Bench philosophy: Multifunctional fibre for optogenetic research
Conductive Rubber Probes
by Steven Buckingham, Labtimes 02/2017
Recent advances in fibre-spinning technology are opening up amazing new opportunities for neuroscience.
The problem has always been that neurones rely on precise connections to do their job. That means that if you want to investigate their function, you would really want to be able to stimulate specific cells and record the effects on other neurones. But then there is the problem that neurones do a lot of their work in large groups. A memory, for example, is not stored in a single neurone but across thousands of them.
So, we need to be able to record from a lot of neurones at once. On top of that, the brain is a very complicated system, and you can't rely on just watching it work and drawing inferences. You have to be able to stimulate and control the behaviour of cells, in order to find out how the thing works. And there lies the huge challenge for neuroscience – the need to stimulate precise neurones and at the same time record individually from a lot of neurones at once.
Nice easy task, eh? No wonder we haven't yet figured out how brains work.
But help is on its way. Radically different kinds of fibres are being developed that allow researchers to deliver a light-sensitive channel into genetically-defined neurones, stimulate the target cells and make parallel extracellular recordings – all with a single fibre. What is more, you can leave the fibres in place in the brain for months at a time, with no adverse effects on the subject.
Why are optogenetics users so excited about these new fibres? First of all, optogenetics is really shaking up neuroscience. A lot of neuroscience is done by looking at how neurones fire during a behaviour or in response to a stimulus. But every undergraduate will remind you that correlation is not causation. Sometimes we fix that by stopping cells from firing (using really subtle approaches, like zapping entire bits of brain with a magnetic field or cutting bits of brain out).
But what neuroscientists would prefer to do, if they could, is to excite a specific group of neurones automatically and look at the downstream effects of stimulating them. The problem is that the brain has such a tight and precise architecture. Chances are, the cells you want to excite are all mixed in with other cells, making it hard to zap one without the other.
That's why neuroscientists are in love with optogenetics. Optogenetics is where two convenient tricks come together. The first trick is to find neurones that not only have similar functions but also share some kind of common genetic feature – such as a specific promoter. Happily, nature has provided us with a decent deck of promoters that define functionally meaningful subsets of neurones. The second is to hijack that genetic control to insert a light-sensitive channel into those cells, relying on the specificity of the promoter(s) to ensure that only a defined subset of neurones express the channel.
Polina Anikeeva's group at the MIT fabricated a flexible, rubber-like fibre that enables both optical stimulation and electrical recording in mice spinal cords. Photo: Chi (Alice) Lu & Seongjun Park
Once you have got that far, stimulating your cells – and only your cells – is in principle at least, very straightforward. All you have to do, is shine a light of the wavelength that excites the photosensitive channel and you can cause a depolarisation that excites the cell. Cells lacking the channel, remain unaffected. Switch on the light and watch to see what your animal does in response.
But there are a couple of problems. First, you have to get your construct into the right part of the brain, so that it can deliver its payload to the right target. Second, shining a light into the deeper parts of the brain is not quite so easy as it may sound. Your brain, and please don't take this personally, is optically dense. And how are we going to record from the neurones that are affected by said excitation?
So it looks like we need three fibres, one to inject the construct, another to act as a light stick and another to do the recording. One part of the solution is to make it all out of plastic: your light-stick, your injection needle and your recording electrodes – or conducting polymers, to be more precise.
Polymer fibres are actually amazingly versatile. You can fine-tune their optical, mechanical and electrical properties by tweaking their composition. Get the optical properties right and you have made your wave-guide for delivering the stimulating light beam. Make a hollow one and there you have your microfluidic channel for delivering your genetic construct. Finally, a conducting polymer will make a fine set of recording electrodes and you can make such a polymer by mixing in some carbon particles.
But the really clever part is getting around the problem of inserting three such fibres, so they all affect the same area of tissue. Just how to do this was shown in a recent paper by Polina Anikeeva and colleagues from the Bioelectronics Group at MIT (Nat Neurosci 20(4):612-19).
They took advantage of the fact that polymers can be spun out into fine fibres quite easily. To do this, you make a larger scale 'preform', which you then heat and draw out as finely as you need it, similar to the way sticks of seaside rock are made.
Anikeeva started with a concentric triple fibre preform. In the middle was a polycarbonate (PC) core that was to serve as the wave-guide. This was then wrapped in a thin cladding of cyclic olefin copolymer (COC). Next, they made the conducting polymer electrodes.
