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We developed a novel calcium (Ca 2ϩ ) channel agonist that is selective for N-and P/Q-type Ca 2ϩ channels, which are the Ca 2ϩ channels that regulate transmitter release at most synapses. We have shown that this new molecule (GV-58) slows the deactivation of channels, resulting in a large increase in presynaptic Ca 2ϩ entry during activity. GV-58 was developed as a modification of (R)-roscovitine, which was previously shown to be a Ca 2ϩ channel agonist, in addition to its known cyclin-dependent kinase activity. In comparison with the parent molecule, (R)-roscovitine, GV-58 has a ϳ20-fold less potent cyclin-dependent kinase antagonist effect, a ϳ3-to 4-fold more potent Ca 2ϩ channel agonist effect, and ϳ4-fold higher efficacy as a Ca 2ϩ channel agonist. We have further evaluated GV-58 in a passive transfer mouse model of Lambert-Eaton myasthenic syndrome and have shown that weakened Lambert-Eaton myasthenic syndromemodel neuromuscular synapses are significantly strengthened following exposure to GV-58. This new Ca 2ϩ channel agonist has potential as a lead compound in the development of new therapeutic approaches to a variety of disorders that result in neuromuscular weakness.

Expansion microscopy1,2 is an increasingly widespread technology for nanoimaging, because its precise physical magnification of biological specimens enables ordinary microscopes to achieve nanoscale effective resolutions. Fluorescent labels such as antibodies can be applied either before or after expansion, with the latter offering the potential for better access to proteins within densely packed environments3–7. We here assess this possibility of epitope decrowding through physical expansion of proteins away from each other, using a 20x expansion protocol that we call expansion revealing (ExR), by labeling, within intact brain circuits, the same set of synaptic proteins both pre- and post-expansion. This comparison shows that post-expansion labeling introduces minimal spatial error and off-target staining relative to pre-expansion staining, while revealing the presence of proteins that are invisible when stained pre-expansion. Using ExR, we show in intact brain tissue the alignment of presynaptic calcium channels with postsynaptic machinery in nanocolumns, which may facilitate precision synaptic transmission, as well as the existence of periodic amyloid-containing nanoclusters containing ion channel proteins in Alzheimer’s model mice, which may help generate novel hypotheses for Alzheimer’s pathology and neural excitability. Thus, the decrowding power of ExR is able to reveal novel nanostructures within intact brain circuitry, and may find broad use in biology and medicine for unmasking nanostructures of importance in normal functions and disease.

No abstract

We have investigated the impact of transmitter release site (active zone; AZ) structure on synaptic function by physically rearranging the individual AZ elements in a previously published frog neuromuscular junction (NMJ) AZ model into the organization observed in a mouse NMJ AZ. We have used this strategy, purposefully without changing the properties of AZ elements between frog and mouse models (even though there are undoubtedly differences between frog and mouse AZ elements in vivo), to directly test how structure influences function at the level of an AZ. Despite a similarly ordered ion channel array substructure within both frog and mouse AZs, frog AZs are much longer and position docked vesicles in a different location relative to AZ ion channels. Physiologically, frog AZs have a lower probability of transmitter release compared with mouse AZs, and frog NMJs facilitate strongly during short stimulus trains in contrast with mouse NMJs that depress slightly. Using our computer modeling approach, we found that a simple rearrangement of the AZ building blocks of the frog model into a mouse AZ organization could recapitulate the physiological differences between these two synapses. These results highlight the importance of simple AZ protein organization to synaptic function. NEW & NOTEWORTHY A simple rearrangement of the basic building blocks in the frog neuromuscular junction model into a mouse transmitter release site configuration predicted the major physiological differences between these two synapses, suggesting that transmitter release site structure and organization is a strong predictor of function.