Brain Topography, Volume 5, Number 2, 1992
71
Optical Imaging of Architecture and Function in the Living Brain Sheds New Light on Cortical Mechanisms Underlying Visual Perception A. Grinvald* Summary: Long standing questions related to brain mechanisms underlying perception can finally be resolved by direct visualization of the architecture and function of mammalian cortex. This advance has been accomplished with the aid of two optical imaging techniques with which one can literally see how the brain functions. The upbringing of this technology required a multi-disciplinary approach integrating brain research with organic chemistry, spectroscopy, biophysics, computer sciences, optics and image processing. Beyond the technological ramifications, recent research shed new light on cortical mechanisms underlying sensory perception. Clinical applications of this technology for precise mapping of the cortical surface of patients during neurosurgery have begun. Below is a brief summary of our own research and a description of the technical specifications of the two optical imaging tectuaiques. Like every tecln~ique, optical imaging also suffers from severe limitations. Here we mostly emphasize some of its advantages relative to all alternative imaging techniques currently in use. The limitations are critically discussed in our recent reviews. For a series of other reviews, see Cohen (1989). Key words: Optical imaging; Brain functional architecture; Living brain; Oximetry; Neuronal assemblies; Real-time imaging; Voltage-sensitive-dyes.
Optical imaging techniques offer unique advantages that are particularly suitable for exploring the architecture and real-time function of primary cortical areas, as well as higher cognitive brain functions. By simultaneously imaging the activity of millions of neurons, rather than one neuron at a time, these optical imaging techniques have shed new light on the organization and function of the cortex.
Development of optical imaging techniques Two optical imaging techniques are involved, realtime optical imaging and imaging based on activity dependent intrinsic signals.
Real-time optical imaging The first technique is based on vital staining of cortex with voltage-sensitive dyes and imaging the activity dependent fluorescence changes. Voltage-sensitive dyes *Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel. Accepted for publication: September 1l, 1992. Correspondence and reprint requests should be addressed to Prof. A. Grinvald, Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel. Copyright © 1992 Human Sciences Press, Inc.
bind externally to neuronal m e m b r a n e s and act as m o l e c u l a r t r a n s d u c e r s , t r a n s f o r m i n g c h a n g e s in membrane potential into optical signals. Investigators in the field have realized the hope of the famous British physiologist, Lord Sherrington who marvelled, some 40 years ago, at "how m u c h easier it would be to understand the brain if neurons would light-up w h e n they talk to one another". The first steps in this direction were taken by the pioneering work of the research groups of Drs. Taskai and Cohen, at the NIH and Yale. Our group contributed to the transformation of this approach from a promising idea into an effective tool, currently used to study numerous model systems used in brain research; from single neurons maintained in culture (Grinvald et al. 1981b; Grinvald and Farber 1981; Anglister et al. 1982), through intact invertebrate ganglia (Grinvald et al. 1977; Grinvald et al. 1981a), mammalian brain slices (Grinvald et al. 1982b) and the living brain (Grinvald et al. 1984; Orbach et al. 1985; Grinvald 1984, 1988, 1991a). Optical imaging has recently undergone an explosion, not only for optical monitoring of electrical activity, but also for the imaging of calcium, sodium, potassium and even important proteins. The most critical part of this technique has been the design of the molecular probes developed by Rina Hildesheim in our laboratory at the Weizmann Institute. More than 600 dyes were synthesized and most of them are the best available for a variety of applications (Grinvald et al. 1977, 1982a,b,
72
1983, 1984, 1986, 1987). Below we briefly discuss the performance of real-time optical imaging, based on voltage-sensitive dyes.
Performance specifications of real-time optical imaging The optical signals reflect the intracellular changes in membrane potential. The spatial resolution is 1 ~tm for two-dimensional preparations and 30-50 ~m for threedimensional, thick preparations (e.g., slices, cortex). Three-dimensional imaging is feasible with optical sectioning and deconvolution, and perhaps also with confocal microscopy. Both evoked activity and on-going activity can be imaged. The method can be used to evaluate pharmacological effects of drugs in-vitro and in-vivo. The optical maps were highly correlated with both anatomical and electrophysiological maps. The smallest size of electrical activity which can be recorded depends on the preparation. For example, in processes of single cells I mV synaptic potential could be detected. Photodynamic damage is the restricting factor of determining the limit of sensitivity of the method. Attempts to enhance the sensitivity of the method by designing better dyes has been a continuous project, and additional improvements are likely. The duration of the recording session can be as short as a few ms, and in many situations, including in-vivo studies, averaging is not required. Once the cortex is exposed, the method is relatively n o n - i n v a s i v e , a l t h o u g h p h o t o d y n a m i c damage is a limiting factor and pharmacological side-effects of the dyes must be evaluated for each dye in each preparation. Such critical evaluation has not yet been made for the application of this approach in human subjects. From animal studies it appears that this optical method may have clinical applications.
