The Next Natural Building Block in the Evolution of Neurotechnology
A Model for Non-Invasive Optogenetics
All technologies undergo evolution over time. Modern day flat screen televisions which produce vibrant colors through thousands of pixels were once dense cube shaped devices with only black and white coloration. The wagons powered by horses that were once used for transportation have been replaced by high tech engine-powered vehicles. The gradual evolution of each of these technologies was very logical, as society a century or two ago was neither ready to develop nor use the present day version of each technology and the rate of gradual change was appropriate for the time period.
However, if a technology doesn’t have appropriate pacing for its evolution it can ruin the success of the product. This is demonstrated perhaps when a product doesn’t evolve fast enough for consumers, but even worse outcomes can stop a product from coming to market at all if it is designed in a way which is simply too advanced for society at that time.
This is exactly the case for an emerging neuroscientific technique called optogenetics. Its applications have the potential to be revolutionary in treating a variety of neurological disorders in a new way. Unfortunately, the way this technology currently exists is too advanced for its time for multiple reasons. It would be horrible to have to watch a technique with such amazing potential fail because it came too early. But rather than giving up on optogenetics and its potentially incredible future, I chose to work on redesigning it. And this version is far more feasible for present day.
Table of Contents
- The Basics of Optogenetics and Action Potentials
- How Its Invasive Nature is Stunting its Development and Acceptance in Society
- The ChRmine and SOUL Models Which Have Worked Towards this Already
- How I Chose the Best Model for Execution Using Molecular Biology, Computational Software, and DNA Alignments
- How Optogenetics is Utilized as a New Approach to Treating Neurological Conditions
- Key Takeaways
The Basics of Optogenetics and Action Potentials
So I told you just now it has great potential and you probably saw that cool visual up there, but what actually is optogenetics? Optogenetics is a neuroscientific technique which uses light and genetic engineering to control the activity levels of neurons (mostly) in the brain.
When neurons fire, it is based on a process called action potential. How often neurons fire is controlled by ion channel receptors, which separate sodium ions from potassium ions on either side. So long as the receptor stays closed, the two types of ions don’t mix. However, if the receptor opens up, the two groups of ions are able to mix. When the sodium and potassium ions get completely mixed up (depolarized), they generate an action potential which causes the neuron to fire.
This is a naturally occurring process, but in the 2000s scientists discovered a way they could manipulate this process using a protein called channelrhodopsin which was naturally occuring in algae. The use of this protein in the plant was to assist in the process of photosynthesis. When sunlight reached the algae, and the channelrhodopsin detected the type of light it is activated by (blue light), then signaled to the plant that it was receiving sunlight and should perform photosynthesis. Scientists realized that this wasn’t the only way the protein could be utilized, which is when they began to experiment with using it in the brain.
In 2009, optogenetics was invented. Channelrhodopsin was utilized in this technique, as it was injected into specific neurons in the brain through surgery along with an invasive brain computer interface (BCI) acting as a light source which was implanted nearby.
On command, the BCI applied blue light in the direction of the genetically modified neurons and just as they had done in the plant, the opsins signaled to the neurons that they were receiving light. However instead of signaling that it was time to perform photosynthesis, this time the opsins signaled to the ion channel receptor to open up and let the ions mix to generate action potentials. This is because channelrhodopsin is a type of opsin which performs excitation (increasing activity levels) of neurons.
After the initial discovery, other types of opsins were also discovered which were receptive to different colors of light. While some of these other opsins were also excitatory, new opsins such as halorhodopsin were discovered to be inhibitory (meaning used to decrease activity levels of neurons).
Its Unique Precise Nature
This discovery wasn’t the first time a technology had been found that could manipulate the activity levels of a neuron, but it was certainly the most precise. Other methods such as deep brain stimulation (DBS) and transcranial magnetic stimulation (TMS) which already existed at the time aren’t precise at all, since when they send a stimulation to the brain it activates every single neuron nearby.
This is not the case in optogenetics, since only neurons which have been genetically modified to express an opsin will have a reaction to the color of light used to stimulate that opsin. All of the nearby neurons which haven’t been modified will have no reaction, and therefore won’t be stimulated at all.
Not only this, but neurons which have been edited to express other opsins won’t have a reaction either since they are receptive to different colors of light. This means that a person can have many different types of opsins utilized in their brain without one type having any reaction to the others being stimulated.
