1. Introduction

Functional brain imaging has become an important tool in exploring both the healthy and the dysfunctional brain in humans and animals. Intrinsic signal optical imaging allows visualization of the active brain at a relatively high spatial resolution (tens of micrometers). There are other methods for studying brain response during behavior, but either they have lower spatial resolution [e.g., functional magnetic resonance imaging (fMRI)], near infrared, surface electroencephalogram (EEG), or are restricted to monitoring single or a small number of neurons (single and multiunit electrophysiology) without providing good spatial organizational information. Such methods can also be more invasive, whereas windows, once implanted, do not require introduction of external materials into the brain.

The intrinsic signal arises from various sources including changes in blood volume due to local capillary recruitment, activity-dependent changes in the oxygen saturation level of hemoglobin (increase or decrease in deoxyhemoglobin), and light-scattering changes that accompany cortical activation.[1] As these components vary in time course, they can be differentially monitored with different illumination wavelengths. Although functional maps can be obtained at a number of illumination wavelengths, in monkey visual cortex, the most commonly used is in the range of 600–630 nm wavelengths at which the oxymetry component is relatively large. The intrinsic signal has been associated with so-called “initial dip” in blood oxygen-level dependent (BOLD) fMRI studies,[2, 3, 4, 5, 6, 7] and its correlation with neural response, both spiking and subthreshhold neural response, is becoming more firmly established. For a recent review see Ref. [8].

The amplitude and time course of the intrinsic signal in macaque visual cortex has been previously described.[1] In the anesthetized animal, the basis of the optical imaging maps is a reflectance signal change of 0.1%–1% peaking at approximately 2–3 s after stimulus onset. Although many orientation maps in visual cortex have been published, there is not much information available regarding the variability of the intrinsic optical signal over groups of functional domains, across multiple experimental sessions, between anesthetized and awake states, and between individual animals.

Why is spatial resolution important? The main computational power in the brain resides in the cerebral cortex, the size and complexity of which serves to differentiate humans from other animals. As each behavior is complex and requires a number of processing stages, the workload of generating sensorimotor behavior is divided among many different areas; some handle vision, others touch, and yet others handle higher cognitive functions such as working memory. Within each cortical area, function is parcellated into fundamental compartments or modules (e.g., Ref. [9]). The organization and function of these modules has been an area of active investigation.

One of the challenges of optical imaging is to understand the modular basis of cognition. How do networks of modules within and across cortical areas achieve vision or memory or emotion? Visualization of such modular activations during behavior is tantamount to watching the brain at work. Over the past decade, researchers have developed methods to visualize activations through implanted “windows on the brain.” By applying these methods to the awake, behaving animal, it is hoped that this method can open new vistas in our understanding of the neural basis of cognitive function. Furthermore, the ability of these studies to forge a critical link between a large body of work on animal models and functional imaging in humans will be invaluable.

Is optical imaging in awake animals feasible? In 1991, Grinvald and colleagues imaged ocular dominance maps in the awake, untrained monkey.[10] This pioneering study demonstrated that optical imaging could be performed in the awake animal. Ocular dominance, orientation, and color maps were obtained in the awake, fixating monkey by Vnek et al.[11] Optical imaging has also been used to demonstrate novel organization in the parietal and prefrontal cortex (Seidemann et al.[12] and Roe et al.[13]). Siegel et al.[14] demonstrated an organization for eye gaze position in Area 7 in macaque monkeys. Preliminary studies from Roe et al. indicate that there may be a spatial topographic map for working memory in monkeys performing delay-match-to-sample tasks (Roe et al.[13]). These and other studies described in this section indicate that optical imaging is emerging as a useful tool for examining cortical function in behaving animals.

