March for science

Where did the concept come from?

Francis Crick in 1979 had first believed that progression of neuroscience was restricted by our inability to independently control one type of brain cell, while others were unaffected. Electrical stimulation is not able to do this because it will stimulate all nearby cells without discrimination. Drugs are too slow to act in comparison to the millisecond timeframe of events in the brain. He suggested one can use light to solve this issue, as it can be turned on and off very quickly. As far back as 1971, microbial biologists had identified the presence of light-activated proteins that control transmembrane ion flow and subsequently influence excitation or inhibition of cells. However, a way to make brain cells sensitive to light was not known at that time.

How does hypothesis become true?

Optogenetics is an intricate yet beautifully simple technology that has transformed the work of scientists in over 800 laboratories around the world since 2005 when the full potential of microbial-opsin optogenetics was first realized.

Zhuo-Hua Pan is a vision scientist at Wayne State University in Detroit, and was the one who invented optogenetics first. Pan was determined to cure blindness. In the early 2000s, he conceived that putting a light-sensitive protein into the eye could bring back vision in the blind — compensating for the death of photoreceptor cells (i.e. rods and cones) by making other cells light-sensitive. In earlier 2004, he was trying to take out channelrhodopsin from ganglion cells and cultured in a dish. They became electrically active in response to light. Pan applied for a grant from the National Institutes of Health (NIH) and received three million USD with the comment that his research was “quite an unprecedented, highly innovative proposal, bordering on the unknown.” Pan didn’t realize that he was racing against research groups across the United States and around the world to put channelrhodopsin into neurons at the same time. Deisseroth and Boyden were working at Stanford, where Deisseroth was completing a postdoc and Boyden was finishing graduate school. The Stanford group had been playing with the idea of controlling neurons with light for quite some time. Deisseroth was in touch with the Georg Nagel who had discovered the channelrhodopsin in March 2004, and requested Nagel to collaborate and share channelrhodopsin DNA so that Boyden could attempt to implement it on neurons. In August 2004, Boyden shined light on a brain neuron in a dish and recorded electrical activity from the channelrhodopsin. Boyden, who is now a professor at MIT, was surprised when told by STAT (also called Stat News) that Pan conducted the experiment first. “Wow. Interesting. I didn’t know that,” Boyden said. Pan replied he might have mentioned the timing of his experiment to Boyden once several years ago, but, Pan said, “I didn’t want to take too much time to talk about this because people feel uncomfortable.”

In 2005, Deisseroth's lab in Stanford published the article "Millisecond-timescale, genetically targeted optical control of neural activity" in the journal Nature. It was the first research paper to completely define optogenetics as a strategy to make neurons sensitive to light and then use light to stimulate them. They showed how, by merging optical skills and breakthroughs in genetics, it is possible for researchers to excite or inhibit specific neurons within the brain and study the outputs. One of the supreme aids of optogenetics is that it can be used to study live, freely moving animals. Karl Deisseroth at Stanford University and Ed Boyden at MIT have collected tens of millions in grants and won millions in prize money as inventors of optogenetics.

How do neurons transmit signals throughout the body?

Neurons are unique because they can send information from the brain to the rest of the body. Your brain communicates with the rest of your body by sending messages from one neuron to the next and ultimately to the muscles and organs of the body.

Fig. 2: A. An action potential is a rapid rise and subsequent fall in voltage or membrane potential across a cellular membrane with a characteristic pattern. Sufficient current is required to initiate a voltage response in a cell membrane; if the current is insufficient to depolarize the membrane to the threshold level, an action potential will not fire. (Image: molecular device); B. Synapses are tiny gaps between neurons, across which the neurons talk to each other. An action potential here (yellow lightning) causes neurotransmitter to be released. The neurotransmitter travels across the gap to activate receptors on the receiving neuron. (Image: Alan Woodruff / QBI)

An action potential (Fig. 2A) is the process that occurs during the firing of a neuron which is also known as nerve impulses, or spikes. When a neuron is not sending signals, the inside of the neuron has a negative charge relative to the positive charge outside the cell. Electrically charged chemicals known as ions maintain the positive and negative charge balance. Calcium contains two positive charges, sodium and potassium contain one positive charge and chloride contains a negative charge. Ion channels are protein molecules that span across the neural membrane of allowing the passage of ions from one side of the membrane to the other. During the action potential, part of the neural membrane opens to allow positively charged ions inside the cell and negatively charged ions out. This process causes a rapid increase in the positive charge of the nerve fiber. When the charge reaches +40 mV, the impulse is propagated down the nerve fiber. This electrical impulse is carried down the nerve through a series of action potentials. When a neuron spikes, it releases a neurotransmitter, a chemical that travels a tiny distance across a synapse before reaching other neurons (Fig 2B). Any time a neuron spikes, neurotransmitters are released from hundreds of its synapses, resulting in communication with hundreds of other neurons.

