PERSISTENCE OF LONG-TERM MEMORY: in Vitrified and Revived Simple Animals

By Natasha Vita-More

First published in Cryonics Magazine

“If the aging process is controlled in a similar way in worms and humans, then we can use
what we learn about worms to speed our study of higher organisms.” — Cynthia Kenyon

Preserving memory after cryonic preservation is a breakthrough science for cryonics, which has been a huge hurdle for cryonics. The research leading to this breakthrough will help to build momentum toward advanced research on information storage within the brain, as well as short-term behaviors of episodic, semantic, procedural, and working memory.

In this article, I will review how I became involved in this research, the guidance along the way, my initial training at 21st Century Medicine, pitching the research project to Alcor and submitting my proposal to its Research Team. I will then take you into the lab, the process of trial and error in our first trials, developing a protocol based on olfactory imprinting and applying several cryopreservation methods, developing the migration index, and the rewards of working with a lab technician who became an admiral colleague.

From this experience, I am more committed than ever to support and help lead scientific research projects that enrich learning about memory after cryopreservation. But this does not come without the insight to imagine, to speculate, and to hypothesize. Observing a gap in the current state of things triggers a desire to understand why there is a gap and to do something about it. From there we can query until one idea sticks and garnishes enough value to move forward. For me, this one idea was all about memory retention.

“The lingering concern: ‘How can something that cannot be demonstrated be scientific?’ found in the Alcor FAQ has now been demonstrated. While the larger question of how can a person’s identity be sustained after cryonics has not been conclusively answered; however, it is a fact that long-term memory is retained in a simple animal. It causes me to think back on Neil Armstrong stated after the Apollo 11 Mission. Certainly not as grand, but nevertheless, ‘This is one small step a [nematode], but a giant leap for [cryonics]” (Vita-More in conversation, 2015).

This research was to put into motion as a project I had been musing about for many years that concerns the outstanding issue of cryonics and memory retention. While the science and technology of cryopreservation has advanced over the past decades, there had been no evidence that an animal could be suspended, revived, and tested for memory retention. During the 25 years I have been a member of Alcor, I have listened to the internal conversations among cryonicists and read public commentary about the viability of cryonics. A core question has been: Will you remember who you are if and when you are revived? While this question can only be answered definitively once the first cryopreserved person is revived, it seemed logical that there needed to be small, baby steps along the way. Several people had begun projects to explore this area, but none had been conclusive, let alone published.

The project I put into action that I was slowing developing over the span of a decade. As a bit of background, no biodesign experiments within the field I pursued my Doctorate in had been developed in the field of cryonics. My colleagues Dr. Edwardo Kac had developed the transgenic “GFP Bunny”, Stelarc succeeded in cloning and transplanting his ear onto his arm, and Dr. Ionat Zurr with Oron Catts had developed tissue culture as “semi-living” sculptures. Yet, there was an identifiable lack of exploration and experiments in the biodesign field of human enhancement and life extension that linked directly to cryonics.

Dr. Greg Fahy, leading cryobiologist, had been an exceptional mentor since the inception of this project. He had told me about a researcher’s work that captured stunning visuals of glycerolized human sperm as they were absorbed and obfuscated by ice formations, and which movements began after the ice receded. Inspired by this, I set out to study what types of life forms I could work with and which exhibited unique physical movement. Based on Dr. Fahy’s advice, I decided to work with C. elegans, a tiny nematode worm that is approximately 1 mm in size. My aim was to learn about this worm and then to explore research that identified its ability to learn and retain information. I also learned about cryopreservation protocols for C. elegans that had been successful.

“Caenorhabditis elegans is one of the most important models used in biology and neurology1 and has countless applications in the area of biological sciences. The simplicity of its size (1mm), the transparency of its neuronal network (hermaphrodites contain 302 neurons),5 and its short but complex life cycle make C. elegans of potential value to studies of memory retention after cryopreservation” (Vita-More & Barranco, 2014).

C. elegans can be trained through nonassociative learning, associative learning, and imprinting. They can habituate to chemical stimuli and learn smells, tastes, temperatures and oxygen levels. They also respond to vibrations, such as tapping on the petri dish. In regards to cryonics, C. elegans have high survival rates, with little to no cryoprotectant, when using ultra-rapid cooling and warming methods. By providing a case where I could use a viable learning environment for the worms, cryopreserve them with their efficacy intact, revive them, and then test their memory of the learning behavior, I might be able to add significant research to the field of cryonics. I spent the next year or so looking for grant money to support the research. Eventually persistence paid off, and Fahy was consequential in my obtaining the grant from Alcor Life Extension Foundation.