2. Challenge

To maintain a clear, unobstructed view of the cortex, the skull and dura mater must be prevented from regrowing over the exposed cortex. Often, as part of a “healing response,” cortex that is exposed for extended periods of time exhibits a growth of “neomembrane,” a new membrane on the surface of the cortex, which can seriously degrade the quality of the optical signal. Exposed cortex is also subject to added risk for infection and mechanical injury. Optimally, one needs to develop an implant method that permits routine, repeated imaging sessions in the anesthetized animal or in the awake, behaving animal. Although in smaller animals (e.g., rats, tree shrews, cats) with thin dura, intrinsic optical signals can be imaged through the intact dura or even through the thinned bone,[15, 16, 17] the thickness of the bone and native dura in old world primates makes this option impractical (Ref. [10], personal observations). Different methods using fiber optics inserted into the brain have also been explored for recording deep structures in the brain.[18]

To achieve the ability to image the brain long term, both issues of biology and instrumentation must be dealt with. Several goals need to be achieved:

Some of the methods currently used to address these issues are summarized here.

3. Chamber and Artificial Dura Design

Two models of chamber and artificial dura have been developed to date. Both have successfully enabled long-term imaging of the cortex. The model developed by Grinvald and colleagues and also used by Siegel and colleagues is derived from the traditional stainless-steel chamber design. In this method, an X cut is made in the dura and the four leaves pulled up and glued to the inner wall of the steel chamber. An artificial silicone-based premolded hat-shaped dura is then implanted and secured with a rubber silicone ring.[19]

The second model is a nylon prototype designed for electrophysiological and MRI recordings[20] (Fig. 1). The top of the chamber is threaded on the inside so that a cap, made of the same material, screws into the chamber securely and provides a good seal (Fig. 1E). The bottom of the chamber consists of a number of flexible flanges (Fig. 1B) that snap snugly into the craniotomy and, once in, are secure and immovable. This chamber has a low profile on the head, is lightweight and therefore suitable for smaller animals, MRI compatible, and requires little dental cement and no or few screws. The artificial dura used in conjunction with this design is a transparent, soft thermoplastic polyurethane (Tecoflex).[21] This material is completely inert and can be implanted for months to years without reactive growth.[13, 22, 23] In this case, the dura is resected and the base of the dura hat is inserted between the native dura edge and the cortex (Fig. 1A). The wall of the hat prevents ingrowth of any native dura into the chamber.

4. Long-Term Maintainance of Healthy Cortex in a Chronic Chamber

Prior to chamber implantation, for purposes of interpreting subsequent optical images, determining the topographic representation of the exposed cortex with a brief electrophysiological mapping procedure is extremely useful (Fig. 2A). Following implantation of chamber and artificial dura, cortex is maintained in a clear and healthy state and vascular patterns remain stable for years (Fig. 2B). Repeated imaging experiments can be conducted on the same cortex in the same animal. For subsequent electrophysiological recordings, the artificial dura is removed, recordings conducted, and dura hat easily replaced.

Chamber maintenance is essential for long-term recording without infection or excessive dural growth. Aseptic procedures are used at all times. Before opening the chamber, the outside of the chamber is first cleaned with an antiseptic solution. The chamber cap is then removed and the inside rinsed with sterile saline. Even though in most instances no removal of dural growth is necessary, at times a small amount of new dural growth will emerge from between the artificial dural wall and the chamber wall. This growth has a gelatinous consistency and is easily removed with either forceps or cotton swabs. To help prevent infection, a sterile piece of gauze soaked in an antibiotic is then placed in the chamber before closing the cap. If these procedures are conducted consistently, problems with dural growth and infection are prevented. In addition, the fluid contents of the chamber should be tested intermittently for bacterial sensitivity and antibiotics are chosen on the basis of this test. These procedures permit long-term maintenance (years) of healthy imageable cortex.[13, 20, 22, 23]

If cortex is carefully maintained, neomembrane growth can be prevented. Neomembrane growth is activated by an insult to the cortex (e.g., mechanical or vascular insult). If significant growth occurs, the new membrane can be surgically removed (durotomy) and the cortical surface reexposed. This procedure, if carefully conducted, reveals a pristine cortex with no apparent change in surface vasculature or degradation of function (as assessed with optical imaging and single unit electrophysiology and animal behavior).

