Lights, Genetics, Action – what are the uses of optogenetics in the field of neuroscience?

Optogenetics is a useful tool employed in many areas of biomedical research including and not limited to neuroscience. But does it have the potential to be a clinical weapon? Since the emergence of this technique, the field of optics in science has evolved. As technology changes science has to keep up and with audacious and revolutionary tools such as optogenetics, it is clear that this is happening. But how useful is it really?

What is optogenetics?

It is a new and revolutionary technique used in bioscience for clinical and laboratory function. Breaking down the word gives more meaning; opto meaning the use of light and genetics is reference to the genome. The basic principle of optogenetics is the use of specific wavelengths of light to control and alter a neurone’s firing potential, be it inhibitory or excitatory. The tools involved in the technique (opsins) are actually proteins that are naturally found in microbes, which when activated by light transport the appropriate ions (Boyden, 2011).

This may in theory seems uncomplicated at first glance, however when you delve into the complicated physics and chemistry of the idea, it is not so simple. These opsins, as mentioned, are naturally found in microbes – not humans. This is where genetics and bioengineering are consulted. In order for there to be any clinical use of opsins in humans, then there has to be an efficient way to alter a person’s DNA in order for them to express these opsin proteins. This is a major challenge currently inhibiting the use of opsins in patients.

However, opsins can be used in the lab setting as research. Transgenic mice can be genetically engineered to express the opsins in different parts of the body. This allows scientists to research the consequences of switching certain channels on and off, thereby allowing them to learn more about a disease, condition or anatomy.

There are actually three types of channels used in the technique (Fig.1). Firstly, are the Bacteriorhodopsins which were discovered by Oesterhelt and Stoeckenius (1971; 1973) in the 1970s and were found in the Halobacterium salinarum bacterium. As shown in the diagram, when activated by green light, protons are pumped out of the cell. The next opsin was discovered in the 1980’s and when exposed to yellow/orange light, inhibits the neurone by causing an influx of negative chloride anions (Boyden, 2011). The final and most commonly used opsin is channelrhodopsin. This opsin, when activated by blue light is excitatory, and allows the influx of various cations into the cell, causing depolarisation. This was discovered in Chlamydomonas reinhardtii (a type of algae) (Sineshchekov et al, 2002), and was found to initiate and control phototaxis (Boyden, 2011).

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Fig.1: diagrams showing the three opsin channels used in current research and how they work when the appropriate light is used to stimulate. The colour of the channels represent the light waves used to specifically stimulate the channel. Diagram from (Boyden, 2011).

How does optogenetics work?

First of all you need to use bioengineering in order for the opsin to be expressed in the genome. As Fig.2 shows, the gene for the opsin in question is coupled to a promoter region, allowing for the protein to be expressed via DNA transcription and translation.

Once the genetic construct has been made, it is then introduced into a viral vector. The virus takes up the genetically modified construct and is merged into its genome. The virus is then injected locally to the target neurones only, in the rodent. Here the virus takes on its normal biological function and hijacks the DNA machinery in the rodent, causing it to transcribe the opsin gene. A fibre-optic electrode is then implanted into the rodent and once turned on, it stimulates the opsin channel.

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Fig.2: a summary of how optogenetics works, from the bioengineering to the stimulation of the opsin channel. http://optogenetics.weebly.com/why–how.html

To allow for variability as well as co-occurrence of different opsin channels, each channel is only stimulated at a certain wavelength of light or colour of light (Fig.3). As shown, channel rhodopsin (ChR2) is stimulated by blue light, in the range of roughly 470nm, whereas channel halorhodopsin (NphR) is stimulated by yellow light in the range of roughly 590nm.

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Fig.3: a graph showing the activation wavelengths of various opsin channels. ChemistryViews, 2013.

Once stimulated, the opsin channel opens, leading to an influx/efflux of positive or negative ions depending on the channel’s function. Taking channelrhodopsin as an example; the optrode is turned on, allowing light to shine on the channel and causing it to open. When the neurone is at rest at roughly -70mV, there is a larger concentration of cations on the outside of the cell with respect to the inside, creating a biophysical gradient as well as a concentration gradient. These two factors favour an influx into the neurone, leading to cellular depolarisation and the generation of an action potential. The optrode, when turned on, causes the neurone to fire. This, alongside a whole population of neurones, concurrently depolarise leading to signal generation (summarised in Fig.4).

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Fig.4: diagram summarising the molecular events that occur once the opsin channel (channelrhodopsin used as example) has been stimulated by light. The black arrows indicate biochemical and electrophysical gradient and the direction in which the cations flow. The red arrow is used as a reference point on the action potential trace. +/ symbols represent cations and anions respectively.