Imaging based on visualization of oxygen delivery The second technique is based on imaging of intrinsic changes in the optical properties of electrically active brain tissue. One source for the activity dependent optical signal is a small change in the color of the tissue, produced by oxygen delivery from oxy-hemoglobin within the capillaries in response to metabolic demand. Other intrinsic signals originate from activity dependent light scattering changes and changes in the oxidation states of intrinsic chromophores such as cytochromes. The pioneering work of Hill, Keynes, Chance, Jobsys and their colleagues began to uncover the existence of these intrinsic signals some 45 years ago. However, since these signals are very small, their use for imaging of the functional architecture of cortex began only in 1986 (Grinvald et al. 1986). This imaging technique permitted the highresolution imaging of the functional architecture of the
Grinvald
visual cortex in the living brain of cats and monkeys (Grinvald et al. 1986; Ts'o et al. 1990; Ratzlaff and Grinvald 1991). In addition, activity maps were also obtained through the intact dura and thinned bone; this should facilitate the direct visualization of the fascinating development of the meticulous, functional architecture of the cortex (Frostig et al. 1990). Furthermore, this technique was successfully applied to the imaging of functional architecture in the cortex of awake behaving monkey (Grinvald et al. 1991b). The latter application suggests that imaging based on intrinsic signals will contribute to the exploration of higher cognitive functions. In addition, clinical brain mapping, as an aid for n e u r o s u r g e o n s , is c u r r e n t l y being i m p l e m e n t e d (Haugland et al. 1992). The finding that activity dependent changes in the microcirculation can facilitate high resolution mapping, indirectly led to the exciting development of MRI functional imaging, based on the same mechanisms underlying the optical imaging (Kwong et al. 1992; Ogawa et al. 1992). Below we briefly summarize some performance specifications of these first optical imaging methods.
Performance specifications of imaging based on intrinsic signals The intrinsic optical signal originates from the oxyhemoglobin to hemoglobin transition that occurs in response to metabolic demands. When using dim red light (605 nm), mostly oxygen delivery is directly imaged. At other wavelengths, changes in blood volume in capillaries and light scattering changes can also be imaged. The spatial resolution of the technique is 50 to 100 ~tm. The rise-time of the intrinsic signals is i to 2 seconds. The smallest region that can be recorded from is limited by optics, but the resolution is about 50 ~tm, depending on the density of capillaries in the imaged volume. Imaging based on light scattering signals may achieve a resolution of about I ~tm,but the signal-to-noise ratio is not as good. High-resolution, two-dimensional imaging was already achieved with a resolution of about 50 ~m while in the third dimension the signal is integrated from the surface of the cortex down to a depth of approximately 500 ~tm. It seems likely that using near infra-red light threedimensional imaging would also be feasible, provided that the signal-to-noise ratio can be improved by a factor of about three. This method is especially useful to record evoked activity, since averaging is required and subtraction of a background image is necessary to visualize the responsive areas. The method can be used to evaluate pharmacological effects of drugs in-vitro and in-vivo. The optical maps were highly correlated with both anatomical and electrophysiological maps. Functional maps of ocular dominance columns were obtained in
Optical Imaging of the Living Brain
73
\\
l Figure 1. Pinwheel-like structures are a prominent feature of the functional organization of orientation preferences (B). The micro-vasculature is clearly visible in this picture (A). Scale bar is 1 mm. B: Colour-coded orientation preference map in cat area 18. The preferred orientation for every location is c o d e d according to the scheme shown at the right of the figure; areas responding best to a horizontal bar are c o d e d in yellow; areas responding best to a vertical bar are c o d e d in blue, etc. It is apparent that the overall organization is continuous rather than banded. (Modified from Bonhoeffer and Grinvald 1991).
about 10 seconds. Imaging of functional domains which are less robust may takes a few hours. However, the duration of imaging sessions with this non-invasive method is probably unlimited. Currently, four groups are attempting the intra-operative implementation of this imaging technique to delineate functional borders in the cortex with a precision of less than 100 ~m. One group already reported functional images in patients during neurosurgery (Haugland et al. 1992)
Major findings related to mechanisms underlying sensory perception F u n c t i o n a l a r c h i t e c t u r e u n d e r l y i n g visual perception
Mountcastle in the somatosensory cortex and Hubel and Wiesel in the visual cortex have found that cortical cells with similar response properties are frequently
Figure 2. Close-up of 4 pinwheels. Two fundamental features of the pinwheels are illustrated in this figure. (a) In all '"pinwheels", each orientation appears once around the center. (b) The pinwheels exist in two forms: a clockwise form (left side), in which the sequence of the colors yellow-green-blue-red is clockwise, and a counterclockwise form (right side), in which the inverse is the case. Scale bar is 300 mm. (Modified from Bonhoeffer and Grinvald 1991).
