A Clear Vision

 

Wed, 12/17/2014 - 9:29am Paul Livingstone For Karl Deisseroth, R&D Magazine’s Scientist of the Year, his two major scientific breakthroughs are just a means to an end: understanding the human brain. At his Stanford Univ. laboratory, Deisseroth leads a team of 30 or more researchers in projects involving both optogenetics and CLARITY-based techniques. Image: Stanford Univ. Around 400 BC, Hippocrates was among the first people in recorded history to postulate the brain as the seat of sensation and intelligence. Yet only in the last 100 years have we identified, and closely studied, its key building block: the neuron. A highly specialized cell found in all but the simplest animals, like sponges, the neuron is one of the keys to understanding the brain. Until recently, studies of the nearly 85 billion neurons in the adult human brain were dependent on imaging methods that either lacked enough optical resolution to study individual neurons, or required the use of dead, frozen, thinly sliced samples placed under the beams of electron microscopes. The results could be useful, but could never provide a comprehensive view of an intact mammalian neural system. Enter R&D Magazine’s Scientist of the Year, Karl Deisseroth. In 2005, just a year after establishing his first laboratory, the D.H. Chen Professor of Bioengineering and of Psychiatry and Behavioral Sciences at Stanford Univ. demonstrated success in applying a technique that allowed direct and precise optical control of individual neurons, which by 2007 he developed into methods applicable to the intact living brain. And in 2013, after spending years improving this first big breakthrough, dubbed “optogenetics”, he worked with his trainees in the lab to fulfill another long-time wish of brain researchers. Together with his expert team, he published the first method for turning brain tissue transparent while making its biomolecules accessible to labeling. Called CLARITY, it allows full brain imaging and molecular analysis, without disassembly, for the first time. These innovations have considerable merit on their own, but their importance will have more to do with the research they stimulate. Since the appearance of optogenetics, researchers in thousands of laboratories around the world have applied these techniques to answer a wide variety of questions in brain science. And in just over a year since his laboratory introduced CLARITY, the technique has been further refined and applied to formal research in hundreds of laboratories, with many resulting publications. The creation of these tools, coming at a time when brain research has taken center stage in biological research, has put Deisseroth in a unique position to contribute significantly to the future of neuroscience. A circuitous path “Very early on, I was definitely interested in understanding the brain. My father was a physician, an oncologist, and he understood the value of reduced systems and getting to the biochemical basis of things,” says Deisseroth. “I was genuinely interested in looking at the intact brain with cellular and molecular resolution.” He arrived at Stanford in the early 1990s eager to work with Richard Tsien, a prominent neuroscientist. Despite Tsien not having any laboratory space for another lab member, Deisseroth overcame his lack of experience to embark on a long-term effort to understand neuronal calcium channel signaling, a path that began with a spot in Stanford’s Medical Science Training Program. Clinical training was a part of that program. “The route I first envisioned, neurosurgery, was exciting. I loved the operating room, it was exciting for me,” he says. Neurons operate by transforming and sending electrical and chemical signals through a complex cellular process. The potential use of light to access and control this natural process was first suggested by Francis Crick, who theorized that light signals would have ideal properties to control the action potentials of specific cell types. “He proposed that if we could use light to control cell types, we could ascertain their function,” says Deisseroth. “A lot of people read that and started trying to work on it.” One of the key tools Deisseroth used in this research, microbial opsins, were actually discovered in 1971. The basic mechanism for neuronal firing is a series of rapid ion flow changes in response to neurochemical events. Microbial opsins also move ions, but in response to photons instead. But it wasn’t believed, nor was it suggested or conceived by Crick, that these proteins from ancient forms of bacteria, which respond to light in a way that is reminiscent of how neurons fire, could be repurposed to sense photons and create ion flow changes in behaving mammals. Sourced from such a remotely related organism, it was assumed these opsins wouldn’t express well and would likely require the addition of cofactors to establish control. As a result, other strategies not involving microbial opsins were attempted by several other researchers, such as Gero Miesenböck at the Sloan-Kettering Cancer Center and Robert Kramer at the Univ. of California, Berkeley. Deisseroth was more optimistic. He anticipated that opsins couldn’t only control neurons, they could do so in a temporally precise fashion. To do this, he initially experimented with an opsin called channelrhodopsin, sourced from algae. He explored how it accomplished the conversion of light into chemical signals through a phenomenon called the phototransduction cascade. Understanding this complex chemical chain of chemical activations and structural changes in molecules caused by photoexcitation is key to harnessing their power to activate neurons, and even after reaching competence in this domain Deisseroth struggled for years to achieve the results in his core domain, “behaving” mammals. But other methods not using microbial opsins hadn’t been practical or adopted by other labs either. The core goal remained to be solved. Deisseroth’s microbial opsin research came after he had concluded a decidedly different pursuit: a residency in clinical psychiatry. The draw to this discipline had motivated his desire to develop technologies, and to find a different way of understanding and possibly treating disorders of the brain. It was also, he explains, a valuable exposure