Uses of optogenetics in the clinical field

A specific reaction occurs depending on the population of neurones, the location and which pathway you want to control. This is particularly useful for researchers as they can study specific neural circuitry and pathways that may be involved in disorders.

Using optogenetics in the treatment of stroke

The selective stimulation of pyramidal neurones in layer V of the primary motor cortex (M1) was shown to induce the repair processes following a stroke in a study conducted by Cheng et al in 2013. Optogenetics was used in the study in order to test whether specific stimulation of a pathway could help stroke-inflicted mice. Not only were the results promising, but you could say groundbreaking. They found that vs non-optogentically-stimulated mice, the stimulated mice performed significantly better in a range of biochemical as well as behavioural tests.

Fig.5 only provides a snapshot to what they found; Fig.5a shows a significant increase in change in CBF (cerebral blood flow) between stimulated and non-stimulated mice. The reason why CBF was measured is because it is a hallmark for repair. When the brain is damaged, an innate reaction called the neurovascular response kicks in and tries to help alleviate the damage. The % change in CBF was used as a function for repair, because a change indicates that this recovery response is occurring.

There are other hallmarks of repair and recovery, which is shown in Fig.5b. There was a significant increase in neutrophic mRNA expression in either the contralesional primary motor cortex (CM1) or the contralesional somatosensory cortex (CS1). The neurotrophins tested all play a part in repair; Brain Derived Neurotrophin Factor (BDNF) is responsible for inducing axonal and dendritic sprouting; Nerve Growth Factor (NGF) controls the growth of nerves and Neurotrophin3 (NTF3) is involved in survival and differentiation of cells.

Finally, Fig.5c shows results from a distance behavioural test. There is a clear significant difference between distance travelled for stimulated and non-stimulated stroke mice, which if the mice can travel further, indicates recovery from the stroke infliction. If these data can be replicated with a better sample, such as increased number of participants or a change in species tested, then this holds a great potential use in stroke patients.

5aA

5b B

5cC

5dD

Fig.5: figures a. and c. taken from Cheng et al’s study, figure b. is a summarised figure of data from the same study. a. = shows the % change in cerebral blood flow (CBF) between stimulated and non-stimulated mice at post-stroke day 15; b. = graph showing the mRNA content of different neurotrophins in stimulated mice in either the contralesional primary motor cortex (CM1) or the contralesional somatosensory cortex (CS1); c. = a behavioural distance test focusing on stimulated and non-stimulated mice.

 

 

Using optogenetics in the treatment of amnesia

A truly fascinating and useful study using optogenetics was conducted by Ryan et al in 2015, in which they used the technique to study engram cells. These cells are believed to play a part in the consolidation of memory and are changed by learning information (storage) and are reactivated once appropriate retrieval conditions are in place (Tonegawa et al, 2015). This theory is used as the basis behind amnesia in this study. The authors wanted to employ optogenetics in order to stimulate these engram cells, in order to retrieve a memory that should be lost due to disruptions in long term potentiation (LTP) protein synthesis by administrating an inhibitor (anisomycin).

As a control, a group of mice were injected with saline, yet were still optogentically altered to respond to pulses of blue light due to ChR2 presence. The mice were habituated with the optoelectrode and after habituation, they were shocked in order to induce contextual fear conditioning (CFC). This is when the mice associate a previously non-harmful situation/environment to a harmful one due to its association with an electrical shock for example. The mice were then habituated again and optogenetic stimulation induced freezing behaviour in both the anisomycin group (ANI) and the saline group (SAL). Mice were then returned to the chamber they had been shocked in, in order to provoke CFC. Freezing behaviour occurred in both groups and the two groups were then injected with either ANI or SAL. 3 days following the injection, the two groups of mice were returned to the CFC chamber, where the SAL group showed significantly more freezing behaviour (P < 0.01) than ANI, suggesting that ANI had disrupted further memory consolidation of the noxious environment. 4 days later, both groups of mice were stimulated in a non-harmful environment and remarkably, both groups showed significant light-induced freezing behaviour (SAL = P < 0.001 and ANI = P < 0.003) (summarised in Fig.6).

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Fig.6: experimental paradigm from Ryan et al’s 2015 study showing the effects of ChR2 activation by optogenetics on contextual fear conditioning freezing behaviour.

This showed the researchers that by specifically stimulating these engram cells with optogenetics can ‘re-form’ a lost memory. This has massive implications in not only amnesia research, but possibly with disorders such as Alzhemier’s disease, where lost memories can maybe be brought back. The implications are immensely promising, however the logistics and practicality of engineering the technique in humans is a big problem.

