Vitrifying the Connectomic Self: A case for developing Aldehyde Stabilized Cryopreservation into a medical procedure

... [b]ut the main point of this paper is to persuade the scientific and medical community
that now is the time to develop this ASC procedure into a reliable medical procedure that can be offered to terminal patients. This is a radical proposal that can easily be misunderstood. This misunderstanding often manifests itself in questions like: ``Why on earth would a terminal patient desire such an option in the first place?'', ``How would such a procedure work on a practical level?'', ``Are patient safeguards even possible for a procedure whose final success won't be known for decades or centuries?'', ``Can we even imagine the technologies that would allow future revival?'' 

Perhaps the best way to answer all of these questions is to offer a speculative short story meant to summarize and clarify this vision. The following fictional story follows a man diagnosed with Alzheimer's dementia in the year 2030 who chooses to undergo ASC preservation in the hopes of future revival. Extensive footnotes throughout this fictional story briefly explain the science behind key steps and point to references that support the science and technology discussed.

HAYWORTH, Kenneth. Vitrifying the Connectomic Self: A case for developing Aldehyde Stabilized Cryopreservation into a medical procedure.

http://www.brainpreservation.org/wp-content/uploads/2018/02/vitrifyingtheconnectomicself_hayworth.pdf

Electron Imaging Technology for Whole Brain Neural Circuit Mapping

The goal of uploading a human mind into a computer is far beyond today's technology. But
exactly how far? Here I review our best cognitive and neuroscience model of the mind and show
that it is well suited to provide a framework to answer this question. The model suggests that our
unique ``software'' is mainly digital in nature and is stored redundantly in the brain's synaptic
connectivity matrix (i.e., our Connectome) in a way that should allow a copy to be successfully
simulated. I review the resolution necessary for extracting this Connectome and conclude that
today's FIBSEM technique already meets this requirement. I then sketch out a process capable
of reducing a chemically-fixed, plastic-embedded brain into a set of tapes containing
20x20 micron tissue pillars optimally sized for automated FIBSEM imaging, and show how
these tapes could be distributed among a large number of imaging machines to accomplish the
task of extracting a Connectome. The scale of such an endeavor makes it impractical, but a
version of this scheme utilizing a reduced number of imaging machines would allow for the
creation of a ``Connectome Observatory''---an important tool for neuroscience and a key
milestone for mind uploading.

HAYWORTH, Kenneth J. Electron imaging technology for whole brain neural circuit mapping. International Journal of Machine Consciousness, 2012, vol. 4, no. 1, p. 87--108.

http://www.brainpreservation.org/wp-content/uploads/2016/01/ElectronImagingTechnologyForWholeBrainNeuralCircuitMapping_Hayworth2012.pdf

High-resolution Whole-brain Staining for Electron Microscopic Circuit Reconstruction

Currently only electron microscopy provides the resolution necessary to reconstruct neuronal circuits completely and with single-synapse resolution. Because almost all behaviors rely on neural computations widely distributed throughout the brain, a reconstruction of brain-wide circuits---and, ultimately, the entire brain---is highly desirable. However, these reconstructions require the undivided brain to be prepared for electron microscopic observation. Here we describe a preparation, BROPA (brain-wide reduced-osmium staining with pyrogallol-mediated amplification), that results in the preservation and staining of ultrastructural details throughout the brain at a resolution necessary for tracing neuronal processes and identifying synaptic contacts between them. Using serial block-face electron microscopy (SBEM), we tested human annotator ability to follow neural 'wires' reliably and over long distances as well as the ability to detect synaptic contacts. Our results suggest that the BROPA method can produce a preparation suitable for the reconstruction of neural circuits spanning an entire mouse brain.

