Optical Neurophysiology Based on Animal Models - IEEE Xplore

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OPTICAL IMAGING

Optical Neurophysiology Based on Animal Models Animal Models Provide Precision and Experimental Control Not Typically Available in Humans

BY JEFFREY J. SABLE, DAVID M. RECTOR, AND GABRIELE GRATTON

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s is often the case in biological science, research in animal models has made substantial contributions to optical imaging of brain function. In this article, we provide a brief, selective review of work that is particularly relevant to noninvasive human optical neuroimaging (with an emphasis on more recent work). Because studies in animal models are often invasive, they provide detail about optical physiology that is not available through human research. These studies provide information about underlying physiology and therefore about the validity of assumptions in noninvasive optical imaging in humans. Considerable work (particularly early optical imaging work) has been done in animals using the administration of exogenous contrast agents such as voltage-sensitive dyes. However, we focus on imaging of intrinsic optical signals, meaning that the changes measured are inherent to the tissue itself and no contrast agents are administered. This work is most directly comparable to research with humans. Although the addition of contrast agents may enhance sensitivity to certain physiological events, there are complications with their use, such as photobleaching and neurotoxicity, and their administration is itself invasive (see [1] for a comparison of the methods). We begin with studies of optical changes that have used primarily in vitro preparations, which provide information about the basic physiology that may underlie various optical signals. We then build up to studies in living animals that examine various optical responses in more natural conditions. Table 1 briefly describes some of the basic terms that apply to the optical methods used in the studies we describe. These studies illustrate the many complexities that are inherent in optical imaging of intrinsic signals and that must be considered when conducting noninvasive imaging in humans. Detailed descriptions of cellular and molecular mechanisms underlying optical changes are beyond the scope of this article. For the interested reader, excellent reviews have been published on such topics as cellular events that determine hemodynamic changes in the brain [2], neurotransmitter control of blood flow [3], and local control of brain homeostasis by “functional units” [4]. For a review of the cellular basis of events that can lead to fast scattered light changes see [5] and [6]. For a thorough review of optical neuroimaging of intrinsic signals up to the mid-1990s, see [7].

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Stimulus-Evoked Scattering Changes in Neural Tissue

Scattering changes in nerve tissue following transient activation have been recognized since the 1940s, when opacity changes to white light were identified in the nerve from the walking leg of the shore crab (which is approximately 0.5 mm in diameter) [8]. These changes did not coincide with the nerve impulses themselves but rather occurred over several minutes following electrical stimulation. Opacity increased during the first few seconds, then reversed with a peak decrease at about two minutes. It took 10 to 20 minutes for the signal to return to baseline. Increasing signal-to-noise levels by averaging many trials made it possible to see scattering changes that followed the compound action potential somewhat closely [9]. By manipulating the refractive index and osmotic pressure, it was determined that axon volume changes probably did not cause the initial transient scattering increase, but it could be responsible for the longer-term change. Optical changes corresponding with action potentials in the squid giant axon were measured with 45-degree light scattering, although the onset of optical changes occurred significantly later than electrical changes. There was also a small, longer-lasting optical change (>10 ms). Changes in 45-degree birefringence (which were approximately 10 times as large as scattering changes) indicated that axoplasm made little if any contribution to the action potential change, placing the mechanism in the membrane. Further examination revealed that birefringence changes were tightly coupled (although with a slight time lag) to membrane potential changes (the authors admitted that this could be due to ionic compression). Subsequent work continued to show that neural activation produces changes in the intrinsic optical scattering and birefringence properties of neural tissue that closely track the electrophysiological response [10]–[15]. There are many mechanisms that may contribute to these changes. Fast optical signals have been difficult to record in intact neural tissue. Experiments, including recordings from cat hippocampus with sensitive photodiodes, showed that the recording of fast optical signals can be optimized with darkfield techniques [14]–[19]. By optimizing a video probe to detect changes in scattered light with dark-field illumination, fast optical responses in vivo can be made evident [15], [17]. 0739-5175/07/$25.00©2007IEEE

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The physiology of the intact cortex has many more variables than isolated nerve tissue, and the larger signals are relatively slow compared to neuronal activity.