Using optogenetics in the treatment of Parkinson’s disease

Parkinson’s disease is a disorder that is characterised by movement deficits, particularly the infamous ‘shuffling gate’. The disease is believed to be caused by the destruction of dopaminergic (DA) neurones within the substantia nigra pars compacta (SNc), leading to atypical neuronal activity in the basal ganglia (BG) (Gradinaru et al, 2009). It is specifically the subthalamic nucleus (STN) and globus pallidus pars interna (GPi) where the irregular neuronal activity leads to the abnormalities including difficulties in initiating and executing motor movements (Albin et al, 1989; Alexander and Crutcher, 1990; DeLong, 1990).

The main advantage of using optogenetics is its selectivity of certain neurones. Deep brain stimulation is a technique used in Parkinson’s disease treatment; however its selectivity for neurones is robust and not very accurate, leading to undesirable effects. This is why Gradinaru et al conducted an optogenetics study in 2009 that primarily targeted the STN by stimulating NpHR opsins, which inhibited the excitatory network. The authors studied the effects of optogenetic stimulation of afferent axonal projections that entered the STN.

Depending on the frequency (be it high or low) determined the therapeutic effects of stimulating the STN afferents using optogenetics. Based on rotational behaviour and head position bias tests, high frequency stimulation (HFS) of the afferents showed remarkable effects (Fig.7). The stimulation ameliorated the parkinsonian symptoms immediately, with significantly reduced rotations per minute (decreased rotations is a sign of Parkinson symptom decrease) compared to pre and post optogenetic HFS. LFS also showed the exact opposite effects.

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Fig.7: the effects of High frequency and Low frequency stimulation (HFS and LFS respectively) on rotational behavioural tests. Note that the Pre and Post indicate before and after optogenetic stimulation. *P < 0.05, n = 5; ***P < 0.001, n = 5 based on t-test.

Based on previous research (Li et al, 2007; Degos et al, 2008) the primary motor cortex M1 and the STN connections may underline the malformed circuitry in Parkinson’s disease. This is why the authors then looked at using optogenetics in the M1. They found that by stimulating from the M1 and recording from the STN (Fig.8a) that similar results were found in reference to LFS and HFS on the rats’ behaviour (Fig.8b).

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Fig.8: a = diagram showing the optogenetic stimulation of the M1 with simultaneous recordings taken from the STN; b = rotational beam behavioural tests at different stages of optogenetic stimulation. **P < 0.01, n = 5 based on t-test.

 

This study helped to show that the selective optogenetic stimulation on certain pathways at high frequency stimulation can help ameliorate Parkinson symptoms, more so than deep brain stimulation. Though hard practically, using optogenetics as a substitute for DBS looks to hold more benefits in treating the movement symptoms.

Using optogenetics in the treatment of Alzheimer’s disease

Alzheimer’s disease (AD) is a lethal neurodegenerative disorder that is the leading cause of dementia and is characterised by memory loss and gradual loss of functional and cognitive processing (Roy et al, 2016). As the disease progresses, neural atrophy gets worse leading to the associated symptoms. Post-mortem brains of patients that suffered AD and those that did not show complete disparity in its structure and general appearance (shown in Fig.9).

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Fig.9: pictures A and B are from patients that did not have Alzheimer’s disease and pictures C and D are from patients that did have the disease. The white arrows are present to compare one picture from another. Note that ventricles in picture D are almost non-existent due to the atrophy. Pictures from Soto-Rojas et al, 2015.

The aforementioned study regarding optogenetic stimulation of engram cells in treating amnesia serves as a basis or as previous research to a study conducted in 2016 by Roy et al. This team built on the ideas and theories found by Ryan et al, as they too used optogenetics to stimulate engram cells (in the hippocampus) in order to retrieve lost memories that occur in the early stages of Alzheimer’s disease (AD).

The team used an AD model which included the over-expression of the delta exon version of presenilin 1 (PSEN1) and also had a mutation in the β-amyloid precursor (APP) leading to increased plaque formations. As well as this model, the team successfully rescued memory recall by optogenetic stimulation in two further models; transgenic mice made from mating c-Fos-tTA mice together (which also had the APP/PSEN1 mutation) and the other model used was a commonly used AD model known as MAPT (Roy et al, 2016). They found that stimulating 7-month-old AD mice could induce memory recall.

It is important to note that a reduction in dendritic spine density has been suggested to play a part in the memory decline of AD patients. Furthermore, the authors detected reduced density of the dentate gyrus (DG) engram spines in 7-month old AD mice against the control (Fig.10).

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Fig.10: the pictures show the difference in spinal density between control mice (left) and the 7-month old AD group (right). There is a clear reduction in the number/density of the dendritic spines in the AD group.