5. Repeated Imaging in Anesthetized Monkeys

Anesthetic choice. Intrinsic signal optical imaging is compatible with a wide range of anesthetics and the anesthetic choice may vary with species, individual, cortical area, and stimulus paradigm. The key to successful optical imaging is maintenance of a stable plane of anesthesia (as measured by heart rate, expired ${\text{CO}}_{\text{2}}$, EEG, blood oxygenation, and blood pressure). This provides conditions optimal for maximizing signal-to-noise ratio. The ideal depth of anesthesia is a light to moderate anesthesia characteristic of sleep. This level is characterized by low-frequency, high-amplitude (up to $150\mu \text{V}$) bursts with periods of suppression less than $1\text{\hspace{0.17em} s}$ in the EEG. In humans in this state, the patient has lost consciousness, has reduced reflex activity, no reaction to painful stimuli, and can undergo surgical procedures. If the subject is too light, the anesthetic plane fluctuates, leading to increased baseline noise; if the subject is too deep, cortical activity is compromised. In our experience, slightly deeper anesthesic planes are preferable over fluctuating anesthetic planes. Imaging in anesthetized monkeys has been successfully conducted with a variety of anesthetics. For example, sodium pentothal provides a stable baseline to which small, rapid adjustments of anesthesia can be made when lightly supplemented with a gas anesthetic (e.g., $0.1\%–1.0\%$ isoflurane). Imaging can also be conducted solely on gas anesthetics, which offer rapid reversibility and fast recovery times for the animal, however, in our experience, this is accompanied by greater baseline fluctuation than with sodium pentothal. Sufentanil has also been used reversibly for studying higher cortical areas (such as V4) that are typically suppressed under anesthesia.[24] Other anesthetics such as propofol,[25] halothane,[26] urethane[16] have also been used successfully in intrinsic signal optical imaging experiments.

Stability of maps. As a measure of the health of the cortex, it is important to establish the stability of the cortical responses to the same stimuli over time (Fig. 3). Stable maps have been reported in both visual and somatosensory cortices.[11, 19, 20, 22] The amplitudes $\left(0.1\%–1.0\%\right)$ and time courses of optical signal over time remains stable. No significant decline in signal amplitude occurs over a 2 year imaging period.

6. Imaging in Awake Monkeys

General strategy. Adult macaque (Macaca fascicularis, Rhesus) and squirrel monkeys (Saimiri sciureus) have been used for awake optical imaging experiments. Monkeys are chronically implanted with optical chambers and head post and trained to sit calmly in a chair. Visual tasks include visual fixation and tasks requiring eye or hand movement responses such as orientation discrimination and delayed match to sample tasks. Eye movement responses are preferred, since manual responses introduce additional movement artifact during imaging. Tactile tasks in squirrel monkeys have required the monkey to hold the hand still. To permit comparison of anesthetized and awake signals, the same monkeys were imaged in both states. Detailed surgical and optical imaging procedures can be found in Refs. [27] and [28].

Monkey selection. In our experience, not all monkeys image equally well. It is widely believed that younger animals have stronger blood-flow related signals, however, there are no systematic studies to test this hypothesis. We thus screen each monkey for imageability before embarking on any training and chamber implantation procedures. We also screen for availability of area of interest. For example, in macaque monkeys, we screen for the availability of V2 exposure (much of V2 is buried in the lunate sulcus and the amount of V2 on the surface of the operculum varies across individuals). In macaque monkeys, we examine the number of trials with which ocular dominance, orientation, and color maps are obtained in V1 and V2. In squirrel monkeys, digit tip activation is a reliable marker for imageability. Typically, most monkeys will produce good maps within 10–20 trials. In stable preparations, it is not uncommon to see ocular dominance in a single trial. Thus, for each monkey, depending on the area of interest, it may be helpful to perform an initial optical imaging procedure conducted under anesthesia. However, if the area of interest is a higher cortical area (such as prefrontal cortex), imaging under anesthesia may not be helpful, since signals in these areas are typically depressed under anesthesia.

Monkey training. Monkeys are implanted with a head post, and then trained to sit calmly in the chair (Fig. 4). The animal is rewarded with a drop of juice to reinforce sitting still in the primate chair during training and recording. After training, monkeys are implanted with a chamber and native dura replaced with transparent artificial dura.