These studies demonstrated the sequential elicitation of brainstem and cortical evoked responses through electrical stimulation of the vagus nerve bundle. Cellular swelling in vivo can lead to profound changes in optical properties of the tissue and may be their principal biophysical mechanism. Total brain volume is composed roughly of 30–40% neurons, 40–50% glial cells, and 15–25% extracellular space [20]–[21]. In cerebral cortical slices, cellular swelling was observed during anoxia and membrane depolarization [22]. Such swelling reduces the extracellular spaces [23]–[24], and affects neural and glial function. Since activation can decrease the extracellular fraction by 67% [25], a reduction in extracellular space increases the excitability of neurons by increasing the proximity of pre- and postsynaptic structures [26]. These profound dynamics in cellular pressure and volume underscore the physical mechanisms that might lead to optical changes in the tissue, possibly due to dilution of intracellular components or a change in refractive index. Since the cellular membrane normally has invaginations, cellular swelling pressure can be relieved by stretching the cell’s membrane, causing changes in light scattering and birefringence. Neurotransmitter-filled vesicles tend to accumulate at the axon terminal, thus forming a high concentration of scattering particles in a localized region. Fusion of the vesicles through activation-induced exocytosis could spread the scattering particles across a larger area. The presence of a complex cytoskeleton within the axoplasm of the neuron creates additional sources of scatteredlight changes. Microtubules exist in all parts of the neuron and

are responsible for creating the shape and structure of the cell membrane. Recent birefringence studies of microtubules show that a significant portion of light rotation can be attributed to bundled microtubules [27]. Since neurons contain an extensive network of microtubules, a large fraction of light-scattering changes may arise from structural alterations in the microtubule lattice. Microtubule formations are particularly dense within dendrites [28]. At least some of the scattered-light changes can occur from direct effects of the gel-like axoplasm that composes the inner layers of the cell [29]. The axoplasm consists of a polymerbased hydrogel that expands with water during activation. As the hydrogel expands, its refractive index and scattering properties change. Through use of artificial hydrogels, Tasaki showed that the propagation of the action potential could be accompanied by a sequence of mechanical events that move along the length of the nerve. Although the cell body of the neuron contains many components that scatter light, most investigators in the field do not consider this part of the cell to be important in producing activation-induced optical changes. The fractional swelling volume change is small for a large object such as the cell body when compared to smaller objects such as dendrites [30]. Mostly by imaging transverse sections of a hippocampal slice, it was shown that increases in the amplitude of the CA1 population spike (evoked by Schaeffer collateral stimulation) were accompanied by a hypo-osmotic increase in light transmission through the slice [31]. Detailed analysis of images from the hippocampal slice revealed that the CA1 dendritic layer

Table 1. Description of general terms for optical imaging methods. Transmission

The amount of light that passes through a volume of tissue.

Reflectance

The amount of source light that is reflected back from the surface (or very shallow depths) of tissue.

Scattering

The amount of light that is refracted by a volume of tissue. Typically measured either as the light detected at a 90-degree angle to the light source (in the case of nerves) or as the amount of light backscattered in the direction of the light source.

Birefringence

Light from the source is passed through a polarizer, which only allows light oriented in a particular plane to pass. A filter is also placed over the detector at a particular orientation relative to the source filter (e.g., 45 degrees), such that the light must be scattered from the original plane to the new plane in order to be detected.

Dark-field illumination

The perimeter of an area of tissue is illuminated, and the backscattered light is detected using sensors inside the light-source perimeter.

Autofluorescence

Light of a specific wavelength is used to excite molecules in a particular state (e.g., oxidized) within the tissue. These molecules then emit light of a different wavelength, which is detected.

