Can we freeze time?

A look into preserving an individual's brain rhythms

Unlike artificial neural networks, which rely on linear, stepwise computations that can be paused and resumed at any time, the brain operates through a complex, time-dependent electrophysiological process. It follows its own unique regime of time, distinct from clock time. The fundamental unit of time in the brain appears to be a cycle, characterized by fluctuating levels of activity. These cycles collectively create rhythms within the brain.

I’ve always found brain rhythms to be confusing. It’s hard for me to think about or visualize them. So when I revisit this topic, I find it useful to go back to the basics.

What are brain rhythms?

Brain rhythms are the natural patterns of neural activity that occur in the brain over time. They can be measured using electroencephalography (EEG) or other neuroimaging techniques, and they are typically categorized based on their frequency.

this picture of someone with an EEG on is in 99% of intro to EEG talks

For example, alpha waves were the first type of human brain rhythm discovered. Back in ye good old days of child experimentation (1920s, Germany), psychiatrist Hans Berger put some electrodes on his kids’ heads to measure electrical activity and discovered these patterns:

to the right of the arrow in the upper tracing, you can see alpha waves

Alpha waves occur at a frequency of 8-12 Hz. Hz, or Hertz, denotes the frequency of oscillation per second, so this means alpha waves oscillate approximately 10 times every second.

Where do they occur? On EEG, alpha waves are seen most prominently over the posterior regions of the brain, like the occipital or parietal cortex.

EEG primarily detects differences in voltage between pairs of electrodes placed on the scalp. These voltage differences arise due to the electrical activity generated by the brain's neurons. The EEG machine records and amplifies these voltage differences, which are then visualized as brain waves.

Since the discovery of alpha waves, all sorts of other brain rhythms have been discovered, at different frequencies, in different brain regions, and at different spatial scales, such as the single neuron, local field potential, or macroscopic levels. These include beta, theta, gamma, and delta waves.

There are plenty of controversies in the field of brain rhythms. Their necessity and sufficiency for various cognitions or behaviors, the roles of specific frequency bands, their interactions, how to distinguish the rhythmic signal from noise, and the degree to which they are associated with various disorders, are all hotly debated. That’s outside the scope of this I don’t really feel like writing about it right now.

a picture of me trying to make sense of the brain rhythm literature

What matters for us is that brain rhythms turn out to be a core aspect of how the brain operates. And that although there are plenty of shared features across people, individuals also have unique signatures to their brain rhythms. So naturally, it’s relevant to brain preservation.

Cryopreserving the brain and rewarming it with rhythms intact

In 1966, Isamu Suda and colleagues published an article in Nature, “Viability of long term frozen cat brain in vitro”, with a pretty audacious experimental design and striking findings.

The study involved perfusing the anesthetized cat brain with a cooled, salt-balanced artificial solution, followed by surgical isolation of the brain within the cranium. The isolated brain was then infused with glycerol in increasing concentrations of up to 15% to protect the tissue during freezing. The brain-containing cranium was subsequently frozen at -20°C and stored for 45 days. For the thawing process, the frozen cranium was moved to a 2°C cold room overnight. Once completely thawed, the brain was extracted from the skull, the glycerol was washed out, and the brain was reperfused with a warm blood solution in an incubator. Electrical activity was recorded from the brain to assess the viability of nerve cells after the freezing and thawing process.

They reported that at the end of all this, when they recorded electrical activity with silver plates on the surface of the cerebral cortex, they were able to record coordinated brain rhythms:

They reported that this “resembled that of the normal in situ brain”. Now, am I able to evaluate whether these rhythms are physiologic? Not at all. But they are obviously not isoelectric (like they are at the end of the second tracing). There seems to be some sort of rhythm maintained after rewarming and reperfusion.

As a side note for clarity, because the skull had been removed, they recorded electrical signals using electrocorticograms (ECoG), not EEG:

They also reported that brain cells had “almost normal cell arrangements” under the light microscope:

“almost” normal, kind of like my interest in brain preservation at a family reunion

Suda’s group followed this up with another publication in 1974, “Bioelectric discharges of isolated cat brain after revival from years of frozen storage”. Basically, this study was an attempt to keep the brains at -20 °C for even longer, up to several years. They found that rewarmed cat brains after long-term frozen storage still had electrocortical activities, but they were simpler, and they gradually lost their rhythmic patterns as the storage time increased:

They also tried storage at other temperatures (-20 °C, -60 °C, -90 °C) and with other cryoprotectants (DMSO, PVP, hydrogenated dextran) for weeks of storage time. They found that preservation with glycerol and DMSO at -20 °C was the best and the second best, while no electrical discharge could be detected after storage at -90 °C or with the use of PVP as a cryoprotectant. This suggests that preserving with at least some sort of effective cryoprotectant was an essential part of their results.