Brain stabilization during imaging. To minimize pulsations during imaging, we typically use either agar (4%) or agar with a transparent silicone wafer. The total time required for chamber preparation can be kept reasonably short (i.e., $\sim 10–15\text{\hspace{0.17em} min}$). Following imaging, the agar is removed, the chamber rinsed with sterile saline, antibiotic applied, and the cap replaced.

Stimulus presentation. Visual stimuli include visual gratings of various orientations and spots at different locations in space. Ocular dominance maps are collected using an electromechanical shutter placed in front of the eye. Tactile stimuli are either cutaneous electrical or vibrotactile stimulation on the fingertips. Stimuli within each set [including blank (no stim) conditions] are randomly interleaved during image acquisition.

Synchronization with heartbeat and respiration. Synchronization with heartbeat and respiration is not necessary; these signal artifacts can be minimized by subtraction of prestim or blank conditions.[11, 19, 22, 29]

Image acquisition. In our experiments, images were collected using the Imager 2001 and 3001 systems (Optical Imaging, Germantown, New York) and a $630\text{\hspace{0.17em} nm}$ illumination. We are currently developing the NeuroCCD-SM256 system (RedShirtImaging, LLC) for use in the awake, behaving monkey; the camera has an extremely low read noise, high quantum efficiency, a large well depth of 10⁶, a large spatial $256\times 256$ array charge-coupled device, and fast readout. In a typical session, depending on the monkey, $50–500$ trials are collected per stimulus condition. Intrinsic signal maps were collected at 5–15 image frames∕s and for 3–5 s starting $200\text{\hspace{0.17em} ms}$ prior to stimulus onset. Interstimulus intervals were 6–12 s.

Comparison of the anesthetized and awake imaging conditions. Identical stimuli are presented in the anesthetized and awake states. However, significant differences remain. In the anesthetized animal, conditions are optimized such that physiological variability is minimized by careful regulation of anesthesia, cortical pulsations are minimized with agar or silicone oil in an optical chamber, and stimuli are presented in a well-controlled, repeatable fashion. In the awake animal, there is much greater variability of the animal's respiration and heart rate, much greater motion artifact due to both body movements and to greater cortical pulsations, resulting in significant trial-to-trial variability of the optical signal. Thus, depending on the behavior of the individual monkey, some portion of the trials are discarded due to excessive movement artifact. Response to repeated presentation of stimuli should, with averaging, produce a consistent signal more reliably than occasional and erratic signals due to movement or changes in the animal's physiology. Appropriate analyses are conducted to ensure that the signal is not due to systematic contribution by sources not related to the stimuli presented (e.g., motion artifact due to licking).

7. Image Analysis

Typically, for each stimulus condition, image frames are summed to maximize the signal-to-noise ratio. Before summation, images are examined either manually[10, 11] or in an automated fashion[14] to remove trials that contain excessive noise. Since noise levels (due to mechanical motion or physiologically related large changes in baseline reflectance) are much larger in the awake animal, elimination of noisy trials is essential. For this reason, individual trials are saved as separate files, rather than saving averaged responses. Blank condition trials are examined for possible nonstimulus related changes in reflectance. In addition, poor behavioral performance (e.g., excessive body movement, sleeping) also leads to trial exclusion. Control analyses include comparison of sums of different blocks of trials; presence of sporadic high-amplitude noise would produce different half-trial maps, whereas consistent nonartifactual signal would produce similar maps. Consistency of single condition (either blank subtracted or first frame subtracted) analyses and two condition subtraction analyses further establish believability of the signal. Blank subtractions not only measure change from baseline, but also reduce blood vessel artifact and minimizes effects of uneven illumination. In addition, comparison of images obtained by summing different subsets of imaged trials can further confirm the validity of results. When used together, we are able to confirm that the imaged signals are consistent and repeatable and not due to noise signals occurring in one or a few of the trials.