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nals. Images of responses to different stimulus types could also be combined to produce differential maps. These methods provided excellent spatial resolution, and are still widely used, although charge-coupled devices (CCDs) are often used as detectors instead of photodiodes. Visual cortex can also be imaged through intact dura or thinned skull [40], although resolution is lost with more tissue. In vivo studies using this type of methodology have shown that various physiological events can be measured in the intact, functioning brain. This opened the door to the optical study of the complex nature of these events and of the relationships among them. Acquisition of scattered light from a single point on the dorsal hippocampal surface with a high-sensitivity photodiode revealed a strong inverse correlation between evoked potentials and light-scattering changes in the hippocampus [41]. Stimulation produced a complex electrical response with at least two components: an early population spike within 10 ms of the stimulus and a population postsynaptic potential within 50 ms. The light-scattering signal showed an inverse relationship to the electrical signal with at least three components: a small, fast response, the onset of which preceded the early population spike by 5 ms; a slow response that corresponded to the population postsynaptic potential; and a very slow response lasting several seconds that may have resulted from vascular

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displayed a much greater change in light transmittance than the cell body layer. In particular, the percent change in the stratum oriens (dendritic layer of the hippocampus) was 5.5%, in stratum radiatum (axonal layer) it was 7%, and in the stratum pyramidale (cell body layer) it was only 2%. The presence of glial cells and small interneurons within the dendritic layers may also contribute significantly to scattered light changes in these experiments—possibly causing confusion as to the source of the scattering signal. Microvessel swelling in direct response to neural activation might also play a role in slow light-scattering changes. Such swelling is responsible for delivering blood to the activated area and may be mediated by direct neural connections to the vascular smooth muscle, or by nitric oxide (NO) release into the interstitial space. Optical changes within smooth muscle can also occur in the absence of blood and may contribute to some of the slower scattering changes seen in hippocampal slices [32], [33]. At least one study did not observe optical changes in vivo in the absence of blood, but neither dark-field nor birefringence illumination was used [34]. A survey of birefringence responses from different nerves of crayfish and lobster showed that the temporal structure of the optical response depended on the type of nerve used and sufficient sensitivity to record from nerves as small as crayfish ventral cord (250 µm) [35]. The larger nerves produced optical responses that could be observed in single trials. A delay in the onset of the 90-degree scattered light change compared to the birefringence signal (see Figure 1) implies different biophysical mechanisms for these two signals. The change in birefringence may be closely related to a change in membrane potential [10], [36] due to reorientation of voltage-sensitive channel molecules and phospholipids in the axon membrane. The 90-degree scattered light changes are presumably caused by swelling from an ionic driven influx of water into the cell during activation. An elastic axon would also delay the swelling effect. The birefringence signal shows significantly more temporal structure and at least an order of magnitude greater amplitude than the 90-degree scattering, making the birefringence signal easier to obtain, and potentially more useful for imaging fast neural events. These studies have primarily used isolated nerve and brain slice preparations to investigate the optical phenomena associated with neural activity at a cellular level. As we have described, they implicate both membrane and structural changes as the basis for scattering and birefringence changes. Ideally, it would be possible to measure these types of neuronal events noninvasively in the human brain. However, these signals are quite small relative to other types of physiological activity occurring in the brain.

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From In Vitro to In Vivo

Of course, the physiology of the intact cortex has many more variables than isolated nerve tissue, and the larger signals are relatively slow compared to neuronal activity. Intrinsic optical signals in intact cortex were initially seen by some as noise in recordings using contrast agents (see [37], [38]). Initial imaging studies involved measurements of exposed cortex with a setup in which a light was shone on the cortical surface and reflected light was detected with a photodiode array [39]. Recordings during an unstimulated period were subtracted from those made during various types of stimulation to increase the dynamic range needed for imaging very small sigIEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE

Fig. 1. Simultaneous recording of changes in birefringence and 90-degree scattered light signals from lobster nerve show electrophysiology (thin lower trace) with corresponding birefringence (thin trace) that occurs 3.0 ms earlier than the 90degree scattered light (bold trace) signal (300 averages). The vertical calibration bar also shows that the birefringence signal (0.25 × 10-4) is nearly an order of magnitude larger than the scattering signal (0.5 × 10-5). The arrow points to the time of stimulation. Reprinted from [35] with permission from Elsevier.