Of course, there is always the possibility of outright fraud in science, but Yuri Deigin reports that Suda sent a copy of his experimental notes to Greg Fahy, who did not detect any signs of falsification.

To the best of my knowledge, nobody has replicated Suda’s studies since. Over the years, they have taken on the status of lore and have been kind of a Rorschach test for cryonicists:

• The field of cryobiology was only really invented in the 1950s, so the 1960s were still early, heady days for the field. Suda’s 1966 study was influential and it was immediately picked up by early cryonics organizations, such as CSNY. This may have helped contribute to their extremely unrealistic revival timelines, even by the standards of cryonicists!

• Suda’s studies were cited at least somewhat approvingly by Ralph Merkle in 1992: “The brain seems more resistant than most organs to freezing damage”.

• Pichugin, Fahy, and Morin 2006 were more reserved in their citation: “Complex neural functions have been recovered after freezing cerebral tissue to relatively high subzero temperatures, but not after cooling to temperatures that would permit long term storage.”

• Mike Darwin has been cynical: “Darwin was and remains the only cryonics activist or professional to publicly criticize the use of positive, dramatic, incredible, and not reproduced published scientific studies to validate and promote cryonics (i.e., data dredging), most notably the claim by Suda, et. al., to have achieved recovery of near normal metabolic and electrical activity in cats’ brains after freezing to and prolonged storage at -20xC and the claim of Blaine C. White reporting neurologically intact resuscitation of humans after an hour of normothermic circulatory arrest via the use of a calcium channel blocking drug.”

• There is still plenty of interest in attempting to cryopreserve and rewarm brains with whole-brain electrophysiological signals intact. For example, Aschwin de Wolf and Chana Phaedra listed it as one of their organization’s goals in 2014.

What do electrophysiologic measurements actually tell us?

But even if some sort of EEG signal was shown to be re-instantiated after cryopreservation, it still wouldn’t necessarily mean that memories or personality are intact. This was pointed out shortly after Suda’s studies were published. Cryobiologist Armand Karow wrote in 1968¹:

The most important question is whether the EEG’s of the revived brains represent organized cerebral functioning. Brain activity can be quite disorganized. The generation of EEG’s is not necessarily an indication of organized, coherent activity. To illustrate, it has been observed that with suitable cryoprotective agents, hearts frozen to -70 degrees C will exhibit electrical activity (EKG), despite the absence of any evidence of mechanical activity. The EEG tracings obtained by Suda probably could have been generated even if a large number of the cells were damaged.

Hussman made a similar point in a 1969 article, “Electroencephalographic Or Biologic Survival”, wondering whether Suda’s results were instead due to an electrical artifact.

And of course, Rouleau et al. 2016 reported that fluctuations in electric potential differences could still be detected in brains stored in fixative for long periods of time at room temperature. Most people would agree that formaldehyde is most definitely going to alter brain rhythms, in the absence of repair far beyond anything possible today (if ever). While this study’s results have also been questioned (a discussion for another day), I view this mostly as a reductio ad absurdum of the idea that measuring brain rhythms alone is indicative of high-quality preservation².

What I think these points indicate is that the presence or absence of electrophysiologic signals alone is not that dispositive. What would be more useful is seeing whether particular types of electrophysiologic signals, such as those indicative of memory retrieval or another form of valued cognitive functioning, are retained after a preservation procedure.

In other words, the consistent demonstration of post-rewarming retention of ECoG or EEG signals, and especially the retention of rhythms associated with memory, would be great progress to consistently demonstrate after cryopreservation. But it wouldn’t be game over.

The only way to really demonstrate a memory is still there would be either some sort of neuroimaging mind-reading experiment (not creepy at all, and also not yet possible) or a behavioral assay. But the latter requires more than just the reanimation of the brain — but rather, the entire organism.

What do I think of Suda’s results?

I’m kind of an inveterate fence sitter and consensus builder. Rather than make any definitive guesses either way, I’d rather try to quantify my uncertainty.

I created a Manifold play-money prediction market on this. You can read the detailed criteria if you’re interested, but basically, it’s a question of whether the core results of the study will be replicated or not by 20 years from now. My current guess is a 60% probability that it would be replicated if tested. But I wouldn’t bet much play money on this, since I have fairly low confidence in my prediction. If nobody tries either way, then the market resolves N/A.

Why do I think 60% chance of replication? My guess is that getting some kind of EEG signal that looks like a brain rhythm is actually not that hard. If the resolution criteria required some sort of meaningful electrophysiologic signal related to something like memory retrieval with Suda’s methods, then my probability estimate would be much lower, < 10%.