Admittedly, you can just buy in conducting polymers for this purpose but they usually come with a couple of problems. First, they tend to break down when they have been sitting in the salty, messy environment you find, even in the finest of brains. Second, they have an inconveniently high resistance. And this resistance issue becomes really problematic when you are trying to make multi-core fibres because you need them to be very thin (otherwise you end up with more of a cable than a fibre – so large, it damages tissue) and that necessarily means a higher resistance.
So, Anikeeva's team made their own polymer by loading it with five percent graphite. This cut the resistance four-fold, meaning they could mill a finer fibre.
Back to our three-ply fibre. Six of these conducting fibres are wrapped in a polymer layer around the wave-guide. This layer has a groove cut along it and that becomes the microfluidic channel, once another COC layer is wrapped around the outside. Finally, the whole thing is cased in an outer polymer layer, designed to get on well with the surrounding brain tissue. So, we have got our preform and at the moment it is about two centimetre in diameter. You then heat the preform to soften it, and then draw it out, just like melting and stretching a plastic syringe (try it, it is fun). The rate of stretching is controlled by an electronic sensor that measures the diameter of the fibre as it is drawn out and adjusts the speed of the stretch to keep it constant. You end up with a fibre of similar cross-sectional shape and with a diameter around 200 micrometer – several kilometres of it!
Now, there are several things that could go wrong with this. For one, can you be sure you can pump fluid down the microfluidic channels that are as thin as that? To test this, Anikeeva pushed their fibre into an artificial brain (nothing exciting, just a lump of 0.6 percent agarose gel) and found that they could squirt a dye ('Blue Juice') into the gel.
But what happens when the fibre gets bent – is it the same as bending my garden hose while I am watering the flowers? Even with the fibre bent through 90°, Anikeeva could still pump up to 100 nanolitres per second of Blue Juice and suck it all back up again through a parallel channel (that last point is important, as it means it is not getting pushed into other parts of the multi-core fibre).
What about the fibre's mechanical properties? Is it flexible enough to move with the tissue? A lot of long-term, multi-channel recordings are plagued by the constant movement of the brain relative to the skull, causing the electrodes to damage the surrounding tissue. Happily, the fibres made by Anikeeva’s team are much more flexible than the fine steel wires used for more traditional, chronic recordings and about 25 percent more flexible than standard silicate optical fibres. Such fibres remain in place with almost unchanged electrical and mechanical properties, even after three months implantation in freely-moving mice.
Does this approach work? First, Anikeeva used a construct consisting of a channel rhodopsin under the control of a promoter specific to excitatory neurones. They injected the construct into the prefrontal cortex and found that expression was detectable a few days after implantation. Pulses of light delivered through the wave-guide evoked action potentials, which could be clearly recorded using the conducting polymer electrodes. What is more, the behaviour of the mice (the paths they followed when exploring an arena) was modified when the optogenetic illumination was switched on.
One of the big advantages with polymer fibres over traditional optical cables or old-fashioned steel wires is their flexibility. This is important because it means the brain tissue does not get damaged as the mouse moves around, so you can record for a very long time. Anikeeva’s team was able to record from single neurones for up to 12 weeks and the level of scarring, as measured by expression of glial scarring-specific proteins, was much less than what you get with steel wire electrodes.
Anikeeva’s setup has one disadvantage, though. You have to run a cable from the recording/stimulating device to an interface. This means that the mice have to be effectively tethered, placing constraints on what you can do experimentally. Other groups have taken the obvious next step of using wireless technology. A group at the University of Illinois at Urbana headed up by Michael Bruchas and John Rogers, for example, described how they combined power supplies, wireless interfaces and cellular-scale LEDs with an array of elastomer microfluidics, to deliver fine-grained stimulation of neural assemblies.
Much of the increasing usefulness of polymer fibres comes not from quantum leaps in technology but from steady growth – tweaking an ingredient here, fine-tuning a parameter there. Anikeeva's lab isn't the only one to hit on the idea of combining electrophysiology with optogenetics, although they are the first to bring transfection into the one-step mix.
Combining optogenetics with electrophysiology will open up brand new avenues for neuroscience. Up to now, you had the choice between the high resolution but narrow field of view offered by electrophysiology, or the wide field of view but poor resolution offered by optical techniques. Polymer fibres, by joining the two approaches together, have bridged that gap.
Last Changed: 26.04.2017