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tive response (P80) that peaked 80 ms after the stimulus, a late and long-lasting positive response (P300), and a slow negative response lasting at least 800 ms (N800). The two faster responses (N30, P80) were limited in their spatial distribution and are likely to represent processes directly associated with neural activation. The two slower components (P300, N800) were more widespread and overlapped with regions corresponding with blood vessels. The time course of the slower components matched the hemodynamic mapping responses reported in earlier studies [39], while the faster responses probably represent direct neural response components not seen in previous studies. Using dark-field illumination, fast changes in the rat cortex were seen with components that paralleled signals observed in rat dorsal medulla [42]. When single whiskers were stimulated and the somatosensory cortex was imaged, the integrated signal across the image showed a dynamic

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processes. The amplitude of the evoked light-scattering change depended on where the stimulus was applied. When the photodiode was replaced with a CCD camera and images captured at the peak of the evoked response, stimulation of different regions of an 8 × 2-mm area of the contralateral dorsal hippocampus produced a topographical map of activation. Fast optical responses to nerve stimulation in the rat dorsal medulla elicited optical response patterns, detected as changes in back-scattered light [15]. A spatio-temporal analysis of the response patterns showed four distinct time courses with components that paralleled fast electrical evoked responses as well as slower hemodynamic signals. Nerve shocks elicited an electrical population spike 30 ms after the stimulus and a population evoked potential peaking at 80 ms. Image sequences showed a distinct spatial pattern of activation within the tissue, with four distinct temporal components: an early negativegoing response (N30) lasting for 20 ms, an intermediate posi-

ERP

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100 ms Fig. 2. Evoked response potential (ERP) and optical signal averaged across all pixels of the imager (OPTICAL) in a CCD experiment showing a response to single whisker stimulation. The optical trace has been inverted to show similarity in temporal structure. An average of 25 baseline images (before stimulation) was subtracted from each frame in an average image sequence based on 1,000 trials. The spatial map constructed during the early peak response shows a discrete region that corresponds to the cortical column associated with the stimulated whisker. A later epoch reveals a spreading of the optical response across space that changes polarity after 500 ms and covers a large portion of the imaged area. In pseudocolor images, warm colors represent decreases in scattered light and cool colors represent scattered light increases. Reprinted from [42] with permission from Elsevier.


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optical response with an early transient corresponding to a time-damped integral of the electrophysiological response (see Figure 2). Optical responses were initially localized to individual cortical columns that represent single whiskers (whisker barrels). Later in time, the optical response spread out across the entire barrel field, with a reversal about 500 ms after stimulation. These response components were similar in spatial and temporal structure to those observed in previous studies in the brainstem. The early peak in the imaged response was in a different location, depending on which whisker was being stimulated. Stimulation of different whiskers and comparison of the regions of apparent activation revealed maps that were consistent in size and location with the expected position of somatosensory columns to which particular whiskers project. High-speed detection by a photodiode also showed evidence for high-speed oscillations (400–600 Hz) in the optical signal that are consistent with oscillations observed in the electrical recordings. While many thousands of averages were required to remove noise, this result suggests that fast optical changes can be observed in close correspondence with high-speed electrical events. Optical measurements directly from the brain established the feasibility of recording various physiological signals from brain tissue in living animals. Activity related to neuronal (and perhaps glial) activity can be measured both directly, as evidenced by responses that correspond with electrical activity, and indirectly, as reflected in measurements of hemodynamic changes. Imaging directly from the brain also showed that optical recording of intrinsic signals could provide high spatial and temporal resolution in mapping dynamic brain function. The relationship between neuronal and hemodynamic activity has proven to be somewhat complex and has been the focus of a great deal of research.

by larger vasculature. However, a more recent study [49] found that focusing on the first 2–3 seconds of the hemodynamic response—oxygenation or blood volume—provided spatial specificity to the level of cortical columns (rat whisker barrel). Their localization was improved with the use of a statistical thresholding procedure that focused on the capillary bed, which has a more consistent trial-to-trial response than the larger vessels. Blood volume and oxygenation changes occurred in an adjacent barrel on the same time scale (within the 250-ms sampling period), but with much lower intensity. Studies using flavoprotein autofluorescence found that transient increases in aerobic metabolism were more localized than subsequent increases in cerebral blood flow (CBF—measured with laser speckle imaging), even early in the responses [50], [51]. The time difference between these two phenomena should cause an initial HbR dip [51]. A recent review of initial dip studies [52] examined numerous possible causes for discrepancies among studies: imaging modality and methodology, depth of anesthesia, general blood oxygen level, hemodynamic differences among species (e.g., blood transit times), and cortical area. In addition, the author proposed a model for the initial dip, based in part on studies showing similar time courses of deoxygenation and NO concentration [53], [54]. Briefly, the model proposes that the early oxygen demand comes from a depletion of mitochondrial oxygen buffers, which is accompanied by other metabolic changes (including NO release). Blood flow increases to replenish oxygen and glucose are then triggered by the release of NO and/or other metabolites, which result in an oxygen overshoot [52]. In support of the role of NO in neurovascular coupling, hemodynamic increases following metabolic activity have largely been eliminated by application of a NO inhibitor directly to the cortex [50], [55].

