown individually in the middle panel. Lower panel shows a homogenous distribution of Iba1 LPs using DBScan analysis. Scale bars: 500 µm on top right panel of a, 50 µm on bottom left panel of a, 25 µm on bottom right panel of a, 20 µm on b, 1 µm on c, i and k, 2 µm on d and f, 10 µm on j.
Article Snippet: STORM imaging was performed for P2Y12R (stimulated by a 647 nm laser) by using a Nikon N-STORM C2+ superresolution system that combines ‘Stochastic Optical Reconstruction Microscopy’ technology and Nikon’s Eclipse Ti research inverted microscope to reach a lateral resolution of 20 nm and axial resolution of 50 nm ( , ).
Techniques: In Utero, Electroporation, Staining, Labeling, Marker, Imaging
