Hello, my name is Karl Deisseroth. I'm a professor of bioengineering and psychiatry at Stanford and the Howard Hughes Medical Institute. And today, I'd like to tell you a little bit about optogenetics. Some of the humble origins of this method in science. Some of its applications and some of the interesting lessons it teaches us about the process of biology. Now, I'm also a psychiatrist and a lot of the inspiration for this technology came from psychiatry. And although this is an important field in medicine, it's got a long way to go. In many ways, we haven't progressed too far in our basic description of the core symptoms and their underpinnings, their causal underpinnings than we had hundreds of years ago. Here is a depiction of some of the earliest medical model type descriptions of depression and its symptom that today we call anhedonia, the inability to enjoy normally rewarding things. And John Haslam in the early 1800s described in melancholy, what we would call depression today, they therefore neglect those objects and pursuits which formerly proved sources of delight and instruction. And this is about as precise as we can be today about what anhedonia really is in psychiatry. And what's a really amazing part of this story is microbial organisms, single celled forms of life, have given us tools that in the last few years have allowed us to come to a deeper understanding of this fundamental and important medical and scientific question. And many others like it. Now this also gives an opportunity to take a broader view about what is optogenetics, where does it stand in the broader context of interventions and experiments that we could do in neuroscience. And in the brain, which as we all know, is a very complicated system with many interconnecting parts, we have historically had the ability to use either magnetic or electrical interventions to stimulate neurons. But one problem is that they cannot discriminate individual kinds of neurons that might be right next to each other. They all look the same to an electrical or a magnetic intervention. What optogenetics allows us to do is to put a particular sensitizers -- antennas of a sort, for external sources of energy and information into specific kinds of neurons. And then we can use light, in this case, if we make light sensitive antennas or transducers, and make neurons of a particular kind respond to light, and we do that to allow different kinds of ions to flow into cells and turn neurons on or off depending on the experiment. That's the fundamental idea of optogenetics and it's something people have wanted to do for a very long time. The first person who seemed to have framed the possible solution to this age old question was Francis Crick in 1999. He described the essence of this problem that we needed to control, turn on or off individual kinds of cells. And he suggested the ideal signal would be light. He didn't have an idea how to do it but in his usual perceptive fashion, he had some pretty good instincts, I would say. And what I'd like to do here is to sort of pay homage to the many people who've thought about this and done experiments, and contributed both historically and up to the present in making all of this ultimately work. Now there were early efforts dating back to 2002, a pioneer in this field, Gero Miesenboeck was able to take individual components from a metazoan eye, the multicellular organisms we all know of course, we have light sensitive cells in our retinas. And they achieved light sensitivity with a cascade of proteins, and he was able to get to the point where he could bring some of those individual components of that cascade together and confer light sensitivity on neurons. And the bright areas of this plot are where the light was on. And there were other groups who also took what you might call a multicomponent approach to try to make neurons light sensitive. Now the only thing that held back these elegant methods was in many ways their multicomponency which contributed to difficulties in targeting, and they of course would be the first state showed beautifully that some delay in the onset of the responsivity, the action potentials that were fired by the cells in response to the light. And what allowed this field to take off was the microbial approach as I alluded to earlier. Now these microbial organisms, they're small, they don't have a lot of space to work with and nor do they have to do as complex processing as other organisms do. And so they had the leeway evolutionarily, it seems, to make very efficient all in one single component systems for both detecting light and generating ion flow. And that allowed the generalizable aspect of this solution to happen. And this is a field that has its roots back, even as far back as the early 1970s. Dieter Oesterhelt and Walter Stoeckenius in 1971 identified bacterial rhodopsin and over decades they and a thriving community of researchers were able to sort out how this amazing protein works, how it responds to light, how its photocycle of the individual protein allows ions to be transported across the membrane. And this pump type class of opsins, bacteriorhodopsin being an example is followed by the discovery of halorhodopsin, where instead of protons being moved, chlorine ions are moved instead. Peter Hegemann and his colleagues beginning with some early predictive work in 1985 leading to a very important paper in 2002 showed that there were also channel type microbial opsins that opened actual pores in the membrane instead of pumping one ion at a time. And then many groups, here Feng Zhang in my laboratory was able to find a red light activated channelrhodopsin in 2008. And a great number of different groups, Feng Zhang, Peter Hegemann, our colleague Ed Boyden, Georg Nagel, Ernst Bamberg, John Spudich, and many others have been identifying amazing, amazingly diverse microbial opsins with different properties from across the phylogenetic spectrum. Now, what is striking as you'll notice from this, is how long we've known about these and this happens to be my biochemistry textbook The beautiful book written by Lubert Stryer in 1988, this was the version that I used, and you can see this is textbook type knowledge. Descriptions of the photocycle of bacteriorhodopsin, for example. Part of the foundational knowledge of