Manipulating the brain with light: overview of optogenetics

Summary #06. Brain stimulation and imaging methods #2 — Optogenetics

If you are a neuroscience researcher, machine-learning engineer, brain-computer interface enthusiast or just curious about these exciting topics — this educational summary is for you.

I will cover:

1. What is optogenetics
2. How does it work
3. What can it be used for
4. Advantages and drawbacks
5. Possible development options

⚡ Shortly

Optogenetics is a set of methods aimed at genetically modifying neurons to interact with light. Light-sensitive proteins allow for both stimulation and recording of neuronal activity with high spatial and temporal precision.

🚀 Motivation

Developing new methods for studying and manipulating brain activity is essential for neuroscience researchers — to understand functioning of neural circuits (“neural code”) and neuroprosthetics engineers — to develop brain-computer interfaces.

In a perfect world, we would have an imaging device that would read activity of every single cell of the brain with high temporal precision, i.e. enough to detect spikes. Stimulation method, similarly, would have to be allow us to manipulate every cell individually.

However, most of the currently available methods have certain limitations. Some only allow to record activity of neuronal populations (EEG, MEG, ECoG, Stentrode), some lack temporal resolution (fMRI, fNIRS), some are damaging to the tissue (microelectrodes).

Discovery of light-sensitive and light-emitting proteins, which link light to brain electrical activity, in combination with genetical modifications and optical devices aim to overcome some of the limitations of aforementioned electrophysiological methods.

Optogenetics allows for precise and well-timed manipulation of brain activity.

⚙️ How does optogenetics work?

But really, how can one record and stimulate neuronal activity with light?

1. Light-sensitive proteins

Electrical activity in the brain is mostly mediated by ion-channels — proteins which allow different ions to flow through the cellular membrane. Ion-channels and transporters are proteins.

Biologists of XX’s century discovered special proteins — microbial opsins, which are ion-channels and can be activated with photons — light particles (Figure 1).

Figure 1. Opening of an ion channel (e.g. chanelrhodopsin) by absorption of a photon (light particle)

Different ion-channels can be permeable to different ions, to produce excitation or inhibition (Figure 2). Nature has already created a number of them in bacteria. So, the first essential component is to find such light-activated ion-channels, which are suitable for the research purposes, i.e. activate, inhibit or allow only a certain ion to flow through the membrane to produce certain effect.

Figure 2. Different electrical effects (excitation/inhibition) elicited by light activation mediated by different light-sensitive proteins.

2. Genetic modification of neurons

The next thing to consider is how to deliver these proteins in the neuronal cells?

The answer is — genetical modification (Figure 3). Proteins are coded by DNA sequences, so it is possible to modify neuronal DNA with additional gene coding the necessary protein. Gene insertion is usually done via viruses, which are naturally acting as genome modifiers. In our case, an empty virus is loaded with a necessary DNA-sequence coding a light-sensitive protein and subsequently injected into the brain. In result, the cell is producing light-activated ion-channel itself!

Figure 3. A general pipeline of making neurons activated by light.

One of the most interesting features of optogenetics, is that it is possible to modify only a specific type of neuronal cells.

Neurons are not all similar, they can express different proteins and have different electrical/chemical/anatomical/genetical features.

For example, some neurons excrete dopamine — that means they have specific genes enabled to code dopamine-synthesizing proteins. If a virus is designed to target these “fingerprint” gene sequences, optically activated proteins will be inserted only in a desired population of neurons.

3. Electrical activation with light

Now, when the selected neurons express necessary proteins it is time to make them fire (activate). Light delivery is usually done via light diode/laser or fiber optic, implanted into the brain tissue (Figure 4, 5).

Figure 4. A mouse with implanted optic fibre.

Figure 5. Schematic illustration of optic fibre implanted through the skull.

The magic of optogenetics is in the details. Of course, you can use a flashbulb (with a necessary light wavelength). However, in that way every modified neuron will be activated. This method is usually called one-photon or whole-field illumination (Figure 6A).

Two- or multi-photon illumination are capable of targeting single-cells or even cell compartments: soma, dendrites — one by one by a focused light beam (Figure 6C).

The other methods include using arrays of photodiodes to induce certain illumination pattern (Figure 6B), or wavefront shaping (Figure 6D) — they allow to activate a subset of cells simultaneously.

Figure 6. Different illumination methods. A — whole field illumination (single-photon), B — array of photodiodes, C — multiphoton excitation of single cells (one by one), D — digital holography, wavefront shaping

🔬 What optogenetics is capable of?

