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To assay neurons change in charge, the most commonly used method is to insert a metal electrode in a petri dish in brain slices, or directly into the brain of live animals, the researchers used this method has been more than 50 years. For a long time, science professor at Boston University School of Medicine and other researchers David Farb also gradually expand the potential of these methods. "I started earlier electrophysiological study, the use of a vacuum tube amplifier." Farb explained, "a researcher may take a whole day to get the record of a cell, and may have no work the next day, so the advance was very slow."
After a series of long-term improvement, Farb and his colleagues are now able to complete the activity in the entire area of the brain in animals, while normal activities. Although this work is still tedious.
Each experiment, Farb laboratories have produced a multi-layer device. First, they want to mount a four electrodes called "tetrode" devices, roughly equivalent to the diameter of a human hair. Giessen, Germany's Thomas Recording Corporation and is located in Alachua, Florida, the Tucker-Davis Technologies, sales prefabricated tetrode and related equipment, but with limited resources can not buy this type of equipment researcher often produce their own these devices. Farb explained that he had a small shop, where all undergraduates in constant production of such disposable tetrode, "because we can not afford them."
However, whether the purchase or production tetrode, using them requires patience, as well as sophisticated surgery skills. Twenty or thirty tetrode connected to the top called a "pre-filter", the device contains a micro-drive (installation of control tetrode bracket). There are some small auger, researchers have only been screwed into a short day, to avoid injury to brain tissue micro drive. This process allows the tetrode slowly into the target area of the brain in rats or other animals.
For multiplexed electrode can control the people, it can generate valuable data. Farb's laboratory has tracked the "place cells" in the network, showing how animals navigate in different environments, this finding can explain everything, from the most basic learning mechanism in the pathogenesis of Alzheimer's disease.
Unfortunately, due to the mouse size too small to accommodate standard equipment in this area, so the animals were conscious electrode analysis will generally be limited to non-human primates, and occasionally can be achieved in rats. Team Leader is located in Ashburn, Virginia, the Howard Hughes Medical Institute Janelia research park Joshua Dudman want to change this situation. "I was really go now deceased grandfather, he was a mechanical engineer designed carburetor, we exchanged some ideas." Dudman said.
Located in Bethesda, Maryland, United States National Institutes of Health (NIH) National Eye Institute of the distinguished scientists who monkey Robert Wurtz developed a similar system, he made some additional recommendations. With Wurtz proposal, plus the cocktail napkin to draw program, Dudman began to explore the brain of mice produced a standard system. His team used 3D printers to produce mounting bracket for the fixed electrode and holding the head of the animal. Their collaboration with another research group Janelia Research Park, followed by improvement in the past based on a silicone chip electrode matrix design, and then create a rodent in vitro / in vivo electrophysiology targeting system (RIVETS). As the name implies, RIVETS using a single, standardized components, it can be studied in live animals or brain slices. It also can take advantage of two-photon microscopy to synchronize imaging.
In addition to the part of the 3D printing, RIVETS also use most of the Neurobiology Laboratory are available ready-made instrument. "One big question is how do you combine it with the other part of the instrument. If we want to be accurate positioning, we do not want to re-create the micromanipulator, because a lot of good companies have produced very high-end products. "Dudman said. Micromanipulator is the result of precision machining equipment, usually together with the microscope stage using the joystick on the handle or allow investigators to move the team points, moved into a position such as the electrodes.
Scientists can download the 3D files and other information printed on Dudman website and from the use of such British East Sussex Scientifica Sutter Novato California company or companies micromanipulator. Dudman went on to say, Scientifica company recently purchased a non-exclusive license to sell sets of RIVETS systems tend to have products commercially supported researchers.
Dudman is not the only who wants to build a better mouse helmet man. "Our main innovation is to produce in the laboratory, but also ultra-light version of the ultra-stable chronic electrophysiological implants way." Located in Providence, Rhode Island, associate professor of neuroscience at Brown University Christopher Moore said.
For the team that wants to study the brains of mice, the weight is critical. "Genetic engineering and in vivo mammalian systems revolution is a huge gift, but it comes from the mice, but they are little." Moore said. He used the new array explains, "Now you can only physiology experiments conducted in rats or monkeys completed, but has been able to better adapt to the mice."
Explore the brain
Whether it is used for in vivo or in vitro, the wire can usually be measured charge variation between neuronal tissue. Patch-clamp as a complementary technology that uses tiny micro capillary to track fluctuations in single neurons in ion channels.
For the first time people using patch-clamp technique, the first step should be recalled that basic electronics. "Do patch-clamp work, eventually you will be seen as a neuron electronic device, an electronic device to learn how this works, and how to apply these principles in biology, I think this is extremely important." Assistant Professor of Biology, University of Oklahoma, Norman, Oklahoma Michael Markham said. Markham is maintenance free software package called "neuronal electrophysiology" for assisted this process.
In all the challenges in electronics, Markham noted capillary deeper into the animal brain, its resistance and capacitance is higher. This means that the deep probe and probe shallow compared with less bandwidth.
The capillary implants patch clamp experiments brain of live animals, the desired action is very difficult, requires special care surgical technique. Cambridge, Massachusetts, the Massachusetts Institute of Technology (MIT) Synthesis of neurobiology research group leader Edward Boyden decided to do the work to the robot. "We developed an algorithm that allows you to patch clamp on neurons operate, and can also make use of computer automation. Without relying on human intuition." Boyden said.