Determining signal significance. Methods for determining signal significance include thresholding,[27, 28] t maps,[30] response correlation,[31] bootstrapping,[14] and principal components analysis[32] methods. In threshholding procedures, images are typically low-pass filtered, pixel distributions established, and the pixels with strongest activations above a certain value (e.g., 15%) identified. This method is appropriate for clear, focal activations where different threshold levels produce a similar region of activation (e.g., Ref. [33]). High-pass filtering is typically used in cases where gradual gradation of pixel values is observed over the image (e.g., due to illuminant unevenness or to cortical curvature). Masks are commonly used to exclude pixels that have aberrantly large reflectance change (e.g., excluding pixels $>0.5\text{\hspace{0.17em} SD}$ from mean of distribution); this is useful for removing the most offensive large blood vessel noise. It is not acceptable to interpolate pixel values across regions of excluded vasculature (see Refs. [17] and [34]). Correlation of pixel time courses have also been used to determine significance in response.[31] In a study on the mapping of gaze direction in the parietal lobe, Siegel et al.[14] devised a method of normalizing every pixel to an initial prestimulus baseline and, following outlier rejection, calculating significance from the mean by Monte Carlo analysis. A useful method developed by Wang et al.[30] generates t maps, which evaluates, pixel by pixel, similarity between trials of prestimulus and poststimulus response using the standard t test.

8. Motion Correction

Linear. At times, monkey's movements may introduce translational noise to the images. It is therefore useful to have a motion correction algorithm to shift back the frames to the original location and thus permit correct frame alignment before image summation (Figs. 5A–5C). This can be done only with original DC frames (not with difference signals typically collected by an 8 bit camera, Imager 2001). As can been seen in Fig. 5D, frame-to-frame shifts in the anesthetized animal (black line) are small and periodic, while those in the awake animal (red line) are larger and more erratic. These procedures improve image alignment, appropriate summing of corresponding pixels, and therefore increase signal to noise in images.

Nonlinear. From day to day and over long periods, the position and curvature of the cortex may change. Deformation algorithms may also be used for nonlinear changes in the cortical map (Fig. 6). These procedures increase the number of trials that can be summed beyond those collected within a single session and therefore help to increase signal to noise.

9. How Similar Are Activation Patterns?

In visual cortex, repeated imaging of ocular dominance and orientation in awake monkeys produce fairly stable maps similar to those obtained in the anesthetized state.[11, 19, 29] In somatosensory cortex, there is significant variability in receptive field size and topography depending on level of alertness and anesthesia, on attentional state, and on activation of local neurotransmitters (especially GABAergic) systems.[35, 36, 37] Indeed, rapid shifts in receptive field organization have been observed (e.g., Ref. [38]) and further suggest the dynamic nature of somatosensory cortex. Given these differences, we asked how similar topographic and areal activation patterns would be in awake and anesthetized squirrel monkeys.

We find evidence that in somatosensory cortex, activation patterns can differ significantly between anesthetized and awake preparations. In the anesthetized state, we generally observe stronger activations in Area 3b than in Area 1 (Fig. 7A). The opposite is observed in the awake state, where Area 1 activations are more prominent (Fig. 7B). In fact, in some cases, only Area 1 activation is observed and no apparent Area 3b activation is seen at all. Topographically, the map in the awake state appears less precise and also different from that in the anesthetized state. The activations to D3 and D4 stimulation are highly overlapped (Fig. 7C). Finally, we observe, within Area 1, a lateral displacement of activation in the awake state from that in the anesthetized state (compare A and B). This result is obtained with both electrical and vibrotactile stimulation.[22] These results suggest significant awake versus anesthetized differences in somatosensory activations, perhaps due to differences in neural response or vacular response.