MIKULA, Shawn and DENK, Winfried. High-resolution whole-brain staining for electron microscopic circuit reconstruction. Nature methods, 2015, vol. 12, no 6, p. 541.

http://dx.doi.org/10.1038/nmeth.3361

Aldehyde-Stabilized Cryopreservation

We describe here a new cryobiological and neurobiological technique, aldehyde-stabilized cryopreservation (ASC), which demonstrates the relevance and utility of advanced cryopreservation science for the neurobiological research community. ASC is a new brain-banking technique designed to facilitate neuroanatomic research such as connectomics research, and has the unique ability to combine stable long term ice-free sample storage with excellent anatomical resolution. To demonstrate the feasibility of ASC, we perfuse-fixed rabbit and pig brains with a glutaraldehyde-based fixative, then slowly perfused increasing concentrations of ethylene glycol over several hours in a manner similar to techniques used for whole organ cryopreservation. Once 65% w/v ethylene glycol was reached, we vitrified brains at -135 degrees C for indefinite long-term storage. Vitrified brains were rewarmed and the cryoprotectant removed either by perfusion or gradual diffusion from brain slices. We evaluated ASC-processed brains by electron microscopy of multiple regions across the whole brain and by Focused Ion Beam Milling and Scanning Electron Microscopy (FIB-SEM) imaging of selected brain volumes. Preservation was uniformly excellent: processes were easily traceable and synapses were crisp in both species. Aldehyde-stabilized cryopreservation has many advantages over other brain-banking techniques: chemicals are delivered via perfusion, which enables easy scaling to brains of any size; vitrification ensures that the ultrastructure of the brain will not degrade even over very long storage times; and the cryoprotectant can be removed, yielding a perfusable aldehyde-preserved brain which is suitable for a wide variety of brain assays.

MCINTYRE, Robert L. and FAHY, Gregory M. Aldehyde-stabilized cryopreservation. Cryobiology, 2015, vol. 30, p. 1--11.

http://dx.doi.org/10.1016/j.cryobiol.2015.09.003

Can I choose how long I'll live?

Bibliotheca Alexandrina

Broca's Brain

It was difficult to hold Broca's brain without wondering whether in some sense Broca was still in there---his wit, his skeptical mien, his abrupt gesticulations when he talked, his quiet and sentimental moments. Might there be preserved in the configuration of neurons before me a recollection of the triumphant moment when he argued before the combined medical faculties (and his father, overflowing with pride) on the origins of aphasia? A dinner with his friend Victor Hugo? A stroll on a moonlit autumn evening, his wife holding a pretty parasol, along the Quai Voltaire and the Pont Royal? Where do we go when we die? Is Paul Broca still there in his formalin-filled bottle? Perhaps the memory traces have decayed, although there is good evidence from modern brain investigations that a given memory is redundantly stored in many different places in the brain. Might it be possible at some future time, when neurophysiology has advanced substantially, to reconstruct the memories or insights of someone long dead? And would that be a good thing? It would be the ultimate breach of privacy. But it would also be a kind of practical immortality, because, especially for a man like Broca, our minds are clearly a major aspect of who we are.

SAGAN, Carl. Broca's brain: Reflections on the romance of science. Presidio Press, 1980.

https://en.wikipedia.org/wiki/Broca%27s_Brain

A Door to the Future

Benjamin Franklin wanted a procedure for stopping and restarting metabolism, but none was then known. Do we live in a century far enough advanced to make biostasis available---to open a future of health to patients who would otherwise lack any choice but dissolution after they have expired?

We can stop metabolism in many ways, but biostasis, to be of use, must be reversible. This leads to a curious situation. Whether we can place patients in biostasis using present techniques depends entirely on whether future techniques will be able to reverse the process. The procedure has two parts, of which we must master only one.

If biostasis can keep a patient unchanged for years, then those future techniques will include sophisticated cell repair systems. We must therefore judge the success of present biostasis procedures in light of the ultimate abilities of future medicine. Before cell repair machines became a clear prospect, those abilities---and thus the requirements for successful biostasis---remained grossly uncertain. Now, the basic requirements seem fairly obvious.

