RESEARCH METHODS Introduction Flourens was the first to identify the region of the brain that controls respiration and the first to correctly identify the motor functions of the cerebellum. To make these discoveries required careful attention to experimental methods. Flourens expressed the importance of research methods clearly and elegantly: In experimental research everything depends upon the method; for it is the method that produces the results. A new method to precise results; a vague method has always led only to confused results. (Flourens, 1842/1987, p. 15) Behavioral Neuroscience Is a Life Science The methods used in behavioral neuroscience today are much advanced over those available in Flourens’s time, but the use to which these methods are put remains the same: to better understand the biology of behavior and of our selves. Thus, behavioral neuroscience focuses on the study of living organisms, both humans and other species. The overriding objective of this research is to deepen our knowledge of the biology of behavior and to use that knowledge in battering the lives and relieving suffering of both humans and animals. Laboratory rats, for example, have been extremely useful in learning about the biology of aging, since rats have a natural life span of two to three years. Thus, behavioral neuroscience is continually enriched by progress in physics, chemistry, and engineering. Visualizing the Human Brain Perhaps most spectacular of the new tools in biological psychology are the recently developed brain-imaging technologies: computerized tomography, magnetic resonance imaging, and positron emission tomography. These techniques make possible the study of both brain anatomy and patterns of brain activation in living, healthy human beings. For this reason, brain imaging is playing a major role in the study of the neural basis of human thought and language. Computerized Tomography: Computerized tomography (CT) was the first of the new brain-imaging technologies, having been commercially introduced in 1973, although the critical patent for the process was issued in 1960. CT is an enhancement of the familiar X-ray procedure. Instead of producing the usual shadow imaging of a conventional X-ray, in CT an image of a horizontal slice of tissue is reconstructed, as shown in Figure 2.2. It is as if a slice of brain were surgically removed and placed on a table for inspection. In CT, narrow X-ray beams are passed through the head in a particular cross-sectional slice from a wide variety of angles. The amount of radiation absorbed along each line is measured. From the measurements associated with each beam, a computer program can determine the density of tissue at each point in the slice. The resulting image is the CT scan, an example of which is shown in Figure 2.3. Magnetic Resonance Imaging: Magnetic resonance imaging (MRI) also provides a mathematically reconstructed image of slices of living tissue, but it does so by using a very different source of information than the X-rays used in CT. MRI exploits a phenomenon known as nuclear magnetic resonance, in which radio-frequency energy in a strong magnetic field is used to generate signals from a particular atom - usually hydrogen - contained within the tissue. Certain properties of the phenomenon of magnetic resonance confer great advantages to MRI as an imaging technique. First, unlike in CT, no ionizing radiation is employed. Second, MR images have extremely fine spatial resolution, providing neuroanatomical images of exquisite detail, as shown in Figure 2.4. Third, because of technicalities in the MRI procedure, it is possible to obtain slices at any angle, not just in the horizontal plane, as is the case with CT. Three-dimensional images of the brain may also be generated, as shown in Figure 2.5. Finally, advanced MRI methods have recently permitted the imaging of brain function as well as structure, measuring both brain blood flow and oxidative metabolism. Previously, functional imaging was limited to positron emission tomography. Positron Emission Tomography: Positron emission tomography (PET) has been used for several decades to provide images indicating the functional or physiological properties of the living human brain. PET involves the injection of a tracer substance labeled with a positron-emitting radionuclide. One common tracer is labeled fluorodeoxyglucose (FDG), a subcourse that is taken up by cells when they need glucose for nutrition. Over the course of a few minutes, metabolically active portions of the brain will accumulate more FDG than well less active regions. By determining where FDG is accumulating in the brain, patterns of differential brain activation can be mapped. PET scanning is now widely used to study patterns of brain activity that underlie higher mental functions. Microscopic Approaches to Brain Anatomy While glamorous computerized brain-imaging machines are opening a new era in the study of the gross (large-scale) anatomy and function of the human brain, microscopy has contributed for more than a century to the analysis of