Special Report: Tools For Neuroscience's Third Decade

Neuroscientists are entering the federally designated "Decade of the Brain" armed with a toolbox bulging with new instruments and techniques. Many have been borrowed from other disciplines; others have been specifically designed to probe the secrets of the nervous system. The variety of tools available reflects the diversity of the researchers who consider themselves neuroscientists. "Neuroscience represents a fusion of several scientific disciplines--biophysics, biochemistry, physiology, anatomy, pharmacology, and psychology--all focused on acquiring an ultimate understanding of the relationships between brain and behavior," explained the National Advisory Mental Health Council to Congress in a 1988 report on opportunities for neuroscience research. The field came into its own only two decades ago, and has grown explosively. When it was founded in 1969, the Society for Neuroscience comprised 500 original members. Today, the society's rolls hold 17,000 names, with 15,000 people expected to attend this week's 20th annual meeting in St. Louis (October 28-November 1). More than 250 exhibitors at this year's meeting are expected to display products of use to the scientists attending, diverse not only in the disciplines they represent, but also in the levels at which they study the nervous system--ranging from molecules to entire organisms. Techniques that neuroscience has borrowed from molecular biology include gene amplification through use of the polymerase chain reaction (PCR), DNA sequence analysis, messenger RNA analysis, in situ hybridization, and the use of monoclonal antibodies for targeting specific cells and cellular elements. The applicability of these techniques to a variety of fields has been partly responsible for the boom in sales of biotech instrumentation over the past few years. The DNA Thermal Cycler, from Norwalk, Conn.-based Perkin-Elmer Cetus Instru- ments, leads the field in PCR equipment. Other major instrumentation companies, such as Applied Biosystems Inc., headquartered in Foster City, Calif., are benefiting from the application of automated DNA sequencers and synthesizers to the field of neuroscience, even though they didn't originally target such products to this field. In addition, many firms that produce reagents and other peripherals are enjoying the added bonus of neuroscience applications. In his studies of spinal cord organizational development and how neurotrophic molecules such as growth factors affect this development, William Snider, assistant professor in the department of neurology at the Washington University School of Medicine in St. Louis, is looking forward to taking advantage of some of the recent advances that have resulted from the application of molecular techniques to neuroscience. "Growth factors have been very difficult to localize in the nervous system because it's hard to measure them," says Snider. Recently, however, a number of groups, including that of Yves-Alain Barde in the department of neurochemistry at the Max Planck Institute for Psychiatry in Germany, have cloned genes that allow them to generate molecular probes for a variety of growth factors, enabling them to localize the synthesis of these factors in the nervous system. For example, using PCR technology and molecular biological techniques, including synthesis of oligonucleotides and re- verse transcription of RNA, Barde's group within the past year reported the full primary structure of two new neuronal growth factors, brain-derived neurotrophic factor (Nature, 341:149-52, 1989) and neurotrophin-3 (Nature, 344:339-41, 1990). According to Snider, "the ability to localize growth factors, both in development and maturity, using in situ hybridization, is going to be a big step toward understanding how growth factors regulate cell survival and organization of the system." In addition, says Snider, PCR technology has allowed almost any laboratory with expertise in molecular biology to fairly easily generate its own probes. Once a sequence is published, short strings of DNA that correspond to parts of the sequence can be made. These synthetic oligonucleotides can be directly applied to tissue by in situ hybridization. Although they are not thought to be as sensitive as RNA probes, the oligos have been found useful for messages that are expressed at high levels. Moving from the molecular to the cellular level, one hot area of research has been that of nerve cell transplantation. But because adult nerve cells in the central nervous system do not reproduce, they cannot be perpetuated in culture for study and transplantation. The use of fetal tissue for such research has shown promise but has been haunted by ethical and subsequent political roadblocks, including a moratorium on federal funding for some types of fetal tissue research. This past summer, Solomon Snyder, director of the department of neuroscience at Johns Hopkins School of Medicine, and colleagues reported finding a potential solution to this research dilemma--a group of cells that has led to the first continuous culture of human brain cells (Science, 248:603-5, 1990). "This breakthrough by Dr. Snyder and his colleagues provides a new neuroscience research tool that should accelerate a broad range of basic research aimed at increasing our understanding of many serious disease problems," wrote Frederick Goodwin, administrator of the Alcohol, Drug Abuse, and Mental Health Administration of the United States Department of Health and Human Services at the time of the discovery. The new cell line will provide pure colonies of brain cells that can be grown in the laboratory for basic research into the biochemistry of the human central nervous system. In addition to transplantation, research applications for the cell line are expected to include testing of drugs used to treat neuronal disorders, the search for receptors to growth factors, understanding how neurons work, and exploring ways of protecting neurons from oxygen deprivation in stroke and degenerative disorders. The cell line is available to interested researchers through the Rockville, Md.