DREXLER, K. Eric. Engines of creation. Anchor Books, 1986.

http://e-drexler.com/p/06/00/EOC_Cover.html

  

A Possible Cure for Death

Chemical preservation of the brain may prevent death. Life for an individual human being is inextricably linked to the existence of his or her mind. It is widely accepted that the mind is a product of the functioning of the brain, which, according to this view, is nothing more and nothing less than a fantastically complicated machine. Chemical preservation of the brain (promptly after the cessation of vital functions) preserves not only the neuronal configuration but also a great deal of molecular structure. Thus, it is plausible that a chemopreserved brain contains within it the information of the design of the "brain machine". If so, then technology of the distant future may be able to extract that information and construct a new functionally identical brain machine (as well as a body), thereby allowing the corresponding individual to wake up and live again. It is argued that one's identity is defined by what the brain does rather than how it does it or what it does it with, and therefore that replacement of one's brain with a functionally identical machine does not affect one's identity. Some advantages of chemopreservation relative to cryopreservation as a possible means of preventing death are discussed.

OLSON, Charles B. A possible cure for death. Medical hypotheses , 1988, vol. 26, no 1, p. 77-84.

https://www.ncbi.nlm.nih.gov/pubmed/3398793

The Nicene Creed (381)

"We look for the resurrection of the dead, and the life of the world to come. Amen"

The Council of Constantinople,  Nicene Creed (381).

Challenges in getting a whole-brain connectome

I've been reading about what is preventing us from getting a whole mammalian connectome scanned today. Here are links to the papers I found:

This is Kenneth Hayworth's 2012 paper describing a facility he calls, a connectome observatory. This facility he estimates can be built for about $200 million and will give us random access to any part of the brain. Scanning the entire brain will still take years though. 

(Hayworth, K. J. (2012). Electron imaging technology for whole brain neural circuit mapping. International Journal of Machine Consciousness, 4(01), 87-108.)

This paper from 2013 reviews the challenges stopping us from getting whole connectomes. I liked this paper because it described, with pictures, some of the difficulties with reconstructing connectomes from stacks of electron microscope (EM) images. E.g. if the dotted line is the plane at which the brain was sliced, it is "difficult" to tell which of the three configurations of neurons the slice represents.

The paper also has this neat graph showing the time it takes to image a brain versus to reconstruct the connectome from the images. We're talking about a 3 or more orders of magnitude difference.

(Helmstaedter, M. (2013). Cellular-resolution connectomics: challenges of dense neural circuit reconstruction. Nature methods, 10(6), 501-507.)




There's a more recent review of the challenges in acquiring a whole-brain connectome by Shawn Mikula (a contestant in the BPF prize). It seems to be his opinion (p.4, last para.) that we'll be able to close the analysis gap in the previous graph from Haelmstadedter's paper. The main computational challenge he identified is how to validate the reconstructed connectome. He says

While consensus of redundantly- and manuallyh-traced neurites and annotated synapses is one appraoch, it relies on model-based assumptions.

I'm not sure what "model-based assumptions" here means but I'm guessing he's saying that a bunch of human annotators look at a reconstruction and agree, "this looks right." Some other (better) options are:

• Imaging a few parts of the brain at a much higher resolution (e.g. 2x2x2nm instead of 10x10x10nm) and checking that all the relevant features visible at the higher resolution also appear at the lower resolution.
• Trace a few neurons unambiguously (e.g. by making them fluoresce) and then compare it with the tracing from the electron microscope images.

(Mikula, S. (2016). Progress towards mammalian whole-brain cellular connectomics. Frontiers in Neuroanatomy, 10, 62.)




Well, it seems like academia is making fast enough progress on studying the connectome. If you want to try your hand at reconstructing (parts of) a connectome from EM images check out the following.

The Eyewire project (videogame where the aim is to reconstruct parts of a connectome) has a Google drive with a bunch of training data i.e. EM images of a mouse retina with the neurons labelled. Here's an example training pair.

The FlyEM group at Janelia farm also publishes a bunch of images and connectome reconstructions from a fly brain. And this powerpoint presentation talks about the analysis problems they're facing as of September 2016. FYI a fly brain may be harder to analyze since it's neurites are smaller than those of a human. But hey, if you can efficiently extract a fly's connectome, you should be able to do the same for a mammal, right?