Optical Spectroscopy and the “Initial Dip”

Rodent Barrels and Neurovascular Coupling

Early in vivo cortical imaging studies indicated that different stimulus-evoked neurophysiological events could be measured at different wavelengths [39], [40]. The advent of spectroscopic imaging of blood oxygenation led to detailed study of the temporal and spatial properties of the “initial dip” in the hemodynamic response [43]. In the visual cortex of an anesthetized cat, a rapid and relatively localized increase in deoxygenated hemoglobin (HbR) preceded the large increase in oxygenated hemoglobin (HbO2) by 1–2 seconds, both in its onset and in its peak. This “initial dip” in HbR provided evidence that brain metabolism was aerobic and was not preceded by an anaerobic phase. Neuronal activity was proposed to induce rapid, highly localized increases in HbR as oxygen is drawn out of the blood to replenish cellular demand. The large, slower, less localized blood volume increase, which is dominated by HbO2, then follows to replenish the depleted oxygen supply [43]. Other researchers have confirmed that the initial dip is confined to specific areas of activation, whereas the large volume response is much less specific (e.g., [44]–[47]). The existence and characteristics of the initial dip have since been debated at length. As originally described, the dip is more highly localized than other hemodynamic signals [43]. One review [48] of studies using a variety of optical methods supported the view that the most spatially specific responses are measured as early changes in the hemodynamic response that correspond with oxygen consumption. Subsequent blood volume and flow changes are less specific and are dominated

The somatosensory cortex of the rat is a popular model for study with optical imaging (e.g., [39]). The whiskers of the rat are distinctly topographically mapped onto the somatosensory cortex and have a relatively large representation on an accessible area of the dorsal surface of the brain [56]. An accurate stimulus control is possible and rats are a relatively easy animal model to use. Relatively high-resolution imaging can be done from exposed cortex or through the thinned skull [57] (and through the intact skull in mice; e.g., [58], [59]). Evidence from this system has called into question the assumption of a uniformly linear relationship between neuronal and hemodynamic activity (i.e., neurovascular coupling). Linearity has been found to exist for some optical measures and stimulus parameters (e.g., blood volume and stimulus intensity), but not for others (oxygenation and number of stimuli) [60]. Low levels of cortical neuronal activity (as measured by extracellular field potentials) may occur without corresponding increases in CBF (measured with laser Doppler flowmetry [61]). Partially due to this, neurovascular coupling was linear only within a limited range of stimulus frequencies and intensities. In contrast, slow reflectance decreases may precede seizure-related EEG activity in rat brain by as much as 1 minute [62]. In the study that found electrical responses without flow increases [61], CBF increased initially in the most superficial cortical layers but was largest in deeper layers that corresponded with thalamic input. Finally, pharmacological manipulations pointed to postsynaptic glutamate