How does optogenetics work in nature?

Green algae Chlamydomonas reinhardtii uses photosynthesis in order to generate the energy it needs to live. These processes become efficient through an eyespot. This light-sensitive part of the cell communicates an unassuming single-celled alga which direction light is coming from so it can move into a better position. C. reinhardtii moves ions across a membrane through ion channels in order to activate the eyespot. When light of the correct wavelength hits these channels, it causes a change in their shape; opening them so that ions can flow across the membrane. The most frequently used ion channel for stimulation in optogenetics is Channelrhodopsin-2 which serves as sensory photoreceptors in unicellular green algae, controlling phototaxis: movement in response to light .

How are neurons engineered to express opsins?

Neurons are activated in the same way, by moving ions from outside the cell to the inside. When a certain number of positive ions have crossed the cell membrane, a threshold is reached which causes the neuron to fire. Light-activated ion channels are expressed on neurons within the brain by genetic manipulation.

At the back of the eye is the retina, a layer of cells that are photosensitive (they respond to light). These photoreceptor cells contain photopigments, light-sensitive molecules that are made up of a protein called opsin. Photopigments change shape when they detect light, triggering a series of chemical reactions and sending a signal to the visual cortex of the brain telling it how much light there is at each point on the retina. It is this that creates a picture of the outside world. Type I opsins (also known as microbial opsins) are found in all three domains of life: Archaea, Bacteria, and Eukaryota. In Eukaryota, type I opsins are found mainly in unicellular organisms such as green algae, and in fungi. In most complex multicellular eukaryotes, type I opsins have been replaced with other light-sensitive molecules such as cryptochrome and phytochrome in plants, and type II opsins in Metazoa (animals).

Efficient delivery and expression of opsin genes is critical for achieving spatiotemporally-resolved cell type–specific manipulation. This can be achieved in multiple ways. One popular method is to permit for tight control over spatial localization of opsin expression, through the use of genetically modified virus to express ion channels within neuron cell. These viruses comprising an opsin gene (for example channelrhodopsin-2 ChR2) are driven by a specific promoter that is injected into the brain region of interest to recombine its DNA with the DNA of the host cells and cell will express the ion channels protein on its cell membrane. Light trigger these ion channels to open and ions enter the cells and cause them to fire (Fig 3).

Spatially targeted and temporally controlled illumination is required to activate the microbial opsins which will modulate the membrane potential or cell's signaling. Two types of microbial opsin exist which can be used to explore neuronal systems; the channelrhodopsin, which excites neurons by causing depolarization when exposed to blue light and halorhodopsin, which inhibits action potentials in response to yellow light (Fig 4). These proteins can easily be introduced into target cells by various techniques, letting scientists to quickly and precisely turn individual neurons on and off without the need for additional drugs or chemicals.

Light-emitting diode (LED's) offer a cost effective, application specific tool for illumination and become the first choice for researchers in place of conventional discharge and incandescent lamps in microscopy. To study the effect of photosensitive proteins elicited by illumination, several methods can be used from electrode recordings of membrane potentials to behavioral studies of free moving animals.

What can we use this for?

Optogenetics approach can be used to transfer light-sensitive transporters or ion channels to retinal neurons to cure retinal diseases like Age-related macular degeneration (AMD) and retinitis pigmentosa. In those cases, retinal neuron cells survive long after photoreceptors have degenerated so one possible way to bring back the light sensitivity in the retina is by using an artificial light sensor. Animal studies have fruitfully reestablished vision in previously blind rodents by ectopic expression of melanopsin which is a type of photopigment belonging to opsin protein family and microbial opsins in surviving cones or bipolar cells.

Ionotropic glutamate receptors are ion channels which are activated by the neurotransmitter glutamate and play a vital role in memory formation and learning. Artificially engineered light-gated ionotropic glutamate receptors have also been inserted into retinal ganglion cells of mice with retinal degeneration, achieving the return of light sensitivity. While animal model indicates the worth of optogenetics for inherited ocular disease, clinical trials are needed to explore its utility in humans.