biochemists and neurobiologists. Now there were people who did try to put these microbial opsins in different settings, in fact, successfully. This is a very interesting paper from 1994, where the bacteriorhodopsin was indeed transferred along with light sensitive flow of ions into a eukaryotic organism. But nobody had yet tried to actually create a tool for neurobiology, to turn on or turn off individual kinds of cells. Neurons are complicated, they're vulnerable, they're sensitive, they're different but in principle, it was an experiment that could be done. And there were many people trying to do it. Now the path to this in my own lab started very near to the time when I had started setting up my independent laboratory. And I had a very humble approach, where as you can see I used very simple tallies to assess how the experiments were working, and it's interesting to look back on the simplicity of this early experiment. But what I was doing here, I had a broader view in the lab to try different kinds of proteins to see if we could turn neurons on or off with different kinds of stimuli. I had acquired the channel rhodopsin gene, I'd written to a group in Germany that had worked on the initial clone from Georg Nagel and he sent me the clone right away, which was very generous of him. I was also looking at different kinds of potassium channels, to see if we could turn on or off, constitutively active or dominant negative potassium channels. And I was putting all of these into separate groups of neurons in culture to see if I could get control over their activity level. And I was using a very simple straightforward read out of membrane depolarization, the phosphorylation of a transcription factor called Creb, which I had done a lot of work on previously. And the amazing result was that in these neurons, you can see by the red staining of the nucleus of the cell in a stimulated compared to an unstimulated neuron. Both of these were expressing the channel rhodopsin coupled to a yellow fluorescent protein, so I could see a number of things here. I could see that the gene that I'd put in was being expressed, it was being transported to the membrane, that the neuron was healthy, it hadn't exploded as a result of putting this high levels of this membrane protein into the cell. Which can happen to be sure. And moreover that the light activated, the light treated neuron revealed that it had been stimulated. That its membrane had been depolarized and calcium flux had occurred. And this biochemical readout was telling us that this was happening. And in neurons that were expression a lot of this channel rhodopsin, I could see a big difference in the number of cells that were reporting on this depolarization event, and overall, this was statistically significant. And although this was a very simple, cheap, humble experiment, this showed us that it was possible. And this was in early July of 2004, but things went very quickly after that. This is what our small group looked like at the time, you can see the two grad students, Feng Zhang and Ed Boyden, now at MIT. Feng Zhang now also very well known for his work on CRISPR-Cas9 systems. But we knew that we had to do a lot more than just show some neurons getting stimulated, we had to show that this was unique, that it was a potentially generalizable or versatile, that it could potentially be applied in broader systems. And there was rightly so a lot of skepticism at the time. What the students did was some really brilliant and creative work. Feng Zhang designed the viral systems that would allow us to put the channel rhodopsin in a very versatile way into different brain regions in vivo, and the fact that it's just a single gene allowed us to be confident that this would work well. Ed did a number of beautiful recordings, showing how fast the process could be, which was another important feature of the system. Both its potential generalizability and its speed. And there were many other groups working on this at the time, Stefan Herlitze, Zhuo-Hua Pan, Yawo's group in Japan. But we were able to put the different pieces together in time to get the paper out, and it was published together with two students, Ed and Feng, as well as Georg Nagel and Ernst Bamberg. And this, though, was, I wouldn't even say this was yet a fully optogenetics because there was a great deal more work that needed to happen. It took about five more years of very intense effort to show that we had actually created something that was generalizable, useful, and broadly applicable, which was the key goal. So optogenetics didn't start overnight, experiments were humble and simple, but it took a number of years to actually say that we finally discovered how to make this work. Part of the problem was how do you get light into big mammalian brains. And this was an early sketch of Feng Zhang's, where he designed the fiber optic interface. And this was from 2006, but then this looks very much like how we now do it today. So as you can see he's a good artist and a great scientist. And this fiber optic interface has since allowed us to do many things, but I'll show you one other first experiment. This was two years later in 2007, Feng was working with some mice that we had made in collaboration with Guoping Feng and George Augustine, and these were mice designed to have channelrhodopsin expressed in the brain and what Feng did was he implanted into this mouse, a fiber optic on the right side of its brain, which as you know would control movement toward or attention to the left side of the world. And when he turned on the light, as you can see here the animal immediately starts moving, starts turning to the left. And this was the moment I would say we finally knew we had made something that was going to be broadly applicable and generalizable. And that was followed quickly by a collaborative set of experiments with Luis de Lecea, Alex Aravanis, Antoine Adamantidis, and others, where we applied this fiber optic interface to target a deep structure in the brain, in lateral hypothalamus. Feng designed some promoters that would fit into a lentivirus and allow us to control just one kind of cell deep in the hypothalamus. And with this work, we were able to play in certain kinds of activity and show some of the neural codes that were causal in sleep-wake