As I’m sure you can see now, this technology is pretty cool. It’s still currently in the mice trials phase of its development, but cool nonetheless. However, the fact that it’s still in mice trials is only one of the effects of the fact that its evolution has been unnatural, or non existent even. Let’s take a look into why this is.
How Its Invasive Nature is Stunting its Development and Acceptance in Society
The part of the optogenetic system which presents significant challenges to its development is the invasive brain computer interface component. Currently, this piece is required in almost all optogenetic systems in order to deliver enough light to receiving neurons that they become activated. However, there are two big problems that this creates.
Stigma
The first is on the societal stigma side. Although they exist in different capacities, invasive BCIs are still very much not accepted by society. This is largely since the devices are implanted on the inside of the skull, aren’t removable, and have access to potentially personal information about brain activity levels at all times. Even when it comes to devices such as the one used for optogenetics which has a sole purpose of providing light to certain neurons and doesn’t require the recording of any neuronal activity, people are still untrusting of the devices.
The main fears regarding invasive BCIs are that they will extract personal information without a person’s knowledge or consent (such as their mental state or honesty), or export personal information to another BCI device through a system of a brain to brain interface (BBI).
Regardless of the fact that each of these are highly unlikely and potentially impossible in the context of the optogenetic BCI, they still exist as large stigmas making people less inclined to purchase products or even receive medical treatments requiring the long term implantment of an invasive BCI. This is only one of many societal concerns surrounding these devices, a lot of which are related to the ethics of invasive computer based devices.
Illogical Evolution and Development
The second, which is more specific to optogenetics, is that the use of the invasive brain computer interface in the product is actually stunting its development. In the initial development of brain computer interfaces as a product, non-invasive brain computer interfaces existed for about 74 years before invasive ones were even invented. This was a very logical development, as non invasive techniques such as EEG became more advanced in their efficiency and different applications until they were advanced to the point that invasive devices were feasible.
However with optogenetics, there never was a non invasive version of the technology. It was invented as invasive, and nobody ever took a step back. Not only does this bring the societal stigma concerns, but it’s simply not logical to jump straight to an invasive model especially after seeing that the timeline of the initial BCI spent so long on non invasive devices before they were ready to make invasive ones.
It’s clear that it makes more sense to take a step back here, and work to create non invasive optogenetics as a starting platform for the technique which should enable less apprehension surrounding the technique and have a more efficient development so optogenetics can become widespread and commercialized.
Executing this requires quite a bit of editing to the preexisting technology, as the initial model was designed under the assumption that optogenetics would be performed invasively. That said, there have been a few models designed in the past for performing “non invasive” or minimally invasive optogenetics, and I used pieces from each of these to design my model. Here’s what already exists.
The ChRmine and SOUL Models Which Have Worked Towards this Already
There are two important models which have been made previously that exhibit less invasive properties than the standard: the SOUL model, and the chRmine model. Neither is a perfect non invasive model, but they each show strong advancements in optogenetics which prove designing an effective non invasive model to be feasible.
SOUL
First up is the SOUL model. SOUL is a type of opsin which was recently discovered in April of 2020, and what makes it special is that it doesn’t require a large quantity or strength of light to be activated. It’s activated by blue light, and is used for excitation of neurons. A group at MIT utilized SOUL in a model for minimally invasive optogenetics where instead of having to place the BCI entirely within the brain, it could be placed in an opening in the skull outside of the brain.
Since SOUL requires less light to be activated, the light source was still strong enough to activate it from this position. This was a big deal because it showed that optogenetics could be slightly less invasive than it had been in the past, but still having the device inside a person’s head at all times is undesirable to many customers and still not quite non invasive.
ChRmine
The second advancement towards non invasive optogenetics was using chRmine, an opsin receptive to red light. Based on its longer wavelength, red light is able to penetrate the skull much better than blue light and this enabled a group of scientists at Stanford to use red light in a non invasive optogenetic model where the BCI wasn’t invasive at all.
This may sound perfect, and it’s still great but unfortunately this model was only able to activate neurons as deep as seven millimeters into the skull. For context, the average volume of a human brain is 1,400 centimeters, or 14,000 millimeters. It’s not that this discovery isn’t still amazing and significant, because it is, but this product is not the end result and there’s still much to be improved.