the cellular structure and - more recently - the cellular function of the nervous system. For most kinds of microscopic investigations, the tissue to be imaged must be this enough for light to pass through it, and portions of it must be of different colors or transparency so that important features are distinguishable. A variety of histological (having to do with the study of the minute structure of tissues) procedures have been devised to meet these objectives. In most instances, the tissue to be examined must first be prepared by fixation, a procedure to preserve the features of interest. Fixation is often accomplished by using an agent - such as formalin - to harden the tissue. Freezing is another useful approach to stabilizing neural tissue. Once hardened, the tissue is sliced very thinly to render it nearly transparent. One typical procedure is to first embed the tissue in a substance such as paraffin to facilitate holding the specimen. It then can be cut by using a microtome, a specialized automatic slicing machine that produces thin, regular sections if the fixed and embedded tissue. The resulting thin sections may then be mounted on glass slides in preparation for viewing. Such microscopic sections are now thin enough for light to pass through them, but - in most cases - they lack sufficient contrast to make different features of the tissue apparent. Staining is a procedure to selectively darken or color particular features of the sectioned tissue. By choosing an appropriate stain, different features of the tissue are highlighted. The Golgi silver stain has the property of completely staining a few individual cells in the specimen. Because only a few cells are stained, they stand out with exquisite clarity. The Golgi method is probably the best histological procedure for visualizing single nerve cells. Nissl staining is useful for visualizing the distribution of cell bodies in the specimen. Myelin stains selectively color this protective coating, a procedure that is useful for mapping connecting pathways in brain tissue. One classical approach to determining where cells in a particular location make their connections is to selectively damage those nerve cell bodies. Since the cell body supplies all the metabolic needs of the cell, the axons then die and begin to degenerate. Silver staining of the tissue - a procedure perfected by Walle Nauta - turns the dying axons dark brown. In this way, specific pathways can be traced through the nervous system. Pathways can also be traced in experimental animals by injecting radioactively labeled amino acids in the vicinity of the cell bodies of interest. The labeled amino acids are taken up by the cell and transported along the axons. After sufficient time for the transport to be completed, the brain is removed, and sections are made. Each section is coated with a sensitive emulsion. The radioactive level then exposes the emulsion that can be developed at a later time, in much the same way as photographic film is developed. This is one example of autoradiography, a term for procedures in which the section in effect takes a radiograph of itself, highlighting areas of intense radioactive label. Pathways can also be mapped in the reverse direction, from the ends of the axon back to the cell body, by infecting the enzyme horseradish peroxidase. The tips of the axon pick up the enzyme, and it is transported back to the cell body. Along the way, the enzyme, and it is transported back to the cell body. Along the way, the enzyme causes reactions in the interior of the axon that may be subsequently visualized by a special staining procedure. Monoclonal antibodies are being developed to recognize and mark particular cellular proteins. Antibodies are proteins produced by lymphocytes - a type of white blood cell - that bind to particular target molecules. Thus, antibodies could be used to locate particular targets, but the problem is to obtain sufficient quantities of identical antibodies to carry out the search. This is accomplished by cloning the antibody . In cloning, a single antibody-producing lymphocyte is joined to a lymphocyte tumor cell. The lymphocyte tumor cell divides indefinitely, producing a strain of identical - cloned - lymphocytes, all of which produce the desired antibody. Monoclonal antibodies - antibodies produced by the same cloned lymphocytes - may then be used to map highly specific biochemical characteristics of specific neural populations. Recombinant DNA procedures are another tool provided by molecular biology that has proven to be extremely useful in studying the brain. Proteins - the building blocks of nerve and other cells - are specified by segments of DNA called genes. The gene transfers its information to messenger RNA that regulates the assembling of the protein. Tens of thousands of different proteins are utilized by the brain, some of them in extremely important ways such as the membrane channels that control electrical signaling in neurons. Molecular biologists have discovered enzymes that act in various ways. These include enzymes