“Memory models that are amendable to testing after cryopreservation are not plentiful.  The best test of memory is behavioral, but there are no easily accessible organisms more complicated than C. elegans that can be cryopreserved whole to enable behavioral tests after rewarming.  So I think Natasha’s proposal is appropriate for pushing the envelope given the constraints involved.  Perhaps success in this project could serve as a jumping off point to testing polar insects or Siberian salamanders down the line, but first things first.  You have to walk before you can fly” (Fahy, 2013).

The question I asked in this research was whether memory could be retained after cryopreservation. The single question became the object of the research. To attempt to answer this question, the C. elegans was the model organism for testing because it is a known model used in biology and neurology, the simplicity of its size, and it had already been successfully vitrified and trained, but there had been no research experiments combining both vitrification and cryopreservation and also training and testing memory after reviving. In short, it was the only simple animal where cryopreservation and revival had been demonstrated and a well-defined assay of learning had been completed.

Starting with the completed research performed in these two areas, my team sought to build upon these experiments in forming what we call the Persistence of long-Term Memory in Vitrified and Revived C. elegans.

Setting up the Lab at Alcor

After receiving the grant to commence the research, the Alcor team worked with me to locate a work area, hood, and then I started ordering supplies. Hugh advised me about basic chemistry and we determined an aluminum mini-dewar was best for holding the liquid nitrogen, we also prototyped several methods for detecting worm migration on plates and petri dishes. Steve Graber created the lab area and set up the hood, and worked with me to test microscopes for depth of field, lens magnification and video recording. Dr. Mike Perry met with me to discuss statistical analyses of trained and tested worms.

Through a colleague of Fahy, Dr. Ramon Risco, I was provided with a particular method for vitrification, known as the slush method. This method uses quartz capillaries that have a specific diameter and require a slush making apparatus. Hugh ran with this and started to build a slush making apparatus.

While we were excited to move forward on the project, one core issue from the beginning of my study was that I needed to hire a lab technician to work with me, since I was not an expert technician. I contacted Crish Rasch, who I knew had worked with C. elegans in the past and invited him to work with me in the lab to test learning protocols for raining the worms, from tapping on petri dishes, to using lighting effects for stimulation, and also chemical attractants.

While we were making some progress, one core issue from the beginning of my study was that I needed to hire a lab technician to work with me, since I was not an expert technician. I was introduced to PhD candidate Daniel Barranco, an expert in the cryotop method of embryo freezing. Since Barranco lives in Seville, Spain and the phone calls and Skype meetings were becoming lengthy, we invited him to work with me in the lab at Alcor. His strong skill set was a key factor in our iterative process of exploring options and testing, retesting, and finally determining both our memory retention protocol and our cryopreservation and vitrification methods.

Establishing the Control Group and Experimental Group for 10 Studies

The research established two groups, the control group and the experimental group. For the control group, we formed eight studies. For the experimental group, we formed two studies. Each of the ten studies contained 100 or more worms (See Table 1).

Methodology: Three Areas of Focus

Our methodology was based on what was already known in the field and what might be the most effective tools and techniques to use. After much deliberation, we decided to incorporate an established method for learning, several methods for cryopreservation, and a chemotaxis assay for observing whether or not the worms had remembered what they learned at the early L1 stage and after cryopreservation and reviving at the adult stage.

1. Learning Method: Using the method of olfactory imprinting method of Remy and Hobert, we established a protocol using the chemical benzaldehyde (C6H5CHO). The studies focused on olfactory imprinting of the nematodes at the L2 stage. This a very early age, just after the nematode develops from the larvae stage. The nematodes were placed in petri dishes, some with the chemical benzaldehyde and some with only water swiped on dish lids where food was placed. In the studies, the benzaldehyde was used as an attractant, which developed an association between food and the chemical smell. The aim was to establish whether or not the nematodes could retain the imprinted experience of the chemical smell of benzaldehyde with food into its adult stage, identifying long-term memory.

2. Vitrification and Cryopreservation Process: The traditional methods for cryopreserving biological samples is through slow freezing and through vitrification, which have different cooling and the warming rates. For our research’s vitrification, we applied the known method of Cryotop, used in the freezing of embryos. While our research experiment’s studies included several methods for cryopreservation, our central focus was the Cryotop protocol indirectly submerging the nematodes into liquid nitrogen using a straw device. One worm at a time was carefully pulled into the straw from the petri dish. From this, we established the effective use of the SafeSpeed closed device, a new technology for ultra-fast warming rates.

3. Testing Results of Long-Term Memory. We used a chemotaxis assay five days after olfactory imprinting, when worms reached the adult stage. Marking 12×12 square agar plates, we drew lines marking off areas with assigned values of from -6 to 6 on the outside of the plates. In the first area of the plates, at value -6, we issued three drops of sodium azide at equal spacing into the agar. In the same areas, with the same equal spacing but on the lid of the plates, we issued three drops of plain water. On the other side of the plate, at value 6, we issued the same three drops of sodium azide at equal spacing; but on the lid of this area, we issued three drops of benzaldehyde, instead of water (Figure 2).