clustered together, forming columns (modules) which often traverse the entire cortical depth, from pia to the white matter. Previous work relied on electrophysiological, anatomical and histological techniques to reveal the organization of each type of module. However, the exact functional organization of different functional modules parallel to the cortical surface still remains an unresolved question for all areas of the mammalian cortex. Optical imaging is a particularly attractive technique for exploring this organization. Our research in cat visual cortex unexpectedly established that the most prominent organizational feature of orientation-preference is a radial arrangement, forming a pinwheel-like structure surrounding a singularity point (figure I and figure 2). Figure I shows how merely by taking a picture of exposed cortex (A), as the animal is viewing gratings of various orientations, the map of orientation preference can be derived (B). The same elaborate organization, underlying the visual perception of oriented lines or edges, was found also in the primate. Our subsequent research on primates explored the relationships between three subsystems playing a major role in visual processing of
74
GrinvaJd
the retina, probably using population coding. In superficial layers these patches exhibit fuzzy borders; the response properties change smoothly across the cortical surface. The size of each of these m o d u l e s is approximately 800 by 400 ~tm. The reported architecture is different from previously described experimental and theoretical models (Bartfeld and Grinvald in press).
i
i
The organization of d y n a m i c patterns of activity in neuronal assemblies
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0 FRAME NUMBER
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Figure 3, Real-time optical imaging of brain waves oscillations, The 3-D images show the amplitudes of the dendritic depolarization in a 3 x 3 mm region of cat visual cortex, To visualize the dynamic behavior of a given neuronal assembly, from the vantage point of a single neuron, 250 action potentials of a single reference cell were used for spike triggered averaging of the corresponding optical data. The result is a movie, in which each frame depicts the pattern of cortical activity at a certain distance in time (in 3 ms intervals) from the occurrence of the spike, A dominant feature of the movie is an oscillation between two recurring patterns, having nearly opposite polarity (top and bottom images), at a frequency of 11 Hz (which is characteristic for the alpha rhythm), The top three 3-D images (frames) show 3 separate occurrences of one of these patterns, and the bottom three show 3 separate occurrences of the other, the latter being a negative image of the former, The graph shows the similarity (as measured by the correlation coefficient) between each frame of the movie and the representative frame of the first pattern (middle frame at the top). The oscillation between the two patterns is clearly seen in the graph,
orientation, depth and color, in striate cortex. The results established the relationships between the three subsystems of CO blobs, iso-orientation domains, and ocular dominance columns; the architecture of basic "processors chips" i.e., fundamental modules analyzing each point on the retinal image, was determined. However it appears that a regular mosaic of fundamental modules does not exist, but instead regular patchy mosaics of all the three subsystems subserve the processing of a single point on
The first use of optical imaging for the visualization of dynamic patterns of electrical activity was reported in 1982 for brain slices (Grinvald et al. 1982b). Optical imaging of responses to a sensory stimulus in the intact brain was first accomplished in 1984 (Grinvald et al. 1984, Orbach et al. 1985). Cortical processing of transformed retinal images is carried out in the myriad dendritic arborizations of i n d i v i d u a l cortical neurons. The processing involves complex dendritic integration of multiple inputs. In cortical tissue, real-time imaging based on voltage-sensitive-dyes reflects mostly the inputs impinging on the fine dendrites of cortical cells, rather than the spike activity in the cell somata. Therefore, optical imaging yields pictures very different from those obtained with electrical recording techniques. Indeed, optical imaging experiments revealed extensive long-range interactions between cortical modules (Grinvald et al. 1991a; Ratzlaff and Grinvald 1991). It appears that distributed but orderly processing in primary sensory areas plays a larger role than that appreciated previously. Recently the hypothesis that coherent activity in large cell assemblies plays a prominent role in encoding and decoding sensory input was tested. By combining real-time optical imaging with electrical recordings from single neurons, neuronal assemblies were visualized for the first time, and the possible role of on-going (spontaneous) brain waves was explored. On-going activity exhibited highly-ordered, recurring spatio-temporal patterns of "Clock-like" cortical waves, scanning the cortex (see figure 3). The results indicated that some recurring patterns of brain waves are an intrinsic property of the cortex and play an important role in sensory perception (Grinvald et al. 1991a).
References Anglister, L., Farber, I.C., Shahar, A. and Grinvald, A. Localization of voltage-sensitive calcium channels along developing neurites; their possible role in regulatin~ neurite elongation. Dev. Biol., 1982, 94: 351-365. Bartfeld, E. and Grinvald, A. Architecture of processing modules in primate striate cortex underlying color, orientation and depth perception. Proc. Natl. Acad. Sci. USA., 1992, (in press).
