Ideally, one would like to know about a given biological process for the entire brain using a technique that has high spatial resolution. Nevertheless, it is generally found that methods with high spatial resolution have low sampling volumes (Fig. 3). For example, depth electrodes that measure electrophysiological events sample tissue in a range of less than 100 µm. While methods that assay global brain func- tion, such as CT, MRI, PET, and SPECT, have spatial reso- lutions in the range of millimeters. While it is obvious that techniques with low spatial resolution provide less detailed data about a given biological process, observations or erro- neous conclusions that occur from low-volume sampling of a given measurement may be more insidious. This is true because the area of interest is small relative to the potentially large network of brain sites that may be involved (Mesulam, 1990; Posner and Dehaene, 1994). A technique that measures only a small volume of tissue in a larger system requires a very well characterized hypothesis with regard to the site of interest compared to techniques with a larger sampling volume. The low-resolution high-sampling-volume tech- nique will miss important events because even a signal of high magnitude but low spatial extent may be diluted and go unobserved using a method with coarse spatial resolution.
Conversely, detailed examination of a local event with high spatial resolution and low volume sampling may lead to con- clusions that would be far different if seen in the context of other participating sites of activity at centers in the brain not sampled in the small measurement volume. It is ideal to have a logical progression of the use of techniques from ones with large sampling volumes and lower spatial resolution to those with progressively higher spatial resolutions and lower sam- pling volumes that are focused on areas of interest identified by earlier steps in the survey. This is analogous to serially selecting objective lenses of increasing magnification when using light microscopy. In brain mapping, this may require
switching to alternate techniques or making composite results with a combination of techniques applied either seri- ally or simultaneously.
V. Sites Accessed
Methods vary with regard to the amount of tissue as well as the site in the nervous system that they can access. While postacquisition data processing may be able to recombine and reposition information acquired from a certain orienta- tion into a more comprehensive view of the entire organ, sites of acquisition may impose an absolute constraint on the applications of a given technique. For example, there are a number of methods that are purely cortical. These include transcranial magnetic stimulation, optical intrinsic signal imaging, and scalp electroencephalography. Note that these techniques span the spatial range of resolution from micro- scopic (optical intrinsic signal imaging) to macroscopic (transcranial magnetic stimulation and surface EEG).
Occasionally, techniques designed to assess one part of the brain can be modified and used for another. An example would be electrophysiology. Originally used intraopera- tively during epilepsy and tumor surgery to locate behav- ioral functions and seizure foci in the cortex, they were modified and used as an implanted depth electrode tech- nique to record seizures as well as to stimulate regions in the hippocampus and parahippocampal areas.
With few exceptions, most brain mapping techniques are not specifically designed to examine white matter. Instead, they are primarily tools to assess neuronal function in gray matter structures both cortical and subcortical. Exceptions to this rule include the evaluation of myelinization using postmortem tissue and specific stains, structural MRI, and PET or SPECT with radiopharmaceuticals that localize in white matter structures. In addition, certain electrophysio- logical techniques measure central conduction time (i.e., evoked potentials) as a means by which to estimate the macroscopic integrity of long fiber tracts in the central nervous system. Identifying neuronal targets by their fiber projection systems is performed by the injection of dyes into gray matter regions during life in animal models and sacrificing the animal at a later date to examine the ultimate site of transport of the compounds (Ralston, 1990). Modern techniques are attempting similar experiments using com- pounds that change local magnetic susceptibility and imaging performed with MRI methods (Jacobs and Frasier, 1994; Li et al., 1999).
As was mentioned in the introduction to this chapter, a critical and much needed set of methods in the brain mapping armamentarium contains those that allow one to bridge large scales in spatial resolution or sampling volume.
That is, techniques tend to cluster in the macroscopic, microscopic, or ultrastructural spatial domains. Few if any
techniques can cross these boundaries and provide the nec- essary linking technologies to appropriately place and scale microscopic data into a macroscopic model. Some excep- tions exist and are being exploited for their unique position in the spatial continuum of brain mapping methods.
Postmortem cryomacrotome studies serve as an example of a bridging technology that spans spatial domains (Quinn et al., 1993; Rauschning, 1979; Toga et al., 1994a,b; Van Leeuwen et al., 1990). This technique allows for the assess- ment of whole human brain (and the brains of smaller species as well) anatomy from a structural point of view.