As series of processes included using a platinum wire to pick up revived worms from the petri dish with food, to a petri dish without food, and after numerous minutes, transfer them onto the square plate to time and observe where they migrated to. This was the Migration Index (MI). The statistical analysis for each study was tested with the Levene test, ANOVA test, and Tahame test (Table 2).

Getting Started: Validation of Memory Retention in Studies

The memory retention protocol we used for learning is known as olfactory imprinting. We distinguished this protocol by using the chemical benzaldehyde for phase-sense imprinting on the young worms, just after the larvae stage. Olfactory imprinting has been studied in many species, including primates, mammals and humans. The key to successful olfactory imprinting is that to be successful, its effect is relative to the period of time (or window of opportunity) when the organism can develop a long lasting learned response. For this research, it was introduce early on so that the worm associated food with the smell of benzaldehyde. This phase-sense imprinting was performed by swiping a very small amount of benzaldehyde on the inside of the petri dish lid every hour for eight hours for worms that were being trained.

Memory retention was validated through a chemotaxis assay of the migration index. The trained worms migrated to areas of the petri dish where the benzaldehyde drops were placed. This showed that they preferred areas of the dish were the chemical smell was detected. Because there is a native reaction to benzaldehyde, the untrained worms preferred other areas of the dish. In sum, the response of the trained worms was double that of the untrained worms, whether they were cryopreserved or not.

A Brief Description of the Research Results

The research shows the first results related to persistence of long-term memory of C. elegans after vitrification and reviving. I, along with Daniel Barranco, describe the results in our paper, in Rejuvenation Research (October issue):

“The survival rates for our study did not show deviation from the expected original slow freezing method of Brenner2 or the SafeSpeed method of Barranco. 32 The survival rate for slow freezing with L2-L3 worms was ∼20%, and for vitrification was ∼100%” (Vita-More & Baranco, 2015).

Are you planning more cryopreservation research with C. Elegans?

I would like to see the Alcor Research Center work with researchers to develop projects relative to cryonics, since we now have a working lab at Alcor.1 With this, I would like to lead a team or advise a team who are far more skilled at the hands-on experiments than I am. The microscopic size of the C. elegans nematode requires agility, patience, and very good vision. Getting one single microscopic worm into a tiny straw is a challenge.

As for extending C. elegans research, I would like to explore alternative learning methods at different maturity stages of the worm. Also, more work is needed to find out if a few or all memory mechanisms are unaffected by the Benzaldehyde and/or vitrification.

Beyond this, I am more interested in testing memory on larger organisms with a more complex central nervous system and leave others to continue the research that I and Barranco completed.

Worm submerged in liquid nitrogen (Vita-More, 2014.)

If you would do similar research in an animal with a more complex nervous system, what would you choose?

I would like to research cold-tolerant species that live suspended in a frozen state during winter seasons and thaw in the warmer seasons. The Greenland Woolly Bear Caterpillar is a species that is active for a mere 30 days of the full 365 days a year, and then goes dormant in self-made cocoons. These cocoons are cleverly attached to rocks and the cocoon coverings form tiny biosphere greenhouses. Another species is the Alaskan Wood Frog, an amphibian that freezes solid through the winter and defrosts in the spring. Nevertheless, after working with C. elegans, who naturally have rhythmic movements that are visually pleasing and emotionally alluring, it would be difficult to work with a leech, which is another option. The ozobranchid leech, is a parasite that attaches itself to freshwater turtles is a highly tolerant organism to freezing conditions and thawing, repeatedly. The downside is that these leeches can carry viruses that form cauliflower-like tumors on the turtles, impeding on their health and survival rate. Here is a note of caution; however, they are known not to affect humans.

How has your research been received?

The first couple of weeks, there were over 11,600 downloads of our paper. I would have been delighted if 600 people downloaded. There is a lot of interest, to be sure. I hope to take the video footage and create a graphic documentary. For more information, download the paper or subscribe to the Rejuvenation Research periodical.

Vitrified and revived worm in orange food coloring (Berranco, 2014).

An Unexpected Incident

An unexpected result from the research was watching a revived worm’s eggs hatch before our eyes. This was one of the most thrilling moments for me personally. We had thought the four oval shapes in the dish were air bubbles and that I had mistakenly emitted them from the straw when I took the vitrified worm I had placed from the warming solution to the petri dish. As I was watching the behavior of the worm to follow its movements and determine if it was surviving the process, I noticed the oval shapes started moving. Then over the next minute or so, all four larvae had hatched and were healthy looking new baby worms.

C. elegans lays four eggs after vitrification and reviving. (Vita-More 2014)

Footnotes

1 Alcor provided a generous grant for this research project. Alcor personnel, including Hugh Hixon, Steve Graber, and Mike Perry worked with me to build a lab that can be used by others in the coming years.