Learning and memory involve neural interactions happening on the order of milliseconds at specific synapses. Electrophysiology permitted recording of neural processing at a sustaining time resolution, but manipulation of inputs was spatially unspecific (electric stimulation) or extremely slow compared to neurotransmission (pharmacological activation or inhibition). Optogenetics overcomes these two major boundaries as this tool has revolutionized the study of learning and memory from the behavioral to molecular level.

Spines (of neuron networks) may emerge, disappear or change in size during learning and memory formation. A neuron receives excitatory signals from other neurons through dendritic spines. When a mouse learns a new task, such as running on an accelerating rotating rod (a rotarod), spines involved in learning this task become potentiated (new spines form and existing spines increase in size). Hayashi-Takagi et al. developed an 'optogenetic construct' based on a light-activatable form of the small signaling protein Rac1, which targets recently potentiated dendritic spines. Blue light activates the modified Rac1, which induces shrinkage of the spines. Ju Lu and Yi Zuo showed that spine shrinkage caused the mouse to forget the skill it had learnt, so it soon fell off the rotating rod (Fig 5). These efforts help us to gain an understanding of the interesting phenomenon of memory simply by shining a light on its physical basis. It would be exciting to develop technology that erases memory. One potential application is to erase “bad” memories — such as trauma experienced in patients with post-traumatic stress disorder (PTSD). Work on this has already been initiated.

Dr. Christine Denny at Columbia University established that they could recall memories in a mouse model suffering from Alzheimer’s disease. They were able to “tag” neurons permanently under certain conditions through genetic programming. The network of neurons connected with one another actually stores long-term memories and activating all of these neurons at the same time could “remind” the mice of that memory. Optogenetics technique can “tag” this set of neurons, stimulating it later to restore those connections and return the memory.

Future direction

Optogenetics (Fig. 6) was elected as the “Method of the Year” across all fields of science and engineering by the interdisciplinary research journal Nature Methods and was also emphasized in the articles on “Breakthroughs of the Decade” in the academic research journal Science in 2010. Although just more than a decade old, optogenetics is already responsible for enormous progress in neuropsychiatric disorders, and its future is undoubtedly bright.

The current development of optogenetic tools over traditional method of electrodes stimulation allow neurons to be turned on or off with bursts of light — promises to develop the study of how neurons function individually and as members of larger networks, and could eventually offer new hope for patients suffering from vision impairment or neurological disorders such as epilepsy or Parkinson’s disease.

Optogenetics will remain to be a priceless tool for clarifying our understanding of complex biological functions; it is also exciting to realize that optogenetics is still in its relative infancy and the casual control it offers for targeted small-scale events has limitless possibilities for future biological and medical advances. For example, light can be used to block pain signals. Large numbers of people worldwide experience chronic pains. All modern medicine has to offer are painkillers, which often cause patients to develop drug addiction or side effects.

Scientists are using the new tool of optogenetics for better understanding in the field of memory research, and it shows a great deal of potential. Even though the construction and demolition of memories may sound perilous, it also has great therapeutic potential for a wide variety of neuropsychiatric diseases. Who knows maybe one day we’ll just go to the doctor to receive our “laser treatments” to combat the memory loss associated with old age?

*Taniya Chakraborty is PhD student in Max Planck Institute for Polymer Research, Mainz, Germany

Reference

[1] Storey, Philip, Lisa Hark, and Julia A. Haller. "Age-related macular degeneration: an overview." Handbook of Nutrition, Diet and the Eye . Academic Press, 2014. 11-20.

[2] Boyden, Edward S., et al. "Millisecond-timescale, genetically targeted optical control of neural activity." Nature neuroscience 8.9 (2005): 1263.

[3] Beyeler, Anna, Christine A. Eckhardt, and Kay M. Tye. "Deciphering memory function with optogenetics." Progress in molecular biology and translational science. Vol. 122. Academic Press, 2014. 341-390.

[4] Lu, Ju, and Yi Zuo. "neuroscience: Forgetfulness illuminated." Nature 525.7569 (2015): 324.

[5] Perusini, Jennifer N., et al. "Optogenetic stimulation of dentate gyrus engrams restores memory in Alzheimer's disease mice." Hippocampus 27.10 (2017): 1110-1122.