One of these models has an opsin which doesn’t require a lot of light, but the type of light used isn’t good enough at penetrating the skull. The other has light good for penetrating the skull, but the corresponding opsin can’t pick up the light unless it’s on the outskirts of the brain. What’s the logical solution here? To combine the two methods to get one supermethod.
How I Chose the Best Model for Execution Using Molecular Biology, Computational Software, and DNA Alignments
The plan for execution which I designed gets pretty involved in molecular biology and requires some protein editing as well. The purpose of this is to edit SOUL plasmids to be receptive to red light.
Given that I am not yet working in any kind of lab and therefore don’t have access to the physical version of these resources, all of my work to reach the conclusions I’m about to share was done using Benchling. Benchling is a popular molecular biology software where you can perform all sorts of functions computationally to experiment with and even edit different bodies of DNA.
Molecular Biology Vocabulary
Another piece of important background knowledge before I dig into this section is to explain some of the terms I will be using. As previously mentioned, the opsins used to perform optogenetics are often found in plants (such as unicellular algae in the case of channelrhodopsin). In order to remove an opsin protein from a larger body of the algae, it is isolated and then cloned into a plasmid. A plasmid is a circular, independent body of DNA which is separate from any larger body such as a plant. (Note: I will use plasmid and protein interchangeably.)
Inside of the plasmid, there are smaller pieces called kinases. In this case since we are looking at opsins which are proteins the kinases are specifically called protein kinases. They are the enzymes that make up the plasmid, and all serve different purposes which work together to help the plasmid function as a whole.
Protein Experimentation
In order to edit my SOUL opsin to be receptive to red light, I knew I needed to take DNA from an opsin which was receptive to red light and edit it into my SOUL opsin. However, this is not an easy task, since you have to be very careful with protein editing as not to break the protein.
One way to decrease the likelihood of the protein breaking and make sure the portion of a protein you edit into your initial plasmid takes well without a strong reaction is to choose a protein as similar to your initial protein as possible. However, this is far from guaranteed success still. The success rate of a designed protein can range from 1 in 1000 to 1 in 10 million which varies depending on the mutant pools.
I chose two popular opsins receptive to red light which I could experiment with online: ChRmine and Chrimson. I found vectors (online interactive diagram models) of plasmids for each of them, and isolated the specific kinases which are the namesakes of each plasmid (the ChRmine and Chrimson kinases respectively) since this is the constant among all opsins belonging to those groups and therefore the provider of the properties the opsins in those groups share (such as being receptive to red light).
Then I needed a SOUL plasmid. I hit a road block here, since there were no SOUL plasmids publicly available anywhere. And believe me, I looked all over. The only SOUL related thing I could find were exons, which are different from genes and you can’t run an alignment between exons and genes. My solution to this was just to use a similar opsin, the parent opsin of optogenetics otherwise known as channelrhodopsin. It is also receptive to blue light, which is the property we needed for the test anyways, so in this regard the genetics of channelrhodopsin were still fit for this test.
I then ran alignments of channelrhodopsin with each of the two red light kinases. In this process, the DNA of each of the two red light kinases was lined up to the channelrhodopsin kinase with the red representing when they differ. The goal is to have as little red highlight as possible.
As you will be able to see from the images, Chrimson was much more similar to channelrhodopsin. Based on this information I can hypothesize that it might be a better candidate for the job. However, it’s important to still test all candidates given the incredibly low success rates of protein engineering. The point of this pre-experiment testing was simply to form a hypothesis and have a general idea of how DNA varies between a blue light opsin and some different red light opsins.
The way the protein editing would be executed is with restriction enzymes. The specific domain of channelrhodopsin which makes it receptive to blue light would be cut out of the plasmid (from the channelrhodopsin kinase itself), and pieces of each different red light opsin kinase which make it receptive to red light would also be cut from their respective plasmids.
When restriction enzymes are used to remove DNA from a plasmid, one small strand of DNA called a sticky end is typically remaining on either side which is able to easily attach on to ends of DNA similar to it.
This is why the similarity of Chrimson is somewhat significant because it will enable the new DNA to latch more easily on to the sticky ends, and from there in the best case scenario one of the the Chrimson edited SOUL opsins is reactive to red light and ready to perform an optimized version of non invasive optogenetics.