• Enables investigation of causal relationships between activity of neuronal populations. It makes possible to measure direct effect of activation of a neuron or a population of neurons (like magnetic and ultrasound stimulation, but with better resolution);
• Possesses specificity for the fast timescales of neural computation — we can stimulate neurons precisely in time and space;
• Allows naturalistic behavior paradigms (moving animals doing stuff with optodes implanted);
• Can be easily combined with calcium imaging by adding the camera to the setup. Ca++ imaging setup shares the same principle as stimulation via opsin activation, but utilizes different protein, which emits light when interacts with Ca++ ions. It allows to see activation of single neuronal cells (Figure 7).

Figure 7. Light-emitting activated neuronal cells, captured by a high-resolution sensitive camera

• With precise stimulation of neuronal activity enables opportunities for closed-loop feedback device (Figure 8)

Combining fast readouts via electrophysiology (voltage-recording electrode) with optogenetics creates the possibility of closed-loop optogenetic interventions, in which optical stimulation is guided by real-time readout of ongoing activity

Figure 8. An example of closed-loop setup with recording of activity with electrode, tuning parameters controlling stimulation and configuring light delivery.

💡 Experimental insights:

• Paz et al. (2013) recorded neuronal activity in the thalamus and cortex to detect seizure initiation in epileptic animal. It was possible to stop the seizure by optogentically silencing (inhibiting) thalamocortical neurons!
• Other study showed that optogenetically stimulating previously identified nucleus accumbens dopaminergic neurons can increase/decrease risk-seeking behavior (Zalocusky et al., 2016)
• Stimulating neurons, which produce serotonin, increased “patience” — willingness to wait (Miyazaki et al., 2014)
• Brain-to-brain interface (upcoming summary)
• Closed-loop fast intracortical brain-computer interface (upcoming summary)
• Retinal prostheses can be made with optogenetic modification of retinal ganglion cells to restore sight (Busskamp et al., 2012)
• A ton of insights for understanding brain function, circuits and computation. Development of optogenetics was awarded with a Nobel prize!

❓ Pros and cons

Advantages & Features 🤩:

• Immediate and reversible effect
• Possible single-cell resolution
• Very high time-resolution
• With fiber optic electrodes (optodes) it is possible to target deep brain structures
• Fully implantable
• Cell-type specific (by activity, by neuronal type, by input, by output)

Disadvantages & Pitfalls 🙁:

• Requires invasive procedure to access the brain tissue via optode
• Requires genetic modification
• Substantial regulatory requirements: FDA approval for genetic modification and use in humans
• With more neurons precise single-cell excitation becomes slow, because stimulation is done in one-by-one manner
• Possible tissue damage by implanted optical devices
• Possible heat damage — energy transmission restrictions — less neurons can be safely stimulated
• For most brain areas, there is no mapping of information — we don’t know how exactly to stimulate to transmit meaningful information into the brain

🧬 Possible development & Perspectives

Invasive procedure is one of the greatest issues regarding implementation of optogenetics in primates and humans. In my view, there are two possible ways to overcome it.

First, develop sonogenetic approches. Sound can be focused to a narrow brain area without a need for cutting the skull (see Summary #02 — tFUS). At the same time, there are proteins, sensitive to the sound (Ibsen et al., 2015).

Second, is to use light of non-visible frequency, which would be capable of penetrating the skull. This might be done using up/down conversion phosphors, which absorb light of one frequency and emit photon of different energy (Matsubara & Yamashita, 2021)

Overall, optogenetics seems to be one of the few approaches potentially capable of full-brain imaging and precise, timed stimulation of single neurons, providing a vast field of opportunities for neuroscience research and brain-computer interface development.

📜 Sources & Further reading

Optogenetics: 10 years of microbial opsins in neuroscience (Nature, 2015)

Integration of optogenetics with complementary methodologies in systems neuroscience (Nature Reviews Neuroscience, 2017)

Prospects for Optogenetic Augmentation of Brain Function (Frontiers in Systems Neuroscience, 2015)

If any of mentioned approaches and experiments are interesting to you, let me know in the comments, I will try to make a short summary about it.

Now that you know basic principles, opportunities and limiations of optogenetics, summaries concerning it will become much shorter! This was a big, tutorial-like methodological summary to clear everything up before jumping into contemporary stuff.

😉 Stay tuned and see you in our medium publication:

the last neural cell

Author: Alexey Timchenko

Collaborator: Alexander Kovalev

Our telegram channel: the last neural cell