that can cut the DNA apart and put in back together again, perhaps altering it in the process. For this reason, these methods are called "recombinant." The very fine structure of nerve cells also can be studied microscopically using electron beams rather than light waves to form the image. The first electron microscope was constructed in Germany in the early 1930s. Today, scanning electron microscopes are routinely used in the biological sciences, producing magnifications of up one million times. An example of a scanning electron micrograph of a part of a nerve cell is shown in Figure 2.8. Recording Brain Electrical Activity Nerve cells - like all living cells - maintain an electrical charge across their outer membrane. Since the electrical signals produced by nerve cells are comparatively small, they must be amplified before they can be measured accurately. Today, this is accompanied by using electronic amplifiers, much like those employed in home audio equipment. It is the size and placement of the electrodes that determine what aspects of neural activity will be recorded. Very large electrodes reflect the activity of larger populations of nerve cells; smaller electrodes can record more localized neuroelectric events. The Electroencephalogram: The electroencephalogram (EEG) is the neurologist’s term for the electrical activity that may be recorded from electrodes placed on the surface of the scalp. When such a recording is obtained from electrodes placed directly on the surface of the brain - usually during neurosurgery - the measure is called the electrocorticogram (ECoG). Several patterns of EEG activity - which he termed alpha, beta, theta, and delta - that differ in their frequency and amplitude. The waking human EEG is characterized by an alteration between two patterns: alpha activity, a rhythmic, high-amplitude, 8- to 12-Hz pattern, and beta activity, a low-voltage tracing at more than 13 Hz. Theta activity is between 5 and 7 Hz and typically is of medium amplitudes. The EEG is generated primarily by the activity of large numbers of nerve cells within the brain. Because the skull, which encloses the brain beneath the scalp, is an electrical insulator, under most circumstances, it is impossible to conclude which portion of the brain is generating any particular part of the EEG signal. The encephalogram has proven to be most useful in studying the sleep-waking cycle and in diagnosing epilepsy. Magnetic Recording: Although, today, magnetoencephalography (MEG) - the magnetic recording of brain activity from the scalp - is strictly an experimental procedure with a great many pitfalls in its application, magnetic rather than electrical recording is of considerable interest. One important difference between MEG and EEG is that the skull is electrically resistant but magnetically transparent. This means that the skull gravely distorts the localizing information that would otherwise be present in the scalp-recorded EEG, whereas much localizing information is preserved in the MEG record. For this reason, magnetic recording may be of significant value in localizing the source of signals produced by populations of nerve cells within the brain if its formidable technical problems can be resolved. Event-Related Potentials: An event-related potential (ERP) is a component of the EEG that is triggered in association with sensory , motor, or mental event. ERPs are used extensively to study the time course of higher-level processes in the human brain, such as perception and attention. ERPs are typically small fluctuations produced by the processing of a sensory stimulus or motor events. Microelectrode Recording: Microelectrodes are very small electrodes with very small tips that can be used to record the electrical activity of single nerve cells. The glass electrodes - called micropipettes - are made from glass tubing that is heated and stretched to narrow the width of the tube. The micropipette is then filled with a conductive solution such as potassium chloride. Microelectrodes may be used for either extracellular or intracellular recording. For extracellular recording, the electrode is placed near the nerve cell. In this position, it can measure the currents flowing from the nerve cell into the extracellular fluid that surrounds it. For intracellular recording, the microelectrode is inserted into the interior of the nerve cell itself. Patch Clamps: Nerve cells regulate their electrical activity by controlling small pores or channels in their outer membrane. A patch clamp is an adaption of the glass micropipette method in which a small amount of suction is applied to the fluid-filled recording electrode. If the tip of the electrode is placed on the outer surface of the cell membrane, a tight mechanical and electrical seal results. The result is that the electrode measures electrical current only from the portion of the membrane that is clamped to the electrode. In this way, the activity of individual membrane channels can be measured. Brain Stimulation Electrical stimulation of the brain (ESB) is an effective means