Boyden team the algorithm used in a robot body, slowly inserted into the target area of the patch clamp capillary animal brain until it detects the resistance increases, which means that the capillary has encountered a neuron. Subsequently, the robot can attach capillary detect not only the activity of ion channels in neurons, but also for biochemical analysis Cytoplasmic extracts.
Posted this technique, the researchers also created a Neuromatic Devices company currently sells automated patch clamp devices, easier for people to copy the technology. Boyden also want to live animals to other aspects of neurophysiology automation. "We believe that we stumbled on the field that people can be called 'living Robotics', we can deploy a range of technologies in this midst, and automate this process and to provide solutions." He said.
Meanwhile, Moore's team is also trying to make the electrode physiologically more convenient. Moore lab graduate student Jakob Voigts create a standard electrophysiological equipment group open source license. The program is called Open Ephys, researchers can only make the equivalent of thousands of dollars in parts, you can assemble a sophisticated neurophysiological devices. Online support can help users to solve any problems that arise.
Whimsy
In addition to make it easier to detect neuronal activity outside, Boyden also helped pioneer in the experimental method to find new ways to activate neurons. This innovation stems from his failure in the traditional pharmacological and electrical stimulation methods, both methods are not very precise. "You can not activate part of specific neurons." Boyden says
Through literature search, he and his colleagues found a good clue. "In the spring of 2000 during our brainstorming, we inadvertently noticed a few so-called microbial opsin article." Boyden explained. The team is particularly interested in the fact that, as the light-sensitive protein, can open ion channels on the cell membrane of microorganisms in the light reaction. When the researchers modified neurons in animals through genetic expression to which a protein, neurons begin light pulses exhibit activity. Since then, Boyden's group and others began to continuously improve the technology - called "light Genetics", this technology has become a standard tool in live animals to explore the neurophysiological and brain slice.
Light genetics latest developments include the red-sensitive channel protein (able to dive into the brain tissue), and a light-sensitive chloride channel (capable of inhibiting nerve in the light of the dollar rather than the reaction of the neurons). Clever mouse genetic techniques, scientists can also express light-sensitive channel is defined in very specific brain areas. The combination of these techniques, researchers are now able to pass through the optical mouse complete skull, and highly accurate activation or inhibition of target neuronal populations. Boyden said, "The tool has really started to get very regular use."
Optical tricks may further explore the possibilities of light genetics. For example, researchers are able to take advantage of multi-photon holograms in an animal brain activate individual neurons. "You can really try to control neural encoding of complex three-dimensional configuration, which will then be used to detect neural coding in very specific assumptions." Boyden explained.
At the same time, light is also very good genetics and electrophysiology integration. A major challenge to the traditional wire electrode is placed in the position of the electrode found in the animal's brain, using light Genetics, researchers can now determine this information. "When you turn on the light, you can clearly see whether or not you use the metal electrodes separated neurons (right type)." Brown University, Moore said.
Moore's lab and even figured out how to combine light Genetics and another neuroscientist favorite tool - Functional magnetic resonance imaging (fMRI). By revealing changes in blood flow throughout the brain, fMRI can be given of stimulus-response profiles. Light genetics now able to provide very accurate stimulation. "You can point to a specific cell type-specific stimuli, for example, say representatives of the hands of the neocortex area; then you can use fMRI to say, 'When I stimulate such cells that location, the What brain regions are activated? ' "Moore added," this is a terrific message. "
In addition to its advantages, there are some flaws light genetics. For most accurate cell activation, the researchers still have to go through an optical fiber inserted into the skull in the mouse brain. Central University of Michigan associate professor of Neuroscience, Mount Pleasant, Michigan, the Ute Hochgeschwender are trying a different strategy. "Avoid using physical optical way is, if we can basically use a biological source." Hochgeschwender said.
Hochgeschwender and her colleagues luciferase fusion protein with a fluorescent light-sensitive opsin protein domains and fireflies. Obtained phosphor opsin (luminopsins) in the photoreactive ion channels open, and providing the appropriate in luciferase substrate can emit light when, in the light emission and detection under a fluorescence microscope. Researchers may apply to animals injected with luciferin substrate to activate the targeted neurons, fluorescent tags will reveal which cells are the expression of these proteins or by light. "Any opsin, any elements developed optical genetics, we can offer another dimension chemically obtained." Hochgeschwender said.
In the most recent iteration technique, Moore and Hochgeschwender are using calcium sensitive luciferase, cooperation also need to create a substrate and calcium ions to activate the system. So that the flow of calcium emitting neurons cytoplasm, so "you can imagine such a system, in the case of the presence of substrate, the real neuronal activity opens bright." Hochgeschwender explained.
Then, researchers can identify the light emitting cells, even while maintaining intact skull case. Hochgeschwender prospect of a new strategy - the scientists conducting the experiment before it is possible to find out which is the correct mice cells expressed proteins exclude inappropriate expression patterns, thereby actually reducing the number of animal experiments required.
However, whether scientists or bioluminescent enzyme electrode, a new generation of scientists and technology deal could not conceal the excitement of a flood of new developments coming. Like Farb said, "Now I feel like a child, we are constantly produce results, I would say, 'wow, I can not believe this, I never thought about it, I could never imagine it . ' "■
(High sea-based Institute of Oceanology one translator assistant researcher)
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