Optical Imaging of the Living Brain
Bonhoeffer, T. and Grinvald, A. Iso-orientation domains in cat visual cortex are arranged in pinwheel like patterns. Nature, 1991, 353: 429-431. Cohen, L.B. Optical approaches to neuron function. Ann Rev. Physiol. 1989, 51: 487-582. Frostig, R.D., Lieke, E.E., Ts'o, D.Y. and Grinvald, A. Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high resolution optical imaging of intrinsic signals. Proc. Natl. Acad. Sci. USA, 1990, 87: 6082-6086. Grinvald, A., Salzberg, B.M. and Cohen, L.B. Simultaneous recording from several neurons in an invertebrate central nervous system. Nature, i977, 268: 140-141. Grinvald, A., Cohen, L.B., Lesher, S. and Boyle, M.B. Simultaneous optical monitoring of activity of many neurons in invertebrate ganglia using a 124 element photodiode array. J. Neurophysiol., 1981, 45: 829-840. Grinvald, A. and Farber, I.C. Optical recording of calcium action potentials from growth cones of cultures neurons using a laser microbeam. Science, 1981, 212: 1164-1166. Grinvald, A., Ross, W.N. and Farber, I.C. Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons. Proc. Natl. Acad. Sci. USA, 1981, 78: 3245-3249. Grinvald, A., Hildesheim, R., Farber, I.C. and Anglister, L. Improved fluorescent probes for the measurements of rapid changes in membrane potential. Biophys. J., 1982, 39: 301308. Grinvald, A., Manker, A. and Segal, M. Visualization of the spread of electrical activity in rat hippocampal slices by voltage-sensitive optical probes. J. Physiol., 1982, 333: 269291. Grinvald, A., Fine, A., Farber, I.C. and Hildesheim, R. Fluorescence monitoring of electrical responses from small neuroris and their processes. Biophys. J., 1983, 42: 195-198. Grinvald, A. Real-time optical imaging of neuronal activity: from growth cones to the intact brain. Trends Neurosci., 1984, 7: 143-150. Grinvald, A., Anglister, L., Freeman, J.A., Hildesheim, R. and Manker, A. Real-time optical imaging of naturally evoked electrical activity in intact frog brain. Nature, 1984, 308: 848-850. Grinvald, A., Lieke, E., Frostig, R.D., Gilbert, C.D. and Wiesel T.N. Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature, 1986, 324: 361-364.
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Grinvald, A., Salzberg, B.M., Lev-Ram, V. and Hildesheim, R. Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes. Biophys. J., 1987, 51: 643-651. Grinvald, A., Frostig, R.D., Lieke E. and Hildesheim, R. Optical imaging of neuronal activity. Physiol. Rev., 1988, 68: 12851366. Grinvald, A., Bonhoeffer, T., Malonek, Shoham, Bartfeld, E., Arieli, A., Hildesheim R. and & Ratzlaff, E.H. Optical imaging of architecture and function in the living brain. In: Memory: Organization and Locus of Change. Oxford University Press, 1991: 49-85. Grinvald, A., Frostig, R.D., Siegel, R.M. and Bartfeld, E. Highresolution optical imaging of functional brain architecture in the awake monkey. Proc. Natl. Acad Sci. USA, 1991, 88: 11559-11563. Haugland, M.M., Ojemann, G.A. and Hochman, D.W. Optical imaging of epileptiform and functional activity in human cerebral cortex. Nature, 1992, 358: 668-671. Kwong, K.K., Belliveau, J.W., Chesler, D.A., Goldberg, I.E., Weisskopf, R.M., Poncelet, B.P., Kennedy, D.N., Hoppel, B.E., Cohen, M.S., Turner, R., Cheng, H.-M., Brady, T.J. and Rosen, B.R. Dynamic magnetic resonance imaging of human brain activity during primary sensory stimulation. Proc. Nail. Acad. Sci. USA, 1992, 89: 5675-5679. Lieke, E.E., Frostig, R.D., Arieli, A., Ts'o, D.Y., Hildesheim, R. and Grinald, A., Optical imaging of cortical activity: a realtime imaging using extrinsic dye-signals and high resolution imaging based on slow intrinsic-signals. Ann. Rev. Physiol., 1989, 51: 543-559. Ogawa, S., Tank, D.W., Menon, R., Ellermann, J.M., Kim, S.-G., Merkle, H. and Ugurbil, K. Intrinsic signal changes accompanying sensory stimulation: functional brain mapping with magnetic resonance imaging. Proc. Nail. Acad. Sci. USA, 1992, 89: 5951-5955. Orbach, H.S., Cohen, L.B. and Grinvald, A. Optical mapping of electrical activity in rat somatosensory and visual cortex. J. Neurosci., 1985, 5: 1886-1895. Ratzlaff, E.H. and Grinvald, A. A tandem-lens epifluorescence macroscope: hunderd-fold brightness advantage for widefield imaging. J. Neurosci. Methods, 1991, 36: 127-137. Ts'o, D.Y., Frostig, R.D., Lieke, E.E. and Grinvald, A. Functional organization of primate visual cortex revealed by high resolution optical imaging. Science, 1990, 249: 417-420.