How Optogenetics is Utilized as a New Approach to Treating Neurological Conditions
Now that we’ve gone through this journey together on why optogenetics needs to be rethought into a non invasive model at least for the time being to aid its development and level of societal acceptance (and then discussed exactly how to do that), let’s go into why this even matters at all. The reason being because optogenetics has potential to have incredible impact for people with a variety of neurological disorders.
As previously mentioned, SOUL specifically is utilized for excitation of neurons. Excitation is useful for treating conditions such as neurodegenerative disorders. The premise of these disorders which include Alzheimer's, Parkinsons, Huntington’s, ALS, and many more conditions caused by the degeneration of neurons. In other words, over time the ability of a certain group of neurons deteriorates significantly and as a result a group of the person’s functions are severely impaired.
Take Parkinson’s for example. This condition is caused by the degeneration of neurons called dopamine neurons in the substantia nigra, an area of the brain which controls movement. When neurons in this area stop working properly or even at all, a person experiences many adverse effects such as uncontrollably shaking hands, trouble balancing, difficulty standing, muscle rigidity, and involuntary movements. These effects present great challenges to the people who experience them throughout their everyday lives, and can be greatly impairing.
However, by utilizing an opsin such as SOUL which has excitatory properties, this can be addressed. When dopamine neurons in the substantia nigra are edited to express opsins and then activated as needed to excite the neurons, they force those neurons to be consistently active once again. Even if these neurons aren’t always active or able to be active on their own, optogenetics acts as a forcing function for them which makes them active and therefore combats both the declining ability of those neurons independently as well as the adverse effects a patient with Parkinson’s suffers from.
The incredibly precise nature and strong temporal control (you can control the exact amount of time optogenetics is performed on the brain with millisecond precision) of optogenetics enables not only a new way to treat brain based conditions, but a more precise way which could have a massively positive impact on millions of lives.
By redesigning this technology to be performed in a way that not only is less stigmatized by society but should also be easier to commercialize as soon as possible, millions of people will have access more quickly to this technology, and in a manner they actually feel comfortable using. The ability of the technology itself to have an impact will be equally as great, but there will be far less apprehension surrounding it which makes the impact far greater.
Optogenetics is the future of treatment for neurological disorders, but society isn’t ready for it in its invasive state. This new non invasive model will help it to proceed through a more natural evolution, and impact millions more people. This may seem like a step back, but really it’s just a readjustment from taking too many steps forward at once initially. This time the goal in sight is far more feasible for the time being, so it really can have the tremendous impact it was always meant to have.
Key Takeaways
- Optogenetics is an emerging neuroscientific technique which uses light and genetic engineering to control groups of neurons in the brain.
- It does so by editing neurons to express opsins and applying light delivered through an invasive brain computer interface to those neurons. This enables the opsins to manipulate the process of action potential (the firing of neurons).
- The invasive brain computer interface aspect of the system presents challenges both given that there’s a lot of societal stigma around it and since the lack of a non invasive model as a stepping stone to get to this ultimately invasive technology has stunted its development.
- In the past, two models for non invasive or minimally invasive optogenetics have been created. One uses SOUL opsins which don’t require a lot of light to be activated, but are activated by blue light which has trouble penetrating the skull. The other uses chRmine opsins which are activated by red light (can more easily penetrate the skull) but based on how much light ChRmine requires it can only work as deep as 7mm into the brain non invasively.
- I wanted to combine the successes of each of these two models by editing SOUL opsins to make them receptive to red light.
- The most likely successful genetic material to edit SOUL with to make it receptive to red light is from Chrimson opsins since they have similar genetic material. However, it is hard to tell since protein engineering has such low success rates, and this makes it important to test all potential candidates. The editing portion can be executed with restriction enzymes.
- Optogenetics is a useful technique because it can pioneer a new age of treatment for a variety of neurological conditions such as Parkinson’s, Alzheimer's, anxiety, depression, etc.
- Creating a non invasive variation of this technology has the potential to positively impact millions.
Thank you so much for reading my article, I hope you enjoyed it! My name is Raina Bornstein and I’m 15 years old. I’m passionate about branches of neuroscience and biotechnology, especially when they connect to treating neurological conditions. I’d love to connect on LinkedIn or Twitter, or you can reach out to me at rainabornstein@gmail.com to talk or collaborate. I can’t wait to hear from you!
