Journal: Science (New York, N.Y.)
Article Title: Astrocyte Ca2+-evoked ATP release regulates myelinated axon excitability and conduction speed *
Figure Lengend Snippet: ATP release from astrocytes may target adenosine receptors on myelinated axons of layer V pyramidal neurons. ( A ) Using luciferin-luciferase to detect ATP puffed into extracellular solution. ( B ) Response to 2-photon excitation uncaging Ca 2+ in astrocytes evoked a luciferin-luciferase signal, unless the excitation failed to raise astrocyte [Ca 2+ ]i (No Ca 2+ u.; see Fig. S2 ). ( C ) Quantification of experiments in B in 7 cells. ( D-H ) A 2a Rs are present in the AIS ( D ) where they overlap with voltage-gated Na + channel (Na v ) expression ( E , mean of 14 Na v and 25 A 2a R profiles) and at the node of Ranvier ( F ) where they overlap with Na v and are flanked by Caspr labeling ( G , mean of 48 Caspr, 48 A 2a R and 24 Na v profiles). ( H ) Percentage of 25 AISs and 134 nodes that express A 2a Rs. ( I-M ) HCN2 channel subunits are present in the AIS ( I ) where they overlap with voltage-gated Na + channel (Na v ) expression ( J , mean of 19 Na V and 16 HCN2 profiles) and at the node of Ranvier ( K ) where they overlap with Na v and are flanked by Caspr labeling ( L , mean of 53 Caspr and HCN2 profiles). ( M ) Percentage of 49 AISs and 98 nodes that express HCN2. Panels A-E and I-J are on rats; F-G and K-L are on mouse; H and M combine rat AIS and mouse node data.
Article Snippet: The slices were permeabilized with 0.2% Triton X-100 (Sigma) in a blocking solution (10% goat serum in 0.01 M PBS) for 1 h. The slices were then incubated overnight at 4 °C with the following primary antibodies, as required: rabbit anti-A2a R (Abcam, ab3461, 1:100), mouse anti-A2a R (Millipore, 05-717 which has been validated as giving no labelling in A2a R KO tissue , 1:200), rabbit anti-A1 R (Abcam, ab82477, 1:100), rabbit anti-A2bR (Cohesion, CPA3755, 1:100), mouse anti-Ankyrin G (Neuromab, N106/36, 1:500), mouse anti-panNav (Sigma, K58/35, 1:100), mouse anti-Caspr (Neuromab, K65/35, 1:100), chicken anti-GFP (Millipore, AB16901, 1:1000), rabbit anti-GFAP (Millipore, AB5804, 1:500), rabbit anti-HCN1 (Alomone, APC 056, 1:100) and rabbit anti-HCN2 (Alomone, APC 030, 1:100).
Techniques: Luciferase, Expressing, Labeling