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Optical Imaging of Architecture and Function in the Living Brain Sheds New Light on Cortical Mechanisms Underlying Visual Perception A. Grinvald* Summary: Long standing questions related to brain mechanisms underlying perception can finally be resolved by direct visualization of the architecture and function of mammalian cortex. This advance has been accomplished with the aid of two optical imaging techniques with which one can literally see how the brain functions. The upbringing of this technology required a multi-disciplinary approach integrating brain research with organic chemistry, spectroscopy, biophysics, computer sciences, optics and image processing. Beyond the technological ramifications, recent research shed new light on cortical mechanisms underlying sensory perception. Clinical applications of this technology for precise mapping of the cortical surface of patients during neurosurgery have begun. Below is a brief summary of our own research and a description of the technical specifications of the two optical imaging tectuaiques. Like every tecln~ique, optical imaging also suffers from severe limitations. Here we mostly emphasize some of its advantages relative to all alternative imaging techniques currently in use. The limitations are critically discussed in our recent reviews. For a series of other reviews, see Cohen (1989). Key words: Optical imaging; Brain functional architecture; Living brain; Oximetry; Neuronal assemblies; Real-time imaging; Voltage-sensitive-dyes.
Optical imaging techniques offer unique advantages that are particularly suitable for exploring the architecture and real-time function of primary cortical areas, as well as higher cognitive brain functions. By simultaneously imaging the activity of millions of neurons, rather than one neuron at a time, these optical imaging techniques have shed new light on the organization and function of the cortex.
Development of optical imaging techniques Two optical imaging techniques are involved, realtime optical imaging and imaging based on activity dependent intrinsic signals.
Real-time optical imaging The first technique is based on vital staining of cortex with voltage-sensitive dyes and imaging the activity dependent fluorescence changes. Voltage-sensitive dyes *Department of Neurobiology, The Weizmann Institute of Science, Rehovot, Israel. Accepted for publication: September 1l, 1992. Correspondence and reprint requests should be addressed to Prof. A. Grinvald, Department of Neurobiology, The Weizmann Institute of Science, Rehovot 76100, Israel. Copyright © 1992 Human Sciences Press, Inc.
bind externally to neuronal m e m b r a n e s and act as m o l e c u l a r t r a n s d u c e r s , t r a n s f o r m i n g c h a n g e s in membrane potential into optical signals. Investigators in the field have realized the hope of the famous British physiologist, Lord Sherrington who marvelled, some 40 years ago, at "how m u c h easier it would be to understand the brain if neurons would light-up w h e n they talk to one another". The first steps in this direction were taken by the pioneering work of the research groups of Drs. Taskai and Cohen, at the NIH and Yale. Our group contributed to the transformation of this approach from a promising idea into an effective tool, currently used to study numerous model systems used in brain research; from single neurons maintained in culture (Grinvald et al. 1981b; Grinvald and Farber 1981; Anglister et al. 1982), through intact invertebrate ganglia (Grinvald et al. 1977; Grinvald et al. 1981a), mammalian brain slices (Grinvald et al. 1982b) and the living brain (Grinvald et al. 1984; Orbach et al. 1985; Grinvald 1984, 1988, 1991a). Optical imaging has recently undergone an explosion, not only for optical monitoring of electrical activity, but also for the imaging of calcium, sodium, potassium and even important proteins. The most critical part of this technique has been the design of the molecular probes developed by Rina Hildesheim in our laboratory at the Weizmann Institute. More than 600 dyes were synthesized and most of them are the best available for a variety of applications (Grinvald et al. 1977, 1982a,b,
72
1983, 1984, 1986, 1987). Below we briefly discuss the performance of real-time optical imaging, based on voltage-sensitive dyes.
Performance specifications of real-time optical imaging The optical signals reflect the intracellular changes in membrane potential. The spatial resolution is 1 ~tm for two-dimensional preparations and 30-50 ~m for threedimensional, thick preparations (e.g., slices, cortex). Three-dimensional imaging is feasible with optical sectioning and deconvolution, and perhaps also with confocal microscopy. Both evoked activity and on-going activity can be imaged. The method can be used to evaluate pharmacological effects of drugs in-vitro and in-vivo. The optical maps were highly correlated with both anatomical and electrophysiological maps. The smallest size of electrical activity which can be recorded depends on the preparation. For example, in processes of single cells I mV synaptic potential could be detected. Photodynamic damage is the restricting factor of determining the limit of sensitivity of the method. Attempts to enhance the sensitivity of the method by designing better dyes has been a continuous project, and additional improvements are likely. The duration of the recording session can be as short as a few ms, and in many situations, including in-vivo studies, averaging is not required. Once the cortex is exposed, the method is relatively n o n - i n v a s i v e , a l t h o u g h p h o t o d y n a m i c damage is a limiting factor and pharmacological side-effects of the dyes must be evaluated for each dye in each preparation. Such critical evaluation has not yet been made for the application of this approach in human subjects. From animal studies it appears that this optical method may have clinical applications.