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mechanisms as critical to both electrical and vascular changes. Thus, neurovascular coupling may depend on the nature of activation and the precision of the measurement. Hemodynamic response amplitudes have been found to increase linearly with stimulus intensity (single whisker deflections), well beyond the saturation point of electrical responses [63], [64]. In these studies, initial decreases in HbR were accompanied by decreases in HbO2 . In one study, HbO2 , HbR, and total hemoglobin (HbT) activity always occurred in an antagonistic center-surround pattern, with the center extending throughout most of the barrel cortex [64]. The surround was outside the barrel field and sometimes even outside the somatosensory cortex and was not accompanied by changes in multi-unit activity or local field potentials (see [65] for a detailed discussion of spreading cortical activation). Inhibitory surrounds have also been observed with optical imaging of mouse primary visual cortex [58]. The nonuniform distribution of cortical blood vessels appears to be an important factor in neurovascular coupling. Capillary density is not uniform across the cortex, and intrinsic signals have been found to correlate highly with areas of high density in chinchilla auditory cortex. This suggests that the specificity and sensitivity of optical imaging of hemodynamic activity may vary across the cortex, specifically favoring more highly vascularized areas such as primary sensory and motor areas. Some association areas may lack such activity altogether [66]. Questions have also been raised about the dynamics of large branches of cerebral vasculature. Stimulation-evoked cerebral blood volume increases in rat whisker barrel cortex are affected by simultaneous forelimb stimulation [67]. In general, forepaw stimulation reduced the optical response in barrel cortex. Most surprisingly, stimulation of the forepaw alone produced blood volume increases in barrel cortex in the absence of evoked potentials. This complex coupling of distinct cortical areas illustrates a dissociation between neural and hemodynamic activity. Note that these two somatosensory areas are fed by different branches of the middle cerebral artery. The use of multiple imaging strategies (intrinsic and extrinsic signals) in cat visual association cortex revealed that vascular responses started in arterioles, followed by capillary and then venous responses. This indicates that upstream communication is involved [68]. However, localized capillary deoxygenation was the fastest response found. The authors also noted a small difference between blood volume and HbO2 signals and suggested that this difference may be due to differential coupling to synaptic and spiking activity, respectively. Recently, hemodynamic activity was found to correlate with local field potential oscillations, particularly at very high (gamma) frequencies [69]. This suggests that synchronous neuronal firing may be critical in determining the hemodynamic response. The increased role of inhibitory cortical neurons in producing gamma-frequency synchronization may mediate this relationship. These studies show the complex relationship between neuronal activity and corresponding hemodynamic changes. Measurement of the early changes in the hemodynamic response is sensitive to many variables, including the methods used for analysis. In addition, there is a complex relationship between neuronal activity and corresponding hemodynamic changes. Neurovascular coupling is a very 22 IEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE

complex phenomenon and researchers should avoid simplistic interpretations of vascular effects in terms of a linear measure of neuronal phenomena. This applies to optical measures, as well as other hemodynamic methods (e.g., functional magnetic resonance imaging and positron emission tomography). The simultaneous measurement of neuronal and hemodynamic events can be very useful in understanding their coupling. These issues must be addressed in order to accurately infer various types of brain activity when measuring hemodynamic changes. It is particularly critical to understand the physiology underlying the various signals if optical neuroimaging is to be used as a clinical tool. Insights from Anesthesia Effects

Optical physiology is further complicated by issues relating to arousal and anesthesia. Again, this is particularly important in cases where optical imaging is used as a clinical tool, as various assumptions based either on anesthetized animals or on awake humans may not hold when humans may be in various altered states. In an imaging study of the primary visual cortex of an awake monkey, reflectance increases peaked and began to decline earlier than in anesthetized monkeys. The responses were 2–3 times larger in amplitude in the awake animal, and the postpeak undershoot was also larger. The authors suggest that these results indicate a slower oxidation response and a faster hemodynamic response in awake animals [70]. Somatosensory responses (HbT, HbO2 , and HbR) in the rat whisker barrel were 2–3 times larger in awake than in urethane-anesthetized rats [71]. The initial dip was larger and slightly earlier in awake animals, after correction for a startle response in the awake rats. In a study of iso-orientation domains in the cat visual cortex, blood volume changes were larger in awake than in isoflurane-anesthetized cats, and the difference was larger (2.7 versus 3.6 times) in stimulus-specific areas than in stimulus-nonspecific areas. No latency differences were found [47]. In imaging studies of squirrel monkey primary somatosensory cortex in response to finger stimulation, overall response areas and amplitudes were much larger when animals were awake than under isoflurane anesthesia [72]. However, the response shifted from being larger in area 3b in the anesthetized monkey to being larger in area 1 in when the monkey was awake. Both baseline and evoked signals were more variable when the monkeys were awake than when they were anesthetized. Consequently, the signal-to-noise ratio was approximately half as high when the animals were awake, despite response amplitudes 2–3 times larger. Similarly, the hemodynamic response in the visual cortex of awake monkeys was 1.5 to 3 times larger than the response in lightly anesthetized monkeys (sodium penthothal and vecuronium bromide [73]) [74]. However, no large differences were found in the timecourses of the responses, including the initial dip. The optical amplitude differences were not accompanied by increases in evoked electrical activity (computed as the ratio of spontaneous to evoked activity), suggesting anesthesia-induced weakening of neurovascular coupling. To summarize, hemodynamic responses were typically much smaller in anesthetized animals than in awake ones. However, noise levels were also much lower. Relative activation may also shift within cortical subregions. JULY/AUGUST 2007