Using these approaches, the entire head, including the brain, meninges, skull, and extracranial tissues, is sectioned after postmortem freezing (Toga et al., 1994a,b). These sections can be as thin as 20 µm. Digital images are made of the entire block face and, currently, have a spatial resolution in the range of 100–200 µm. Higher magnifications can be achieved, with resolution as high as 30 ×30 µm per pixel by zooming the optics onto a small area of the block face. A series of these higher resolution images can be assembled as a patchwork tessellation that recreates the entire macro- scopic surface of the tissue. New digital cameras have been announced that may increase the spatial resolution by a factor of 16 (4 ×4) in the plane of section. At these spatial resolutions, identification of the individual cells will be possible. Thus, with a single technique one can obtain microscopic data and place it in the appropriate macroscopic reference system. Since tissue sections are actually col- lected, they can be stained to provide information about cyto-, chemo-, and myeloarchitecture. Further, as atlases are built using these methods, the high-resolution cryomacro- tome brain images can be stained with conventional or
“landmark” stains (e.g., Nissl stain). With such an approach, an investigator who studies, for example, GABA receptors in the human hippocampus can incorporate data into an appropriate macroscopic atlas. In preparing the tissue, this investigator would process every Nth section using one of the landmark stains that are part of the atlas. The investiga- tor would then digitize the information from both the GABA receptor sections and the landmark-stained sections. Using alignment, registration, and warping tools, the investigator would then register the landmark-stained sections with the atlas and then use the same mathematical transformations to enter the GABA receptor information into the appropriate region of the atlas. Once referenced, the appropriate visual- ization of these new data could be performed in the macro- scopic domain.
Another, less direct but in vivo, bridging technology is the marriage between PET or SPECT imaging and auto- radiography. In both, an identical compound can be used to trace a specific process in the brain. For macroscopic in vivo imaging, this compound would be labeled with the appro- priate positron- or single-photon-emitting radioisotope and external imaging would be obtained demonstrating the
macroscopic behavior of the compound (Huang et al., 1980;
Phelps et al., 1979). For the microscopic counterpart, the compound would be labeled with an appropriate autoradi- ographic radioisotope (Sokoloff et al., 1977), the same experiment performed in an animal model, and autoradiog- raphy of the tissue performed to obtain the resultant images.
Image sets obtained in this parallel fashion can be used to validate or refute animal models. For valid animal models, invasive or more detailed experiments can be performed using approaches that would be logistically impossible or unethical in human subjects.
MRI techniques are applicable in both the macroscopic world of human subjects (Prichard and Brass, 1992) and microscopically (Damasio et al., 1991; Jacobs and Frasier, 1994). This is true for both structural and spectroscopic MRI and probably will be applied to all aspects of MR imaging within the capabilities of its resolution. Finally, electrophys- iological techniques are equally applicable macroscopically in human subjects (e.g., surface EEG) and in local micro- scopic studies of animals (e.g., depth electrodes).
VI. Invasiveness
As was discussed in the section on the frequency of sam- pling, the degree of invasiveness and the practical/logistical aspects of performing a brain mapping measurement are important variables that should be discussed in the context of space and time. Obviously, the most invasive experiments require exposure or surgical manipulation of the nervous system itself, up to and including sacrifice of an animal involved in the measurement. Other techniques are designed to be employed in the purely postmortem setting.
Intraoperative techniques that are utilized in human subjects add significant time to surgical procedures and, as such, their value with regard to patient care must be weighed against the added risk of increased exposure to anesthesia and intraoperative complications. Some intraoperative tech- niques such as awake questioning of the patient during cor- tical stimulation also add stress and anxiety in addition to prolongation of the procedure. Human intraoperative methods are, by definition, not performed in normal subjects and, therefore, do not provide pure information about normal brain function. This caveat is important to remember when comparing human data from normal subjects collected noninvasively with data from patients assessed intraopera- tively who suffer from epilepsy, brain tumors, or vascular abnormalities. Since many of these pathologic states may have been long-standing, developmental or compensatory reorganization of the brain may have also taken place, adding further variability to the data and making it less rep- resentative of the normal condition. Further, both human and animal brain mapping experiments that involve intra- operative manipulation of the brain or its surrounding struc-
tures will perturb the underlying function to varying degrees. This also will add noise or variance to the data and make them less representative of the function of the organ in the natural state.
Methods that use exogenous compounds, whether they are radioactive or nonradioactive, add a degree of invasive- ness because of the exposure of the subject or animal (and at times the investigator) to that agent. Nevertheless, these methods are typically applied in normal subjects and do not directly perturb brain function unless the mass of the admin- istered agent is so large as to interfere with the natural chem- ical or physiological processes. Often the exogenously administered agent is not blood-borne but can be in the form of energy. This is true of some of the in vivo human tech- niques such as CT (X-irradiation) or MRI (magnetic fields and radio-irradiation). It is taken for granted that those tech- niques that use ionizing radiation are more invasive and have more restrictive boundary conditions for exposure in specific subject groups. Transcranial magnetic stimulation and, possibly, high-field fMRI techniques directly excite neuronal tissue in ways other than those that naturally occur under physiologic conditions. Care must be taken in this situation to determine what effects such perturbations produce and that their comparison with physiologic states may not always be valid.
A number of techniques require anesthesia. This is typi- cally true of larger animal experiments that involve in vivo tomographic methods such as PET, SPECT, MRI, and CT.