Imaging based on visualization of oxygen delivery The second technique is based on imaging of intrinsic changes in the optical properties of electrically active brain tissue. One source for the activity dependent optical signal is a small change in the color of the tissue, produced by oxygen delivery from oxy-hemoglobin within the capillaries in response to metabolic demand. Other intrinsic signals originate from activity dependent light scattering changes and changes in the oxidation states of intrinsic chromophores such as cytochromes. The pioneering work of Hill, Keynes, Chance, Jobsys and their colleagues began to uncover the existence of these intrinsic signals some 45 years ago. However, since these signals are very small, their use for imaging of the functional architecture of cortex began only in 1986 (Grinvald et al. 1986). This imaging technique permitted the highresolution imaging of the functional architecture of the
Grinvald
visual cortex in the living brain of cats and monkeys (Grinvald et al. 1986; Ts'o et al. 1990; Ratzlaff and Grinvald 1991). In addition, activity maps were also obtained through the intact dura and thinned bone; this should facilitate the direct visualization of the fascinating development of the meticulous, functional architecture of the cortex (Frostig et al. 1990). Furthermore, this technique was successfully applied to the imaging of functional architecture in the cortex of awake behaving monkey (Grinvald et al. 1991b). The latter application suggests that imaging based on intrinsic signals will contribute to the exploration of higher cognitive functions. In addition, clinical brain mapping, as an aid for n e u r o s u r g e o n s , is c u r r e n t l y being i m p l e m e n t e d (Haugland et al. 1992). The finding that activity dependent changes in the microcirculation can facilitate high resolution mapping, indirectly led to the exciting development of MRI functional imaging, based on the same mechanisms underlying the optical imaging (Kwong et al. 1992; Ogawa et al. 1992). Below we briefly summarize some performance specifications of these first optical imaging methods.
Performance specifications of imaging based on intrinsic signals The intrinsic optical signal originates from the oxyhemoglobin to hemoglobin transition that occurs in response to metabolic demands. When using dim red light (605 nm), mostly oxygen delivery is directly imaged. At other wavelengths, changes in blood volume in capillaries and light scattering changes can also be imaged. The spatial resolution of the technique is 50 to 100 ~tm. The rise-time of the intrinsic signals is i to 2 seconds. The smallest region that can be recorded from is limited by optics, but the resolution is about 50 ~tm, depending on the density of capillaries in the imaged volume. Imaging based on light scattering signals may achieve a resolution of about I ~tm,but the signal-to-noise ratio is not as good. High-resolution, two-dimensional imaging was already achieved with a resolution of about 50 ~m while in the third dimension the signal is integrated from the surface of the cortex down to a depth of approximately 500 ~tm. It seems likely that using near infra-red light threedimensional imaging would also be feasible, provided that the signal-to-noise ratio can be improved by a factor of about three. This method is especially useful to record evoked activity, since averaging is required and subtraction of a background image is necessary to visualize the responsive areas. The method can be used to evaluate pharmacological effects of drugs in-vitro and in-vivo. The optical maps were highly correlated with both anatomical and electrophysiological maps. Functional maps of ocular dominance columns were obtained in
Optical Imaging of the Living Brain
73
\\
l Figure 1. Pinwheel-like structures are a prominent feature of the functional organization of orientation preferences (B). The micro-vasculature is clearly visible in this picture (A). Scale bar is 1 mm. B: Colour-coded orientation preference map in cat area 18. The preferred orientation for every location is c o d e d according to the scheme shown at the right of the figure; areas responding best to a horizontal bar are c o d e d in yellow; areas responding best to a vertical bar are c o d e d in blue, etc. It is apparent that the overall organization is continuous rather than banded. (Modified from Bonhoeffer and Grinvald 1991).
about 10 seconds. Imaging of functional domains which are less robust may takes a few hours. However, the duration of imaging sessions with this non-invasive method is probably unlimited. Currently, four groups are attempting the intra-operative implementation of this imaging technique to delineate functional borders in the cortex with a precision of less than 100 ~m. One group already reported functional images in patients during neurosurgery (Haugland et al. 1992)
Major findings related to mechanisms underlying sensory perception F u n c t i o n a l a r c h i t e c t u r e u n d e r l y i n g visual perception
Mountcastle in the somatosensory cortex and Hubel and Wiesel in the visual cortex have found that cortical cells with similar response properties are frequently
Figure 2. Close-up of 4 pinwheels. Two fundamental features of the pinwheels are illustrated in this figure. (a) In all '"pinwheels", each orientation appears once around the center. (b) The pinwheels exist in two forms: a clockwise form (left side), in which the sequence of the colors yellow-green-blue-red is clockwise, and a counterclockwise form (right side), in which the inverse is the case. Scale bar is 300 mm. (Modified from Bonhoeffer and Grinvald 1991).