transitions. And then, even then though this required some very specific fragment of a promoter, the hypocretin promoter, which would fit into a virus. And there was still greatly so some skepticism as to how broadly generalizable this would be. But between 2009 and 2010, we finally were able to show that there were a broad array of different tools you could use to generalizably target different kinds of cells. And by this point, I would say by 2010 or so, people were able to see that this could be applied to virtually any system. A good example of how that all worked was in our studies in anxiety. Now anxiety is a normal part of life, it's a healthy part of life, of course it can become excessive and it can be pathological in some cases. But what we were able to do is to use optogenetics to play in patterns of activity or suppress patterns of activity in particular cells or projections in and around structures relating to the basolateral amygdala, and the bed nucleus of the stria terminalis. And the way we did some of these experiments is we injected a virus, an adeno associated viral vector into the structure of interest, the GNST, but then we were able to position our fiber optics in other downstream structures. By doing so, we were able to recruit cells to find by having a particular projection pattern, which as you can see is a generalizable strategy. You don't need a special kind of animal or a special promoter, you just need to know your anatomy. And this works because the opsins are trafficked down the axons of the cells, particularly if we help them out with little trafficking motifs that were discovered in the lab by Viviana Gradinaru and her colleagues. And I'll show you an example of how this sort of thing works. This is an experiment, I'll play this movie, Kay Tye in the lab's work which was published in 2011. This is a what's called an elevated plus maze, this so-called closed arm has these elevated walls and you'll see that the animal prefers to spend its time in the closed arm of the maze. Very rarely venturing out into the open arms of the maze, which is like walking the plank for the animal. Both in our studies of anxiety related behaviors, what we found and what Kay was able to show, and we followed this up with a great deal of other work, was that there were anti-anxiety pathways buried within the brain. When the blue letters come on here, you'll see when she started driving one of these, as we discovered, anti-anxiety pathways. And you'll see for the first time, the mouse is willing to go out into the open arms and explore, which it never had done before. Nothing else changed about the animal, the speed or anything else, it simply was willing to accept this risk. And this was instantaneously reversible and this of course was only one feature of anxiety, apprehension in the absence of immediate threat. But there are other features, too. Respiratory rate changes, subjective feeling, anxiety feels bad and resolution of anxiety feels good. And in later work, we were able to show different projections coming from the bed nucleus of the stria terminalis recruit different features of anxiety using this projection targeting approach, where we position the fiber optic in a different location than the injection of the virus. And this sort of thing allowed the generalizability of optogenetics to become clear. And so, in this sort of work, if you think back to those very early humble experiments in culture, you can see it took a number of years to finally put all the pieces together. I'm often asked what's the potential clinical impact of optogenetics, and although I'm a psychiatrist, I run a purely basic science laboratory. I'm not working on clinical translation in my lab. But it's a natural question to ask, and other people are working on it. The group of Antonello Bonci found in 2013 that they could turn down cocaine seeking behavior in rats with optogenetic stimulation of the medial prefrontal cortex. A very striking result that they published in 2013. And that was then followed up with Antonello Bonci and some clinical colleagues with a optogenetically guided clinical intervention. They used transcranial magnetic stimulation, which can be non-invasively delivered to awake human beings. Of course, human brains are a lot bigger but they have a lot of the same basic reward circuitry and what they found was guided by optogenetics, they were able to have a group of cocaine addicted human beings no longer seek cocaine. They had an experimental group and a control group, and they had a very powerful effect. This sort of thing shows how far we've come, that we can directly affect with a principle-guided approach, these very powerful and important fundamental behaviors in human beings. And this makes, of course coming back to the initial inspiration that we have starting to go down this path, now we do understand in a causal and precise fashion many of the cells and connections across the brain that do affect these very important and both basic and disease relevant behaviors. Now this, of course, had very humble beginnings, as you recall, those initial tallies in the lab notebook. And we've come a long way since then. But in a way, it's even more striking when you think how these very simple organisms themselves have created these tools that let us make this progress. There's a whole other world that has happened as well, in terms of discovering how these basic microbial opsins work. We've been able to adjust the speed of the opsins. Their sensitivity to light. We've been able to engineer them to conduct different kinds of ions to turn cells on or off. Guided by getting to the crystal structure of one of these channel rhodopsins. And we can change the color that they respond to, as well. This has allowed us to do something very important which is to tune the amount and the timing of the activity that we play into precisely what's naturally occurring in the organism, even that same individual during a very well-defined behavior. And so this elegance and precision of the method is all the more striking when you think back to how crude some of the early experiments were. And how long it took to get the whole system working. But in the end, that's what makes biology fun and beautiful. And I hope to have conveyed some of that to you today.