Conclusions

Optical imaging is a powerful tool in the study of brain function, and the progress that has been made in noninvasive optical neuroimaging is impressive. The potential for further applications is perhaps even greater. Animal models are critical in understanding the physiology that underlies the optical signals measured noninvasively. They allow a level of precision and experimental control that is not typically available in humans. Studies in isolated preparations from animals have provided important information for understanding the neuronal mechanisms that are generally of ultimate interest in optical imaging studies. In addition, much has been learned from animal models about the relationship between neuronal activity and the larger hemodynamic signals that generally accompany them. It is apparent that there is not a simple relationship among the many physiological events that contribute to optical signals. It is important to remember this when conducting human experiments. The implementation of noninvasive optical imaging in clinical and applied settings makes awareness of the complexity of optical physiology even more important. Acknowledgments

Jeffrey J. Sable’s work on this manuscript was supported by a University of Illinois Critical Research Initiative grant. David M. Rector’s work is supported by MH60263, a SRS J. Chris Gillin Junior Faculty Award, the Murdock Foundation, and a Junior Faculty Development Award from the Beckman Foundation. Gabriele Gratton’s work was supported by NIBIB grant # EB002011. The authors thank James Lee and two anonymous reviewers for their helpful comments on an earlier version of this manuscript. Jeffrey J. Sable is currently an assistant professor in the Division of Neurosurgery at Saint Louis University, where he performs presurgical functional mapping of cortex. He received his Ph.D. in psychology from the University of Missouri–Columbia in 2003 and then was a postdoctoral associate in the Department of Cell and Developmental Biology, Beckman Institute for Advanced Science and Technology, and Neuroscience Program at the University of Illinois at Urbana-Champaign. He has engaged in optical imaging research in humans, cats, rats, songbirds, and isolated lobster nerve preparations. He has worked with several laboratories to develop applications of optical measures in awake, behaving animals. David M. Rector, assistant professor in the VCAPP Department at Washington State University, received his bachelor’s in biology with a strong emphasis on electrical and computer engineering from the University of California at Davis in 1988. He subsequently spent one year developing a complete pulmonary function testing system for research and diagnostic use in premature infants at the Stanford University Medical Center. He went on to work on his doctorate in neuroscience with Ronald M. Harper at the University of California at Los Angeles where he developed an implantable video system for imaging scattered light changes in neural tissue from freely behaving animals and IEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE

studied mechanisms behind sudden infant death syndrome (SIDS). He completed his Ph.D. in 1995 with honors and started a Director’s funded postdoctoral fellowship and eventually became a technical staff member at Los Alamos National Laboratory where he continued to develop high-speed electronic equipment for imaging scattered light changes from neural tissue. Gabriele Gratton is professor of psychology and neuroscience at the University of Illinois at Urbana-Champaign, where he is also a full-time affiliate of the Beckman Institute for Advanced Science and Technology. He received his M.D. from the University of Rome in 1980 and his Ph.D. from the University of Illinois in 1991. He has led the development of the event-related optical signal (EROS), a noninvasive method of imaging fast activity in the human brain. Address for Correspondence: Jeffrey J. Sable, Ph.D., University of Illinois at Urbana-Champaign, Beckman Institute, 405 N. Mathews Ave., Urbana, IL 61801. E-mail: [email protected]. References [1] A. Grinvald, R.D. Frostig, E. Lieke, and R. Hildesheim, “Optical imaging of neuronal activity,” Physiol. Rev., vol. 69, pp. 1285–1366, 1988. [2] M. Lauritzen, “Reading vascular changes in brain imaging: Is dendritic calcium the key?” Nat. Rev. Neurosci., vol. 6, no. 1, pp. 77–85, 2005. [3] D. Attwell and C. Iadecola, “The neural basis of functional brain imaging signals,” Trends Neurosci., vol. 25, no. 12, pp. 621–625, 2002. [4] C. Iadecola, “Neurovascular regulation in the normal brain and in Alzheimer’s disease,” Nat. Rev. Neurosci., vol. 5, pp. 347–360, 2004. [5] A. Watanabe, “Mechanical, thermal, and optical changes of the nerve membrane associated with excitation,” Jpn. J. Physiol., vol. 36, no. 4, pp. 625–643, 1986. [6] I. Tasaki, “Rapid structural changes in nerve fibers and cells associated with their excitation processes,” Jpn. J. Physiol., vol. 49, no. 2, pp. 125–138, 1999. [7] D.W. Hochman, “Intrinsic optical changes in neuronal tissue: Brain mechanisms,” Neurosurg. Clin. N. Am. vol. 8, no. 3, pp. 393–412, 1997. [8] D.K. Hill and R.D. Keynes, “Opacity changes in stimulated nerve,” J. Physiol., vol. 108, no. 3, pp. 278–281, 1949. [9] L.B. Cohen, R.D. Keynes, and B. Hille, “Light scattering and birefringence changes during nerve activity,” Nature, vol. 218, no. 5140, pp. 438–441, 1968. [10] L.B. Cohen, “Changes in neuron structure during action potential propagation and synaptic transmission,” Physiol. Rev., vol. 53, no. 2, pp. 373–418, 1973. [11] B.M. Salzberg, A.L. Obaid, and H. Gainer, “Large and rapid changes in light scattering accompany secretion by nerve terminals in the mammalian neurohypophysis,” J. Gen. Physiol., vol. 86, no. 3, pp. 395–411, 1985. [12] I. Tasaki and P.M. Byrne, “Rapid structural changes in nerve fibers evoked by electric current pulses,” Biochem. Biophys. Res. Commun., vol. 188, no. 2, pp. 559–564, 1993. [13] D. Landowne, “Molecular-motion underlying activation and inactivation of sodium-channels in squid giant-axons,” J. Membr. Biol., vol. 88, no. 2, pp. 173–185, 1985. [14] D.M. Rector, G.R. Poe, P. Redgrave, and R.M. Harper, “A miniature CCD video camera for high-sensitivity light measurements in freely behaving animals,” J. Neurosci. Methods, vol. 78, no. 1-2, pp. 85–91, 1997. [15] D.M. Rector, R.F. Rogers, J.S. Schwaber, R.M. Harper, and J.S. George, “Scattered-light imaging in vivo tracks fast and slow processes of neurophysiological activation,” Neuroimage, vol. 14, no. 5, pp. 977–994, 2001. [16] D. Rector and R.M. Harper, “Imaging of hippocampal neural activity in freely behaving animals,” Behavioral Brain Research, vol. 42, no. 2, pp. 143–149, 1991. [17] D.M. Rector, R.F. Rogers, and J.S. George, “A focusing image probe for assessing neural activity in-vivo,” J. Neurosci. Methods, vol. 91, no. 1-2, pp. 135–145, 1999. [18] R.A. Stepnoski, A. LaPorta, F. Raccuia-Behling, G.E. Blonder, R.E. Slusher, and D. Kleinfeld, “Noninvasive detection of changes in membrane potential in cultured neurons by light scattering,” Proc. Natl. Acad. Sci. USA, vol. 88, pp. 9382–9386, 1991. [19] K. Holthoff and O.W. Witte, “Intrinsic optical signals in rat neocortical slices measured with near-infrared dark-field microscopy reveal changes in extracellular space,” J. Neurosci, vol. 16, pp. 2740–2749, 1996. [20] K. Ballanyi and P. Grafe, “Cell volume regulation in the nervous system,” Ren. Physiol. Biochem., vol. 11, no. 3–5, pp. 142–157, 1988.

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