ASC, (aldehyde-stabilized cryopreservation) is a brain-banking technology that can preserve all of the synaptic connections in a whole mammalian brain for longer than a century. ASC uses glutaraldehyde and ethylene glycol to stabilize and preserve the brain's structure.

No, the process of chemical fixation with glutaraldehyde irreversibly crosslinks proteins and prevents meaningful metabolic activity from occurring. It is not possible with current technology to reverse aldehyde fixation, and it would be extremely difficult to develop such a technology.

ASC is only concerned with preserving the brain's detailed nanoscale structure, not it's capacity for metabolism.

I see connectomics as a bottom-up approach to understanding the brain which treats the brain as a network that performs computations. A "complete connectome" would be whatever level of detail you need to fully describe the computations performed by the brain. Connectomes can also exist at multiple scales; for example a large-scale incomplete connectome might provide general connectivity information referenced between major brain regions, while a much more fine-grained incomplete connectome might be a connectivity matrix for all 302 neurons in the C. elegans.

Dr. Hayworth, President of the Brain Preservation Foundation, contends that the sort of information you can get from detailed electron micrographs of preserved synapses, combined with "general" knowledge of how neurons work, is likely enough to serve as a complete connectome. There's QUITE a lot of information contained in electron micrographs, much more than might first be apparent. For example, in an electron micrograph series you might be able to tell what type of neuron a particular neuron is by looking at its 3D shape, then you would be able to infer from general knowledge about that neuron type that all of its synapses are excitatory. In this case Dr. Hayworth’s connectome, generated form these electron micrographs, contains information about the types of molecules present in that neuron’s synapses because that information can be inferred from the detailed structure of the neuron along with deep knowledge of that general type of neuron won from other experiments.

See the Brain Preservation Foundation's "What is a connectome" page for further discussion about the definition of "connectome".

ASC preserves much more than just the information available in electron micrographs (which Dr. Hayworth claims to be sufficient), because almost all proteins such as ion channels must still be present in their original locations in brains preserved with ASC, even though they are not visible under an electron microscope.

If it is true that the level of detail preserved with ASC can serve as a complete connectome, then that means that all the brain's memories as well as all the computations that the brain was able to originally perform must still be accessible in the ASC preserved brain. "Accessible" in this case means that it is possible to build an accurate model of the original brain's computations by using information gathered from the brain after it is preserved with ASC.

In general, whenever I talk about "the connectome" without further qualification, I mean the information you can get using detailed electron microscopy.

The key here is "traceability": can you easily tell where every neural process is going, and do they all terminate in reasonable structures, or is there morphological damage? If you look at Mikula's paper, he does a very formal evaluation of his technique by comparing the judgments of expert human tracers; if they all agree on which synapses trace back to which neurons, then they must have been clearly preserved. Practically, you can pretty easily tell if brain tissue has any major connectivity damage just by looking at single electron micrographs. Traceability becomes MUCH easier to determine when looking at a FIB-SEM video.

In particular, when I look at at these ASC brains, I'm looking for things like shriveled up neurons with retracted axons, vacuolated processes that have lost all their internal cytoskeletal structure, damaged mitochondria, "holes" in various membranes, and "unraveled" myelin.

There's a lot of examples of control electron micrographs to compare to, many of which are available on the Open Connectomes website: http://openconnecto.me/catmaid/

We use 65% ethylene glycol as our CPA and ensure that it's fully delivered to the brain by perfusing for an entire hour at full concentration. 65% ethylene glycol is amazingly effective at suppressing ice and actually forms a thermodynamically stable glass during vitrification. This means that even if you put a ice template crystal into the mix, the cryoprotectant mixture would simply destroy the crystal. The cryoprotectant is intrinsically stable and won't ever freeze regardless of the cooling and warming rate.

Fixed brains, even without being stored at -135°C, are already quite stable themselves. I've kept a fixed brain in a fridge at 4°C for several weeks without any structural damage! (an unfixed brain would suffer severe structural damage during this time). So we know that at least a week at 4°C is tolerable (and actually I imagine a month at 20°C is probably also tolerable). Naively considering the decrease in diffusion times using the Arrhenius equation, we see that 1 second at 4°C is equal to around 5 hours at -135°C. In fact, the time dilation will be much more extreme due to vitrification and a second at 4°C will be more like a year at -135°C. However, even with these VERY conservative assumptions we get 100 years @ -135°C = 2.2 days @ 4°C, which is well within our week timeframe. (100 years is the target for the brain preservation prize).