clustered together, forming columns (modules) which often traverse the entire cortical depth, from pia to the white matter. Previous work relied on electrophysiological, anatomical and histological techniques to reveal the organization of each type of module. However, the exact functional organization of different functional modules parallel to the cortical surface still remains an unresolved question for all areas of the mammalian cortex. Optical imaging is a particularly attractive technique for exploring this organization. Our research in cat visual cortex unexpectedly established that the most prominent organizational feature of orientation-preference is a radial arrangement, forming a pinwheel-like structure surrounding a singularity point (figure I and figure 2). Figure I shows how merely by taking a picture of exposed cortex (A), as the animal is viewing gratings of various orientations, the map of orientation preference can be derived (B). The same elaborate organization, underlying the visual perception of oriented lines or edges, was found also in the primate. Our subsequent research on primates explored the relationships between three subsystems playing a major role in visual processing of
74
GrinvaJd
the retina, probably using population coding. In superficial layers these patches exhibit fuzzy borders; the response properties change smoothly across the cortical surface. The size of each of these m o d u l e s is approximately 800 by 400 ~tm. The reported architecture is different from previously described experimental and theoretical models (Bartfeld and Grinvald in press).
i
i
The organization of d y n a m i c patterns of activity in neuronal assemblies
1
i 8 -1
-100
0 FRAME NUMBER
100
Figure 3, Real-time optical imaging of brain waves oscillations, The 3-D images show the amplitudes of the dendritic depolarization in a 3 x 3 mm region of cat visual cortex, To visualize the dynamic behavior of a given neuronal assembly, from the vantage point of a single neuron, 250 action potentials of a single reference cell were used for spike triggered averaging of the corresponding optical data. The result is a movie, in which each frame depicts the pattern of cortical activity at a certain distance in time (in 3 ms intervals) from the occurrence of the spike, A dominant feature of the movie is an oscillation between two recurring patterns, having nearly opposite polarity (top and bottom images), at a frequency of 11 Hz (which is characteristic for the alpha rhythm), The top three 3-D images (frames) show 3 separate occurrences of one of these patterns, and the bottom three show 3 separate occurrences of the other, the latter being a negative image of the former, The graph shows the similarity (as measured by the correlation coefficient) between each frame of the movie and the representative frame of the first pattern (middle frame at the top). The oscillation between the two patterns is clearly seen in the graph,
orientation, depth and color, in striate cortex. The results established the relationships between the three subsystems of CO blobs, iso-orientation domains, and ocular dominance columns; the architecture of basic "processors chips" i.e., fundamental modules analyzing each point on the retinal image, was determined. However it appears that a regular mosaic of fundamental modules does not exist, but instead regular patchy mosaics of all the three subsystems subserve the processing of a single point on
The first use of optical imaging for the visualization of dynamic patterns of electrical activity was reported in 1982 for brain slices (Grinvald et al. 1982b). Optical imaging of responses to a sensory stimulus in the intact brain was first accomplished in 1984 (Grinvald et al. 1984, Orbach et al. 1985). Cortical processing of transformed retinal images is carried out in the myriad dendritic arborizations of i n d i v i d u a l cortical neurons. The processing involves complex dendritic integration of multiple inputs. In cortical tissue, real-time imaging based on voltage-sensitive-dyes reflects mostly the inputs impinging on the fine dendrites of cortical cells, rather than the spike activity in the cell somata. Therefore, optical imaging yields pictures very different from those obtained with electrical recording techniques. Indeed, optical imaging experiments revealed extensive long-range interactions between cortical modules (Grinvald et al. 1991a; Ratzlaff and Grinvald 1991). It appears that distributed but orderly processing in primary sensory areas plays a larger role than that appreciated previously. Recently the hypothesis that coherent activity in large cell assemblies plays a prominent role in encoding and decoding sensory input was tested. By combining real-time optical imaging with electrical recordings from single neurons, neuronal assemblies were visualized for the first time, and the possible role of on-going (spontaneous) brain waves was explored. On-going activity exhibited highly-ordered, recurring spatio-temporal patterns of "Clock-like" cortical waves, scanning the cortex (see figure 3). The results indicated that some recurring patterns of brain waves are an intrinsic property of the cortex and play an important role in sensory perception (Grinvald et al. 1991a).
References Anglister, L., Farber, I.C., Shahar, A. and Grinvald, A. Localization of voltage-sensitive calcium channels along developing neurites; their possible role in regulatin~ neurite elongation. Dev. Biol., 1982, 94: 351-365. Bartfeld, E. and Grinvald, A. Architecture of processing modules in primate striate cortex underlying color, orientation and depth perception. Proc. Natl. Acad. Sci. USA., 1992, (in press).
