Figure 1. 3D-STORM imaging of CB₁ receptors on hippocampal GABAergic axon terminals.

(a) Immunofluorescence labeling of CB₁ receptors in the CA1 stratum pyramidale of CB₁^+/+ mice showed basket-like arrangement of CB₁-positive axon terminals encircling a CB₁-immunonegative pyramidal cell somata. (b) These axon terminals could also be visualized by immunofluorescence staining for the neuropeptide CCK, a neurochemical marker of CB₁-expressing interneurons. (c) STORM imaging of CB₁ receptors revealed identical basket-like orientation of the same perisomatic GABAergic axon terminals. (d,e) Magnifying the epifluorescence images failed to delineate the morphological structure of the respective profiles located in the boxed region or the precise nanoscale position of CB₁ receptors with the axon terminals. (f) In contrast, the improved spatial resolution of 3D-STORM microscopy made it possible to reliably discern individual CB₁-positive boutons located adjacently to one another, as well as to reveal localization points representing the nanoscale position of CB₁ receptors. (g,h) To validate the specificity of our approach, hippocampal sections from littermate CB₁^−/− mice were also processed and imaged under the same conditions. In these sections, we found an almost complete absence of STORM localization points representing CB₁ within CCK-immunolabeled boutons. (i) Histogram of CB₁ NLP in individual CCK-positive varicosities in the CA1 stratum pyramidale of sections derived from littermate CB₁^+/+ (n = 3 animals; n = 208 axon terminals) or CB₁^−/− (n = 2 animals; n = 200 axon terminals) mice. Note that the drastically reduced CB₁ NLP value in CB₁^−/− samples validates the specificity of the antibody, the staining process and the STORM imaging protocol.