of demonstrating functional neural connections between two brain regions. If an electrical stimulation of one area evokes an electrical response in another, there must be some functional pathway linking the two regions of the brain. Using specialized techniques, stimulation may be confined to a single nerve cell. Usually, however, a population of cells is activated in the region of the electrode. It is generally believed that using electrodes with 1 mm2 exposed tip and passing about 1 milliampere (mA) of current stimulates about 1 cubic millimeter of brain tissue, although a larger region may be affected under many circumstances. Usually, the effects of ESB are apparent immediately following stimulation. Electrical stimulation of the lateral hypothalamus - an area deep within the brain related to feeding - may have no immediate effect but will increase the amount of food that a cat will eat the following day by as much as 600 percent. Human Brain Stimulation: The problem for a neurosurgeon attempting to remove diseased tissue in regions of the brain that support higher mental functions - such as language - is that those functions are not always carried out by exactly the same brain areas, particularly in individuals with a long history of brain disease. ESB provides the "gold standard" by which the functional properties of a region of brain may be determined before the tissue is surgically removed. For this reason, ESB is often carried out with the surface of the brain exposed during neurosurgery. The patient is conscious, since it is necessary to determine whether stimulation of a particular cortical region affects speech perception of production. Such an operation is possible because the brain itself does not have pain receptors; only a topical anesthetic is required to deaden the nerves of the scalp and skull. As different regions of the brain are stimulated, speech perception and production are tested. Electrical stimulation of the human brain is also carried out - although much less frequently - by using electrodes that have been surgically implanted within the brain of patients undergoing prolonged (e.g. several weeks) of monitoring often for the localization of epileptic disorders. Studies of such patients have also contributed to the understanding of certain higher mental functions. Magnetic Stimulation: Recently, a new - completely noninvasive - procedure for stimulating the neurons has been developed, using focused magnetic fields rather that electrical current. By using a small (10-15cm) coil placed against the surface of the scalp, a 1- to 2-tesla focused magnetic field may be generated. This field is capable of locally exciting the regions of the underlying brain and inducing electrical discharges from that tissue. In this way, functional activity of the brain can be determined in normal individuals who are not undergoing neurosurgery. Neurochemical Approaches Chemical Stimulation: Similar analysis can be carried out on a much more localized basis, by specifically introducing the chemical to a particular brain region. Using a small injection tube, called a cannula, any pharmacological agent can be placed in a restricted brain region. In animal research, a sturdy, large-bore guiding cannula is surgically implanted before testing is to take place. After the animal has recovered, a small injection cannula can be inserted through the guide to deliver the agent to the target structure. Microiontophoresis: Injecting chemical into the brain using a cannula affects - of necessity - a large number of cells, since a cannula is comparatively large with respect to the size of these cells. Microiontophoresis provides a more precise means of stimulating single nerve cells with chemical agents. In this method, a cluster of micropipettes is employed. One is used as a microelectrode to record the electrical activity of the target cell. The other pipettes are filled with specially prepared solutions of the chemicals to be tested. These solutions are ionized, or electrically charged. BY passing a small electrical current through a pipette containing an ionized solution, molecules of the substance can be released from the pipette onto the target cell. Iontophoresis means literally "carried by ions." Microiontophoresis is the most precise form of chemical stimulation of the brain possible today. Microdialysis: Microdialysis is a related procedure by which chemicals are extracted from the fluid surrounding nerve cells for purposes of analysis. Dialysis is a process by which molecules are separated from a solution by using a special membrane that allows molecules of a particular size to cross the membrane freely. A neutral solution is slowly flushed through this apparatus, where it mixes with molecules surrounding nerve cells in the region of the special dialysis membrane. As the solution is circulated, it is removed from the probe and made available for chemical analysis. In this way, ongoing chemical activity can be measured. Brain Lesion Analysis A lesion is an abnormal disruption of a tissue, produced by injury