-based American Type Culture Collection, which collects, preserves, and distributes cultures of microorganisms and plant and animal cells (The Scientist, Aug. 20, 1990, page 1). For neuroscientists already involved in fetal cell transplantation work, cryopreservation technology perfected by reproductive biologists and adapted to neuroscience has become useful. "We have been able to cryopreserve cells and hold them for extended periods of time, to essentially put them in the bank and then bring them out of free storage for grafting," says Timothy Collier, assistant professor of neurobiology and anatomy at the University of Rochester School of Medicine. Collier and colleagues at Rochester and at Yale University modified protocols that had been used for cryopreserving eggs and embryos and were able to preserve human fetal tissue and show viability after up to two months of storage (Science, 242:768-71, 1988). In their study, the group successfully grafted the stored human fetal tissue into the brains of monkeys. Researchers could create a tissue bank for human fetal neural tissue using these cryopreservation and storage techniques, which would allow them to test cells for cellular identity, viability, and bacteriological and virological safety before clinical use. "The capacity to collect and maintain viable human fetal neural tissue would also facilitate research efforts to understand the development and function of the human brain and provide opportunities to study neurological diseases," the researchers reported. "We borrowed a technology that existed in another area and applied it to neuroscience," says Collier. "It has been a good thing, and I think that it will be used significantly in the field." Another tool with origins in a different field but with exciting applications in neuroscience is the confocal microscope (The Scientist, July 23, 1990, page 1). Confocal microscopes bring one plane of a three-dimensional object into focus by manipulating either laser or white light, plus reflected light, through pinholes that block extraneous light. Only single points of the sample are illuminated at a time. One user of this instrument, Meredith Halks-Miller, senior neuropathologist at SRI International in Menlo Park, Calif., believes that her new machine will be a powerful tool for studying spheroidal reaggregate cultures of neurons and glia, a half-millimeter in diameter, that she derives from fetal brain cells. A conventional microscope could never see into the middle of the balls of cells. Whereas previously she was compelled to use classical and time-consuming histological methods involving serial sections through the spheroidal cultures, Halks-Miller and her technician can now perform a simple, two-step immunostaining procedure on whole cultures. Then, using the confocal microscope, they optically section the same balls of cells without ever making a real slice through the tissue. "This is exciting for me because my work has always been three-dimensional," says Halks-Miller. "Neurons don't normally grow in flat planes. They normally grow in three dimensions and have glial cells all around to protect them from the environment." To take advantage of confocal and other types of microscopy for observing nerve cell structure and function, a group of new techniques of dye labeling for cells and cell parts has emerged. Many new fluorescent dyes and markers are helping researchers to see things in both fixed and living tissue that they could never see before, even with the most powerful microscopes. "So much of what is done in neuroscience has to do with the way in which cells communicate," says Rodolfo Llin s, chairman of the department of biophysics at the New York University Medical Center. "Historically this had been measured using voltage recording systems to measure the voltage across the membrane. Now people can use [visual] techniques in addition to electrical recording." A wide range of dyes has been developed. These can be injected inside cells to quickly reveal measurement of events such as membrane voltage. "Using voltage-dependent dyes," says Llin s, "you can basically see what a voltage profile of a cell looks like when it generates nerve signals." Other dyes can reveal ions such as calcium, potassium, or sodium. Changes in concentration of these ions are important events in the transmission of neural signals. "When ion-dependent dyes are used intracellularly, they allow us to see the distribution of ions in the cell," Llin s says. "For instance, using fura-2, which detects calcium concentration changes, the localization of the points of entry for these ions may be directly observed during cell function." The distribution of the dyes not only allows researchers to see and measure levels of calcium and other substances, but also, for the first time, gives researchers a direct view of what is happening in these cells. "Before, you could only just `see' in a very abstract sense. Now you can really peer right into the cell's interior, so to speak," adds Llin s. Two popular dyes for tracing neuronal pathways were developed in a field other than neuroscience. The fluorescent carbocyanine dyes known as diI and diO (pronounced die-eye and die-oh) had been used extensively in membrane biology before their first neuroscience applications (Trends in Neuroscience, 12:333-41, 1989). "Until 1985 or so, these dyes had been used almost exclusively for the study of cell membranes, so neurobiologists tended not to be aware of them," says Marcia Honig, assistant professor, department of anatomy and neurobiology, at the University of Tennessee, Memphis. Since then, these lipid-soluble dyes have proved increasingly popular and versatile as retrograde and anterograde