10. How Big Is the Signal?

Magnitude, time course, and signal to noise in anesthetized versus awake monkeys. As shown in Fig. 8, the time course of optical signal in both anesthetized and awake squirrel monkey SI cortex peaks at approximately 2.0–2.2 s poststimulus onset and is sustained longer than in the anesthetized animal. Consistent with signals obtained in awake visual cortex,[19] the peak reflectance change in the awake animal is several times larger in the awake than in the anesthetized animal. In an average of 12 awake monkey experiments, signal amplitude was $0.92\%+0.38\%$ at the digit center, and $0.38\%+0.22\%$ on a control location away from activation center. In an average of 13 anesthetized monkey experiments, the corresponding numbers are $0.32\%+0.068\%$ on digit center, and $0.014\%+0.017\%$ on control location. If one takes the ratio of signal size to error size, this factor is $2.4\left(=0.92/0.38\right)$ in the awake state and $4.7\left(=0.32/0.068\right)$ in the anesthetized state. As a measure of discriminability from unstimulated cortex (center∕control), the factor is $2.4\left(=0.92/0.38\right)$ in the awake state and $22.8\left(=0.32/0.014\right)$ in the anesthetized state. Thus, while signal size is larger in awake animals, noise is also larger by at least a factor of 2 and discriminability decreases by an order of magnitude. The basis of these differences remains to be investigated.

11. Future Directions

Optical imaging has been used to elucidate the functional organization of sensory cortical areas in primates. A future goal is to use this methodology to explore functional organization of cognitive functions. For example, optical imaging methodology has been used to study the organization for spatial working memory in monkeys performing an oculomotor delayed response task.[13] The results suggest possible spatial organization for memory related signals in prefrontal cortex (see also Refs. [12] and [39]). Other studies using intrinsic signal imaging in awake, behaving monkeys have examined the organization of gaze direction[40] and spatial attention[41] in parietal cortex. In addition, the development of exciting new voltage sensitive dyes, which increase the temporal resolution of optical imaging to the millisecond time scale, increases even further the power and possibility of studying brain function in behaving animals.[29]

Many thanks to Haidong Lu and Limin Chen for assistance with this document and to Haidong Lu for generating the motion correction algorithm. This research was supported by the National Eye Institute, the National Institute of Neurological Disorders and Stroke, and the Packard Foundation.

 

Fig. 1 A, Profile of chamber and artificial dura over the brain; B, chamber; C, artificial dura; D, chamber open; E, chamber closed. (From Chen et al.[20])

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Fig. 2 A, Dura over SI somatosensory cortex (Areas 1, 3b, and 3a) in the squirrel monkey has been resected and a detailed electrophysiological map recorded. B. Appearance of cortex remains healthy and vascular pattern remains stable for over a 2 year period.

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Fig. 3 Cortical responses to tactile indentation on Digit 3 (D3) is imaged repeatedly on three different days. Response region overlain on blood vessel map (indicated by outlines in overlay) reveals stable topography over time. Signal amplitudes were stable over time ($0.77\%+0.1\%$, $0.83\%+0.07\%$, and 0.59% + 0.14%).

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Fig. 4 Optical imaging setup of squirrel monkey with implanted chamber.

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Fig. 5 Motion correction algorithm. A. Time sequence of image frames. B. Select one frame as base frame (usually the very first frame of the run). Shift each frame in x and y coordinates in 0.2–0.5 pixel steps. For each shift, calculate the correlation coefficient between the base frame and the current frame. C. Repeat for every pixel in a small area (e.g., $10\times 10$ comparison will give 100 correlation coefficients). Find the best coefficient and use corresponding shift offset $\left(\mathrm{d}x,\mathrm{d}y\right)$. D. Magnitude of correction in an anesthetized monkey (black) and an awake, fixating monkey (gray) (140 frames over $28\text{\hspace{0.17em} s}$).

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Fig. 6 Ocular dominance (B, E) and orientation (C, F) maps from V1 and V2 in macaque monkey on two different days (A–C versus D–F). Black crosses in A and D indicate the alignment points used to generate transformation function between images. F–I Application of transformation to images in A–C produce images similar to D–F. (From Chen et al.[20])

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Fig. 7 Cortical response to vibrotactile stimulation of D3 and D4 in the squirrel monkey. A. Anesthetized activation prominent in Area 3b (see time course in Fig. 8). B. Awake activation prominent in Area 1 (see time courses in graphs below). C. Electrophysiologyical mapping of Areas 3a, 3b, and 1.

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Fig. 8 Time course of optical signal in Area 3b of the squirrel monkey in response to vibrotactile stimulation of digits D2 (circle) and D4 (triangle). Awake (A) and anesthetized (B) signals were obtained from identical location in same monkey to identical stimulus. All signals measured at the D4 location.

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