or disease. The study of naturally occurring brain lesions in human beings has formed the cornerstone of train research in the field of neurology. The discovery of the sensory and motor areas of the human brain, for example, was the result of localizing lesions in individuals who suffered a disruption of sensory or motor functions as a result of brain damage. This approach to the study of brain and behavior is called lesion analysis. A number of different procedures may be used to produce brain lesions. One of the simplest, at least for brain areas that are easily accessible, is surgical removal of the targeted tissue. A related procedure for producing a lesion in accessible brain tissue is aspiration, in which tissue is removed by suction applied through a glass pipette. Aspiration is particularly useful for the removal of tissue of the surface of the brain. Lesions targeted for deeply embedded brain structures are often produced by using an electrode through which high-frequency current is passed. A stereotaxic apparatus is a mechanism that fastens to the head in a fixed position relative to standard features of the skull. From these skull landmarks, the approximate location of hidden brain structures can be determined. A stereotaxic atlas, a map of the typical brain and skull for the species, is used to calculate the coordinates of the tissue to be lesioned. Stereotaxic procedures are also used in human neurosurgery - using radiographic rather than skull landmarks - to produce therapeutic brain lesions in deep regions of the brain that are inaccessible for visually guided dissection. Chemical procedures are also useful in producing specific brain lesions. In this approach, a neurotoxin, or nerve poison, is injected through a stereotoxically positioned cannula. Some very special types of neurotoxins destroy only cells that have particular chemical properties. Finally, temporary lesions can be made by using a refrigerating probe, or cryoprobe. A cryoprobe lowers the temperature of nerve cells that it contacts so that they can no longer function. During this time, a functional brain lesion is produced. When the probe is turned off or removed, the nerve cells warm up and function normally again. No matter how a brain lesion is produced, interpreting the behavioral effects of the lesion can be tricky. First, even experimental brain lesions are not perfectly made. A second, related issue is the inadvertent damage to fibers of passage. Fibers of passage are nerve fibers passing through the region of the lesion that neither originate nor terminate in that structure. A third, perhaps more fundamental problem in lesion analysis is that specific functions are often distributed through a number of brain areas. Summary: The study of the biological basis of behavior depends critically upon the integrity of the experimental methods by which theoretical ideas are tested. Fortunately, the physical, chemical, and engineering sciences over the past several decades have provided increasingly powerful and precise tools for the study of the nervous system and its functions. Perhaps the most spectacular of these new methods are the brain-imaging technologies. Computerized tomography utilizes multipass X-ray data to construct images of horizontal slices though the human brain. Magnetic resonance imaging, a more recent development, provides images with higher resolution, in any arbitrary plane, without the use of ionizing radiation. Positron emission tomography permits the imaging of the functional and chemical activity of the nervous system in addition to providing data about brain structure. Microscopic methods and histological procedures clarify the structure and functions of the nervous system at the cellular level. Staining methods are now routinely available to visualize many specific properties of individual neurons, including their metabolic demands and chemical properties. Recording the electrical activity of the brain and its cells also has contributed greatly to understanding nervous system functions, since neurons process information by altering their electrical potential. The electroencephalogram recorded from the scalp has been particularly useful in the study of sleep and certain neurological disorders, such as epilepsy. Magnetoencephalography may provide a way of obtaining more information concerning the sources of EEG signals. Brain responses to specific sensory stimuli may be extracted from the ongoing EEG by event-related signal averaging. Recording may also be performed to study the electrical activity of single nerve cells by using microelectrodes or even portions of a cell by using patch clamp methods. Yet another approach to analyzing brain functions is to study the behavioral effects of either brain stimulation or brain damage. Electrical or chemical methods may be employed to stimulate the brain. Similarly, a variety of procedures are useful in producing restricted brain lesions. In either case, careful behavioral analysis is required to understand the precise effects of the experimental treatment.