to the variety of expressions of brain activity dysfunction. His residency, simultaneous with a fellowship under Prof. Robert Malenka at Stanford, had exposed him not only to psychiatric theory, but also to real people who demonstrated disorders of the brain daily without any real hope of improvement. He grew to appreciate the vastness of the divide between neuroscience and clinical psychiatry. “That was really transformative for me, to get that experience of seeing and treating people with different psychiatric diseases. To witness their suffering and to see how different their reality was, it showed me how vast was the set of unknowns,” says Deisseroth. Unlike much of his work in the laboratory and in the operating room, psychiatry doesn’t easily create an approachable path, he says. “There’s a lot of mystery there. Inadequate treatments are common, and there’s no way of getting into the complex structures of the human brain.” But what psychiatry did do was give Deisseroth extra drive in his opsin research. In 2004, he formed the Deisseroth Lab with the intention of making microbial opsins useful in neuroscience. He enlisted several talented graduate students to his group, including Feng Zhang, who has since gone on to further fame with his discovery of genome editing with the CRISPR-Cas9 system. In a flurry of work over the next year, Deisseroth’s team achieved a breakthrough that resulted in a 2005 paper published in Nature Neuroscience. Deisseroth had, the previous year, found that microbial opsins could be safely expressed and functional in the cellular membranes of mammalian neurons in culture. This major step was made possible by learning how to leverage gene delivery methods—basically infecting brain cell cultures—to effectively express opsins and place them in a position to stimulate mammalian neurons using high-speed photoswitching. In 2013, Deisseroth’s team published a paper that described the first successful effort to remove lipids from brain tissue, leaving neurons and proteins intact for 3-D fluorescent imaging. Image: Stanford Univ.Not long after, Deisseroth and his team did just that, delivering flashes of blue light to cultured rodent neurons in a petri dish. The team was surprised at how well it worked when all of the required elements were brought together, including molecular, viral, optical and electrophysiological methods. But this was only in a dish, and was not “optogenetics” yet, as Deisseroth would soon call control of specified events in defined cells in behaving animals with millisecond precision. As with any new method, says Deisseroth, a number of bugs had to be ironed out. “We had to overcome about five or six major challenges to really make optogenetics work,” says Deisseroth. “The goal with the early research was slow to be achieved, to show that light sensitivity can be conferred, by microbial opsins, to defined cells in behaving mammals via a robust, practical approach.” In 2010, Deisseroth’s team eventually solved the key problems of opsin targeting, expression in mammals and deep brain light delivery. Optogenetics gained traction in research settings as more robust opsins were developed. “Now, we’ve gotten even better at guiding light,” says Deisseroth. Today, clones of effective opsins are sent to thousands of laboratories, and development of better tools continues. Tissue transformation As optogenetics developments continued, Deisseroth tackled another longstanding problem: visualizing structural detail throughout the entire intact brain. High-resolution information about complex biological systems is difficult to obtain. Optical solutions rarely penetrate more than a few tenths of a millimeter in living samples, and special sample preparation techniques are required with seemingly inevitable sample damage. Deisseroth’s dream was to do what had been thought impossible—creating transparent tissue without disrupting it or losing information. He recruited to his laboratory, as a postdoctoral fellow, Kwanghun Chung, a chemical engineer by training. With the help of Chung and other colleagues, Deisseroth’s team converted fully intact brain tissue into an optically transparent form, supported by a nanoporous and macromolecule-permeable hydrogel. Cross-linked to 3-D network of hydrophilic polymers, this transformed tissue retains almost of all of the brain’s original neural wiring information and biomolecules, including neurotransmitters, nucleic acids and subcellular structures. His 2013 paper describing the method goes on to describe the use of lightsheet microscopy to image the transformed samples, as well detail potential clinical tissue applications. Although developed more than 100 years ago, lightsheet microscopy had not been widely adopted in biology until a few years ago. Because CLARITY removes lipids, it allows the “sheet” of laser light to scan through the sample with far less scattering. “One of the issues that we faced early on in developing CLARITY was the fact that commercially available light-sheet systems were engineered to analyze samples at about 10- to 20-µm resolution. For neuroscience, we needed to study individual axons, which meant an order of magnitude increase in resolution, so we had to build our own system an order-of-magnitude better,” says Deisseroth, and this achievement became known as “CLARITY-optimized lightsheet microscopy”. Compared with his work to develop to optogenetics tools, the pace of development for CLARITY has been far more rapid and more quickly applied by others. He credits this progress in part to the availability of better instrumentation, but also with the effectiveness of the team he brought together to make it happen. Deisseroth is quick to stress that neither technology represents a treatment in its own right, even when they do enter clinical settings. But the potential, in fundamental research and commercial domains, is considerable. In 2013, the U.S. government committed hundreds of millions of federal dollars to map the tens of billions of neurons in the human brain. One of the BRAIN Initiative’s top advocates, Deisseroth is now one of the 15 advisory committee members. Innovations like CLARITY and optogenetics tools will likely contribute significantly to progress in this effort. Origene di questo articolo : www.rdmag.com