The authors also induced long-term potentiation (LTP) by using optogenetics in order to help consolidate a memory and restoring spine density. Fig.11 shows how after optogenetic-induced LTP, the AD mice group that also had 100 Hz showed an increased freezing response relative to normal AD group. This suggests that optogenetics induced the LTP, which helped the mice recall contextual memory.

This study is relatively recent, yet holds many potential useful future projects, as this way of tackling AD is unique and different from the standard way of administering Acetylcholinesterase inhibitors, which are relatively useless.

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Fig.11: outline of natural memory recall test in which rodents were contextually trained to noxious stimulus and

Future for optogenetics: a conclusion

Optogenetics is a brilliant and innovative tool that has clearly shown promise based on the studies mentioned. The exciting prospect of this tool is that it will only get better and more expansive, as over the next few decades it will surely be used in frontline medicine, as well as in research. The brilliance of using optogenetics in order to try and regain a lost memory is second to none, and is surely a step forward in combating Alzheimer’s disease; a problem that is on the rise in the western world due to increased health procedures.

Though it is very useful in the lab doing research on circuitry, it is harder to employ in in vivo subjects, as the practicality of bioengineering is no easy task. This surely is the biggest obstacle facing optogenetics. Once a way has been found e.g. a possible mixture of implantation by surgery or even using lentiviral vectors, then optogenetics will surely be one of the greatest tools in modern medicine.

 

Author: Ryan Gourlay

References

Albin, R.L., Young, A.B. and Penney, J.B. (1989). The functional anatomy of basal ganglia disorders. Trends Neurosci, 12, 366-375.

Alexander, G.E. and Crutcher, M.D. (1990). Functional architecture of basal ganglia circuits: neural substrates of parallel processing. Trends Neurosci, 13, 266-271.

Boyden, E. S. (2011). A history of optogenetics: the development of tools for controlling brain circuits with light. F1000 Biology Reports3, 11.

Cheng, M.Y., Eric, H., Wanga, E.H., Wooson, W.J., Wanga, S., Suna, G., Leed, A.G., Araca, A., Fennoc, L.E., Deisseroth, K. and Stainberg, G.K. (2014). Optogenetic neuronal stimulation promotes functional recovery after stroke. Proceedings of the National Academy of Sciences, 111, 12913-12918.

Degos, B., Deniau, J.M., Le Cam, J., Mailly, P. and Maurice, N. (2008). Evidence for a direct subthalamo-cortical loop circuit in the rat. Eur. J. Neurosci, 27, 2599-2610.

DeLong, M.R. (1990). Primate models of movement disorders of basal ganglia origin. Trends Neurosci, 13, 281-285.

Gradinaru, V, Mogri, M, Thompson, K. R, Henderson, J. M. and Deisseroth, K. (2009). Optical deconstruction of parkinsonian neural circuitry. Science, 324, 354–359.

Li, S., Arbuthnott, G.W., Jutras, M.J. Goldberg, J.A. and Jaeger, D. (2007). Resonant Antidromic Cortical Circuit Activation as a Consequence of High-Frequency Subthalamic Deep-Brain Stimulation. J. Neurophysiol, 98, 3525-3537.

Oesterhel, D., Stoeckenius, W. (1971). Rhodopsin-like protein from the purple membrane of Halobacterium halobium. Nat New Biol, 233, 149–52.

Oesterhelt, D., Stoeckenius, W. (1973). Functions of a new photoreceptor membrane. Proc Natl Acad Sci U S A, 70, 2853–7.

Roy, D.S., Arons A., Mitchell, T.I., Pignatelli, M., Ryan, T.J. and Tonegawa, S. (2016). Memory retrieval by activating engram cells in mouse models of early Alzheimer’s disease. Nature, 531, 508-512.

Ryan, T.J., Roy, D.S., Pignatelli, M., Arons, A. and Tonegawa, S. (2015). Engram cells retain memory under retrograde amnesia. Science, 348, 1007-1013.

Sineshchekov, O.A., Jung, K.H., Spudich, J.L. (2002). Two rhodopsins mediate phototaxis to low- and high-intensity light in Chlamydomonas reinhardtii. Proc Natl Acad Sci U S A, 99, 8689–94.

Soto-Rojas, L.O. et al. (2015). Neuroinflammation and Alteration of the Blood-Brain Barrier in Alzheimer´s Disease. Chapter 3.

Tonegawa,S., Pignatelli, M., Roy, D.S. and Ryan, T.J. (2015). Memory engram storage and retrieval. Current Opinion in Neurobiology, 35, 101-109.

Optogenetics procedure picture: Available from: http://optogenetics.weebly.com/why–how.html.

Wavelengths of light picture: Available from: http://www.chemistryviews.org/details/news/1360543/Optogenetics__Revolutionizing_Biochemistry.html

 

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