"An anesthetized adult mouse was perfused transcardially with a fixative solution containing glutaraldehyde, paraformaldehyde, and CaCl2 in cacodylate buffer. The brain was removed and maintained overnight at 4°C in the same fixative solution."
I don't know much about neurobiology. But i know that in two other fields of cell biology - cell motility and vesicle trafficking - something that became clear in the last decade is that the way you fix samples for electron microscopy has a huge impact on what you see. Cells are made of highly dynamic structures that exist in fine-tuned environments; soaking them in formaldehyde and some random buffer salts is pretty much guaranteed to disrupt those environments, and let the structures drift away from the states they have in live cells over the time it takes to fix them, even if it's only fractions of a second.
The gold standard for fixation is now extremely fast freezing, achieved by plunging a sample into liquid nitrogen under very high pressure, so that the water solidifies as a glass rather than growing crystals, which would disrupt structures inside the cell:
In my old field, cell motility, when someone finally tried this, they found that a thing called the lamellipodium, which has been an object of intense study for decades, had a rather different structure to what everyone had thought:
As a result, it turns out that the prevailing model of how this thing worked was complete fantasy. Since numerous successful professors have built their careers on that model, this discovery has not yet really been taken on board by the field, but it's inevitable that it will.
Which is a very roundabout way of saying that when the authors of this brain paper write:
"For example, by tracing the trajectories of all excitatory axons and noting their juxtapositions, both synaptic and non-synaptic, with every dendritic spine we refute the idea that physical proximity is sufficient to predict synaptic connectivity (the so-called Peters’ rule)."
I am skeptical, because they are looking at the positions of the spines and synapses in their formaldehyde-soaked sample, not live tissue, or tissue prepared in such a way as to preserve the structure of live tissue.