tracers in the developing nervous system and for labeling axonal projections in fixed tissue. "DiI has caught on so much because it is so useful for studying neural development," says Honig. "It has been very hard to trace these pathways with other techniques." According to Washington University's Snider, lipid-soluble fluorescent dyes have brought to the study of mammalian nervous systems a level of detail previously available only in studies of insects or lower vertebrates. "One can see the developing azonal arborizations and developing dendritic arborizations of a variety of different neuronal systems," says Snider. "Since they can be used in fixed tissue, one has a chance to study in mammals, and even in humans, the development of neural connections in a way that has simply not been possible previously." In one newer application of diI, a photo-oxidation method by which the fluorescent dye is excited in the presence of diaminobenzidine converts diI to a stable, insoluble reaction product. "The advantage of doing this is that the marker then becomes permanent," says Honig. "It's electron dense, which means it can be visualized by electron microscopy." Honig, who developed the first neuroscience applications of diI and diO with Richard Hume of the department of biology, University of Michigan, says that in the past two years, they have become aware of at least a dozen laboratories in which the photo-oxidation process for labeling has become routine. When used in fixed material, applications of fluorescent dyes are limited in mature nervous systems, says Snider. "For reasons that are not entirely clear," he says, "they are not as good at tracing connections in the mature nervous system as they are in the embryonic or early postnatal nervous system." This may be related to the lack of neurons in the insulating myelin sheath of mature cells. In addition, because they diffuse in lipids and are not actively transported by the cells, the distance the dyes can travel is limited to a few centimeters. "As development proceeds and the animal gets bigger, or if you're trying to trace a long pathway, [these dyes] just wouldn't be appropriate." Molecular Probes, of Eugene, Oreg. (The Scientist, Oct. 15, 1990, page 1), which manufactures diI and diO, has released additional lipophilic dyes. One of these is an alternative to diO, called diA, which some researchers are finding useful for overcoming solubility limitations of diO. Future products of interest to neuroscientists that Molecular Probes plans to release over the next few months include a series of fluorescent antibodies, a new cell viability assay, substrates and kits for measuring gene fusion, and a line of fluorescent micro-spheres for use in phagocytic studies and as neuroanatomical tracers, according to sales manager Warren Colvin. An additional advance for looking at nerve cells in action is the patch clamp method, which allows the recording of synaptic currents and membrane properties in neurons. In this technique, a small patch of membrane is isolated from the rest of the cell by the tip of a fire-polished glass micropipette filled with a saline solution. A small amount of suction is applied to the electrode, forming a tight seal of high electrical resistance between the membrane and the inside of the pipette. When ion channels in the patch of neural cell open, current flows into the pipette and is measured by a sensitive current monitor. Although the technique is almost a decade old, alterations are making it an increasingly popular tool. Manufacturers of this equipment include Dagan Corp., located in Minneapolis; R.C. Electronics Inc. of Santa Barbara, Calif.; and Warner Instrument Corp., headquartered in Hamden, Conn. "Various modifications of patch clamp methodology allow you to record [activities such as those] of single ion channels in cell bodies [and] axon growth cones," says Eric Kandel, director of the division of neurobiology and behavior at the Columbia University College of Physicians and Surgeons. "This methodology has been modified in various ways so you can now record from cells that are very small and previously would have been difficult to record from." In one of the newly developed patch clamp techniques, known as whole cell recording (Journal of Neuroscience Methods, 30:203-10, 1989), a relatively large (5-10 micrometers in diameter) glass microelectrode is pressed against a cell. A small amount of suction is applied, creating a perfect seal between the membrane and the electrode. Then a small slit in the cell membrane is made, allowing researchers to access the synapses directly. According to Llin s, whole cell recording allows the researcher to introduce chemicals and manipulate their reactions inside the cell. In addition, he says, it "allows measurement of electrical currents with very low noise, because this electrode, by being large, has very low resistance and low intrinsic noise." A range of tools for research beyond the molecular and cellular levels includes extracellular physiologic sensors, typically linear arrays of thin film microelectrodes enabling researchers to study ensemble activity of nerve cells; motion analysis, a technique involving sequential still photographs of objects in motion, for the study of sensory motor coordination; and computer software for modeling neural systems through a marriage of mathematics and neurobiology. Several companies have recently developed physiologic sensors for extracellularly monitoring changes in metabolic activity that signify the behavior of populations of nerve cells. For example, multiple sensor probes developed by OttoSensors Corp. of Mayfield Village, Ohio, for measuring oxygen, temperature, and electrical potential within living tissue, have been used in a variety of neuroscience research applications, including stroke, ischemia, and response to drug treatment. For neuroscientists who use motion analysis to study such areas