This is because of the immobilization requirement of these methods combined with ethical issues about producing mus- cular paralysis in an awake animal. Intraoperative tech- niques in human subjects and most animal models also require some degree of sedation although the patient is often awake during the actual measurement. Nevertheless, drugs are required for the preparatory phase of craniotomy, and their lasting effects, or required continued use at lower levels, undoubtedly distort the signal from the natural phys- iological one. Some techniques induce a certain degree of sedation or anesthesia by virtue of the method itself. An example is stable xenon CT blood flow measurements. As the inhaled concentration of stable xenon increases, pro- gressive sedation and, ultimately, anesthesia can occur. In this case, the actual contrast agent used to make the blood flow measurement induces a perturbation in the system to be measured, in this case, anesthesia.
VII. Conclusions
As can be seen, a wide range of brain mapping tech- niques currently exist for use in human subjects and animal models. Each has its own unique advantages and disadvan- tages and all vary on the continuum with regard to spatial and temporal resolution as well as sampling frequency and
volume. Special issues with regard to the sites that these methods can access, their degree of invasiveness, require- ment for anesthesia, and repeatability all contribute to the selection of the appropriate approach for a given neurobio- logical situation. As has been stressed throughout this chapter, it is important to understand the limitations and constraints for each method so as to interpret the results appropriately and to use the resultant data to build reliable hypotheses that can be rigorously tested with further exper- imentation. The ideal brain mapping technique would have extremely high spatial and temporal resolution with the capacity to sample a large volume of the brain continuously.
Its costs would be low as would its invasiveness, making it applicable in many settings, in human subjects as well as animal models. At present, no such method exists.
Nevertheless, the combination of data sets acquired from many different techniques synthesized into an ever-growing atlas of brain structure and function provides the most uni- fying means by which to span the spectrum of all these vari- ables. Current and developing tools as well as the increasing power and decreasing cost of digital approaches to the man- agement of such data sets make this goal appear to be not just a possibility in the near future but an actual requirement as the volume of information and the need to standardize it across laboratories, experiments, and species continues to grow.
Developing tools and mapping the brain are important challenges for neuroscience. The increase in the quality and variety of techniques that provide input for brain mapping experiments is rapidly expanding. Simultaneously, the speed and memory capacity of computing devices are advancing at an ever-accelerating pace while cost is dropping, making powerful desktop manipulations of brain mapping data not only feasible but a reality in most laboratories. The appro- priate mathematical and statistical models are being devel- oped to provide advanced population-based probability atlases of the human brain and the databases to use them.
Once a proper framework for the organization and storage of neuroscientific data across spatial scales and tem- poral domains is available the results of every experiment and clinical examination involving the nervous system could ultimately have an appropriate place for future refer- ence. This depends on the ingenuity and farsightedness of the creators of the reference system to provide an approach that is flexible, compatible with existing as well as future technologies, and presented in a manner that is acceptable, in both the technical and the sociological sense, to the neu- roscience community at large. Such a system will be neither easy to create nor inexpensive. Nevertheless, when one examines the amount of data collected in both clinical and research settings today that becomes inaccessible soon after acquisition, one quickly realizes the economy of developing a system for storage and reference of this untapped and yet very costly information. Time and funds spent to organize
and store these data that reflect the convenience of the inves- tigators as well as the confidence and credibility ratings for the quality of each data set will provide a usable system that will stand the test of time.
Finally, one should return to the concept of using these systems not simply as libraries or databases but rather as rich sources of neuroscientific information upon which one can base hypothesis generation and test such theories against actual data that one need not personally collect.
Similarly, the ability to rigorously correlate in vivo human data acquired tomographically or by other methods with postmortem tissue that is available for the myriad of immuno-, histo-, and biochemical stains provides a two-way system to develop a more thorough understanding of the microscopic anatomy of the brain that is driven by hypothe- ses generated from human in vivo experiments. Given the rapid growth and the amount of neuroscientific information, and the pace with which it increases, such organizational, storage, and conceptual systems should no longer be con- sidered a luxury but rather a necessity.
Acknowledgments
I thank Arthur Toga, Ph.D., for his thoughtful comments in the review of this chapter, Andrew Lee for preparation of the graphic materials, and Laurie Carr for preparation of the manuscript itself. Partial support for this work was provided by a grant from the Human Brain Project (P20- MHDA52176), the National Institute of Mental Health, the National Institute for Drug Abuse, the National Cancer Institute, and the National Institute for Neurological Disease and Stroke. For generous support, the author also thanks the Brain Mapping Medical Research Organization, the Brain Mapping Support Foundation, the Pierson–Lovelace Foundation, The Ahmanson Foundation, the Tamkin Foundation, the Jennifer Jones- Simon Foundation, the Capital Group Companies Charitable Foundation, the Robson Family, the Northstar Fund, and the National Center for Research Resources, Grants RR12169 and RR08655.
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