My guess is that Suda’s results also probably won’t be tested, even with these stipulations that it doesn’t have to be a direct replication. That’s because it’s not a very long-term preservation method. Brain vitrification followed by rewarming is a much more interesting project because (a) if it does work, it could work for the long-term and (b) if it can be achieved, it seems more likely to preserve meaningful whole brain electrophysiology.

Preserving brain structure to potentially recapitulate brain rhythms

Maybe a core aspect of Suda’s experiments was correct, maybe people will be able to extend those studies alongside cooling to lower temperatures for longer-term preservation, and maybe further research will suggest that the particular rhythms retained are a good proxy for important cognitive functions like memory retrieval. But my personal guess is that this is going to be a relatively thorny problem.

An alternative option, which also has a lot of other advantages, is to just focus on structural preservation quality.

This doesn’t get us around the problem of brain rhythms, though. Sadly, you can’t just look at the structure of a cell or synapse and say “yeah, that looks well enough preserved to produce coherent theta-gamma coupling when embedded in an appropriate neural network.” We don’t know. So we have to guess whether the information that would eventually be required to reinstatiate brain activity with its natural rhythms intact is still present in the structurally preserved brain. That introduces considerable uncertainty to the project.

There has been plenty of computational neuroscience work trying to figure out the structural correlates of brain rhythms. In the most recent publicly recorded Aspirational Neuroscience Prize Journal Club, Randal Koene focused on one such effort.

Koene’s presentation focuses on a paper that examines the serial representation of items during working memory maintenance at letter-selective cortical sites. The authors propose a model based on nested theta-gamma oscillations, which can explain the phenomena observed in working memory tasks.

To recapitulate this model or one of its successors in silico, numerous electrophysiologic properties of the brain would need to be preserved and measured. This includes the biomolecules and morphology necessary for neuronal spiking activity, the interactions between brain regions, the synaptic plasticity mechanisms underlying learning and memory, the modulation of oscillatory activity by neurotransmitters and neuromodulators, and the dynamics of local and global network connectivity. One of the bold claims of structural brain preservation is that this seems potentially doable.

Just because I’m ending this article with a section on structural brain preservation doesn’t mean I think that research on directly cryopreserving the cells responsible for brain rhythms is a bad idea. I think it’s super valuable and I wish the people working on it total success.

References

Suda I, Kito K, Adachi C. “Viability of long term frozen cat brain in vitro,” Nature, 212, 268-270 (1966).

Suda I, Kito K, Adachi C. “Bioelectric discharges of isolated cat brain after revival from years of frozen storage,” Brain Research, 70, 527-531 (1974).

Bree S van, Melcón M, Kolibius LD, Kerrén C, Wimber M, Hanslmayr S. The Brain Time Toolbox, a software library to retune electrophysiology data to brain dynamics. bioRxiv. Published online May 13, 2022:2021.06.09.447763.

Jensen O, Spaak E, Zumer JM. Human brain oscillations: from physiological mechanisms to analysis and cognition. In: Supek S, Aine CJ, eds. Magnetoencephalography: From Signals to Dynamic Cortical Networks. Springer; 2014:359-403.

Van De Ville D, Farouj Y, Preti MG, Liégeois R, Amico E. When makes you unique: Temporality of the human brain fingerprint. Sci Adv. 7(42):eabj0751.

Merkle RC. The technical feasibility of cryonics. Med Hypotheses. 1992;39(1):6-16.

Pichugin Y, Fahy GM, Morin R. Cryopreservation of rat hippocampal slices by vitrification. Cryobiology. 2006;52(2):228-240.

Hussman LH. Electroencephalographic or biologic survival. JAMA. 1969;207(1):153-153.

Johnson EL, Knight RT. Intracranial recordings and human memory. Curr Opin Neurobiol. 2015;31:18-25.

Rouleau N, Murugan NJ, Tessaro LWE, Costa JN, Persinger MA. When is the brain dead? Living-like electrophysiological responses and photon emissions from applications of neurotransmitters in fixed post-mortem human brains. PLOS ONE. 2016;11(12):e0167231.

Bahramisharif A, Jensen O, Jacobs J, Lisman J. Serial representation of items during working memory maintenance at letter-selective cortical sites. PLOS Biology. 2018;16(8):e2003805.

1. Interestingly, as Mike Darwin writes, Karow got into some trouble for this association: “Armand Karow was chastised for listing the Cryonics Society of New York as a financial supporter of his research on rat heart freezing, as well as his involvement with CSNY. Karow once expressed his opinion to the author that he “was passed over for a position on the Editorial Board of the Society’s journal Cryobiology because of his association with cryonics.” Karow followed these remarks with an observation to the effect that he had “learned his lesson” and did not intend to get tangled up with cryonicists again (Armand Karow, personal communication)”

2. I’m quite sympathetic to the idea of long-term fixative storage as a method in brain preservation, but not because of the electrophysiology data.