Optical Imaging of the Living Brain
Bonhoeffer, T. and Grinvald, A. Iso-orientation domains in cat visual cortex are arranged in pinwheel like patterns. Nature, 1991, 353: 429-431. Cohen, L.B. Optical approaches to neuron function. Ann Rev. Physiol. 1989, 51: 487-582. Frostig, R.D., Lieke, E.E., Ts'o, D.Y. and Grinvald, A. Cortical functional architecture and local coupling between neuronal activity and the microcirculation revealed by in vivo high resolution optical imaging of intrinsic signals. Proc. Natl. Acad. Sci. USA, 1990, 87: 6082-6086. Grinvald, A., Salzberg, B.M. and Cohen, L.B. Simultaneous recording from several neurons in an invertebrate central nervous system. Nature, i977, 268: 140-141. Grinvald, A., Cohen, L.B., Lesher, S. and Boyle, M.B. Simultaneous optical monitoring of activity of many neurons in invertebrate ganglia using a 124 element photodiode array. J. Neurophysiol., 1981, 45: 829-840. Grinvald, A. and Farber, I.C. Optical recording of calcium action potentials from growth cones of cultures neurons using a laser microbeam. Science, 1981, 212: 1164-1166. Grinvald, A., Ross, W.N. and Farber, I.C. Simultaneous optical measurements of electrical activity from multiple sites on processes of cultured neurons. Proc. Natl. Acad. Sci. USA, 1981, 78: 3245-3249. Grinvald, A., Hildesheim, R., Farber, I.C. and Anglister, L. Improved fluorescent probes for the measurements of rapid changes in membrane potential. Biophys. J., 1982, 39: 301308. Grinvald, A., Manker, A. and Segal, M. Visualization of the spread of electrical activity in rat hippocampal slices by voltage-sensitive optical probes. J. Physiol., 1982, 333: 269291. Grinvald, A., Fine, A., Farber, I.C. and Hildesheim, R. Fluorescence monitoring of electrical responses from small neuroris and their processes. Biophys. J., 1983, 42: 195-198. Grinvald, A. Real-time optical imaging of neuronal activity: from growth cones to the intact brain. Trends Neurosci., 1984, 7: 143-150. Grinvald, A., Anglister, L., Freeman, J.A., Hildesheim, R. and Manker, A. Real-time optical imaging of naturally evoked electrical activity in intact frog brain. Nature, 1984, 308: 848-850. Grinvald, A., Lieke, E., Frostig, R.D., Gilbert, C.D. and Wiesel T.N. Functional architecture of cortex revealed by optical imaging of intrinsic signals. Nature, 1986, 324: 361-364.
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Grinvald, A., Salzberg, B.M., Lev-Ram, V. and Hildesheim, R. Optical recording of synaptic potentials from processes of single neurons using intracellular potentiometric dyes. Biophys. J., 1987, 51: 643-651. Grinvald, A., Frostig, R.D., Lieke E. and Hildesheim, R. Optical imaging of neuronal activity. Physiol. Rev., 1988, 68: 12851366. Grinvald, A., Bonhoeffer, T., Malonek, Shoham, Bartfeld, E., Arieli, A., Hildesheim R. and & Ratzlaff, E.H. Optical imaging of architecture and function in the living brain. In: Memory: Organization and Locus of Change. Oxford University Press, 1991: 49-85. Grinvald, A., Frostig, R.D., Siegel, R.M. and Bartfeld, E. Highresolution optical imaging of functional brain architecture in the awake monkey. Proc. Natl. Acad Sci. USA, 1991, 88: 11559-11563. Haugland, M.M., Ojemann, G.A. and Hochman, D.W. Optical imaging of epileptiform and functional activity in human cerebral cortex. Nature, 1992, 358: 668-671. Kwong, K.K., Belliveau, J.W., Chesler, D.A., Goldberg, I.E., Weisskopf, R.M., Poncelet, B.P., Kennedy, D.N., Hoppel, B.E., Cohen, M.S., Turner, R., Cheng, H.-M., Brady, T.J. and Rosen, B.R. Dynamic magnetic resonance imaging of human brain activity during primary sensory stimulation. Proc. Nail. Acad. Sci. USA, 1992, 89: 5675-5679. Lieke, E.E., Frostig, R.D., Arieli, A., Ts'o, D.Y., Hildesheim, R. and Grinald, A., Optical imaging of cortical activity: a realtime imaging using extrinsic dye-signals and high resolution imaging based on slow intrinsic-signals. Ann. Rev. Physiol., 1989, 51: 543-559. Ogawa, S., Tank, D.W., Menon, R., Ellermann, J.M., Kim, S.-G., Merkle, H. and Ugurbil, K. Intrinsic signal changes accompanying sensory stimulation: functional brain mapping with magnetic resonance imaging. Proc. Nail. Acad. Sci. USA, 1992, 89: 5951-5955. Orbach, H.S., Cohen, L.B. and Grinvald, A. Optical mapping of electrical activity in rat somatosensory and visual cortex. J. Neurosci., 1985, 5: 1886-1895. Ratzlaff, E.H. and Grinvald, A. A tandem-lens epifluorescence macroscope: hunderd-fold brightness advantage for widefield imaging. J. Neurosci. Methods, 1991, 36: 127-137. Ts'o, D.Y., Frostig, R.D., Lieke, E.E. and Grinvald, A. Functional organization of primate visual cortex revealed by high resolution optical imaging. Science, 1990, 249: 417-420.