as motor behavior and motor control, a number of advances are increasing the equipment options (The Scientist, July 9, 1990, page 29). Motion Analysis Corp., of Santa Rosa, Calif., offers a range of products useful for research on whole bodies as well as on a cellular level. For ex-ample, in the two-dimensional, PC-based GaitTrack System, a television camera is pointed at someone who is asked to walk. Markers are placed on the person's joint centers, and the system tracks the position of the markers. It creates a stick figure of the person and measures the velocities, angles, and forces at all the joints that it can see. The system can be directly tied to electromyographs, giving a picture of how abnormalities in the nervous system are manifested in gait. Peak Performance Technologies Inc., head- quartered in Englewood, Colo., also offers several options for whole-body motion analysis. The Peak 2D/3D/Automatic Video/ Computer Motion Measurement System is a modular system that can be upgraded to various levels. The basic setup is a two-dimensional (single-camera), manual system, which requires the user to digitize measurement points manually on each frame; an automatic digitizing module can be added to eliminate this need. With the addition of up to four cameras and the appropriate module, the system can be upgraded to record three-dimensional data. Points to be digitized can be selected on the video monitor with a mouse after the recording is made, eliminating the need for markers, especially useful if the subject is a wild animal. At the cellular level, the Cell-Track System, a product Motion Analysis Corp. originally targeted to other researchers, has proved valuable to neuroscientists as well. "The CellTrack System would be useful at the cellular level for measuring the change in size and shape and growth rates of neural growth cones," says Bill Hand, vice president of the company's Life Science Group and one of the cofounders of the company. "This tool was specifically designed originally to look at ameboid cells, but a neural growth cone grows very much like an ameboid cell, so it's directly applicable there as well." Unlike a conventional image processor, which analyzes shape and size of a static image, the CellTrack System grabs video pictures at frame rates up to 60 images per second for up to 1,500 consecutive video images. Most neuroscientists use frame rates of about one frame per second. "You could collect 1,500 seconds of consecutive information about a neural cone," says Hand. "The system then traces the outline of the process that's growing or changing shape. It measures the change in curvature, the direction of travel and speed, it's orientation, and it does this dynamically as a function of time." "In the past you'd look at something like this through a series, possibly of photomicrographs, and make manual measurements, and then have to figure out what the time span is between these and interpolate. In this case, you can actually end up with a data set that gives you all the variables of the shape change and the motion variables." Neuroscience research has also been enhanced by new physiological monitoring systems, such as Experimenter's WorkBench and Discovery, both from Brain-Wave Systems Corp. of Broomfield, Colo. Discovery is a recording software package with multiple probes, measuring the firing of individual neurons in the vicinity of an electrical probe. In addition to recording data, Discovery can be used to control experiments. BrainWave also provides a more general-purpose package in the form of Experimenter's WorkBench, used for data acquisition and experiment control. Originally tailored to neuroscience applications, BrainWave is now developing versions for other fields as well. Experimenter's WorkBench can be tailored to individual experiments. Information the package can collect includes population responses, which measure the response of a group of cells to a stimulus, electroencephalograms, and electroretinograms. In addition, BrainWave sells audio and visual stimulators that can be hooked up to these packages. Finally, the noninvasive imaging technologies of positron emission tomography (PET), magnetic resonance imaging (MRI), and neuromagnetic imaging have proved of significant use to neuroscientists. These techniques are valuable for studying the relationships between activity and behavior and also for studying pathology. They are allowing links to be made between what scientists know at the basic science level and what they need to determine at a clinical science level. These imaging tools are used for both basic and clinical research. PET, MRI, and neuromagnetic imaging equipment are big-ticket items requiring extremely expensive equipment and facilities. Combining a cyclotron, a computer, and a scanner, PET can quantitatively measure physiological, biochemical, and pharmacological processes in the living brain. Through the use of huge, superconducting magnets, MRI provides excellent anatomical detail, as well as the opportunity to detect previously unrecognized changes in the brain. Neuromagnetic imaging is a noninvasive technique that records the weak magnetic signals emitted by the human cerebral cortex. "The understanding that noninvasive techniques are the wave of the future has really come to us clearly now," says Llin s. "It is every day in people's minds, `What can we do to find out what is wrong with this person without opening the head and without having to speculate wildly?' " Charged for the upcoming decade with revealing the biological basis for many major mental illnesses and neural deficiencies, neuroscientists will use these tools and others to improve their understanding of the basic functions of the brain and nervous system. Laurel Joyce is a freelance science writer based in Palo Alto, Calif.

Special Report: Tools For Neuroscience's Third Decade

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