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Infrared neural stimulation is a promising tool for stimulating the brain because it can be used to excite with high spatial precision without the need of delivering or inserting any exogenous agent into the tissue. Very few studies have explored its use in the brain, as most investigations have focused on sensory or motor nerve stimulation. Using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f, here we show that the application of infrared neural stimulation induces intracellular calcium signals in Layer 2/3 neurons in mouse cortex in vivo. The number of neurons exhibiting infrared-induced calcium response as well as the amplitude of those signals are shown to be both increasing with the energy density applied. By studying as well the spatial extent of the stimulation, we show that reproducibility of the stimulation is achieved mainly in the central part of the infrared beam path. Stimulating in vivo at such a degree of precision and without any exogenous chromophores enables multiple applications, from mapping the brain’s connectome to applications in systems neuroscience and the development of new therapeutic tools for investigating the pathological brain.

International audience; Recording brain activity at the mesoscopic scale has a strong potential to unveil many new fundamental neuronal operations. Optical imaging offers a unique opportunity to measure brain activity over a large area with high spatio-temporal resolutions (20 µm x 1 ms). However, two major limitations of this imaging technique partially explain the lack of development in this field. The cortex being non-planar, the field's depth limits the region in focus to a small region close to the center of the field of view. This is particularly significant for the highly curved lissencephalic small cortex of non-human primates that are becoming popular in neuroscience experiments. The ideal technique would be a method that compensates for such curvature; it would enable imaging the whole visual system at once, from the primary to the fifth visual cortices, in small non-human primates. Additionally, the signal-to-noise ratio is strongly degraded by the dynamic evolution of the brain curvature due to physiological rhythms (heartbeat, breathing, etc.). This strongly limits the ability to work at a single-trial level and to unravel the real dynamics of neuronal processing, such as spatio-temporal waves. Here in this project, we present an interdisciplinary approach for imaging of the non-human primate cortex, using technologies from astronomical instrumentation to overcome current technological limits. This will be of interest to a wide neuroscientific audience but also will impact the clinical community interested in mapping the nervous activity at the mesoscopic scale. Our current preliminary development involves redesigning the illumination source and the optical design.

International audience; Functional mapping of the connectivity of the brain is an essential procedure for neurosurgeons aiming for maximal resection of a brain tumor, while minimizing damage to the eloquent cortex. Being able to delineate the location of critical cortical areas in individual patients allows the surgeon to preserve sensorimotor and cognitive functions. Several methods exist for functional mapping, however, none are capable of delivering fast, label free mapping of the cortex with millimeter precision. In this work, we investigate the potential use of infrared neural stimulation (λ=1470nm) as a means of focal stimulation of the mouse cortex in vivo. Here, we show that the application of infrared neural stimulation induces in vivo cortical intracellular calcium signals in Layer II/III mouse neurons, using intravital calcium imaging with the genetically encoded calcium indicator GCaMP6f.

Visual Abstract, Transparent and flexible materials are attractive for a wide range of emerging bioelectronic applications. These include neural interfacing devices for both recording and stimulation, where low electrochemical electrode impedance is valuable. Here the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) is used to fabricate electrodes that are small enough to allow unencumbered optical access for imaging a large cell population with two-photon (2P) microscopy, yet provide low impedance for simultaneous high quality recordings of neural activity in vivo. To demonstrate this, pathophysiological activity was induced in the mouse cortex using 4-aminopyridine (4AP), and the resulting electrical activity was detected with the PEDOT:PSS-based probe while imaging calcium activity directly below the probe area. The induced calcium activity of the neuronal network as measured by the fluorescence change in the cells correlated well with the electrophysiological recordings from the cortical grid of PEDOT:PSS microelectrodes. Our approach provides a valuable vehicle for complementing classical high temporal resolution electrophysiological analysis with optical imaging.

A successful outcome of epilepsy neurosurgery relies on an accurate delineation of the epileptogenic region to be resected. Functional magnetic resonance imaging (fMRI) would allow doing this noninvasively at high spatial resolution. However, a clear, quantitative description of the relationship between hemodynamic changes and the underlying epileptiform neuronal activity is still missing, thereby preventing the systematic use of fMRI for routine epilepsy surgery planning. To this aim, we used a local epilepsy model to record simultaneously cerebral blood flow (CBF) with laser Doppler (LD) and local field potentials (LFP) in rat frontal cortex. CBF responses to individual interictal-like spikes were large and robust. Their amplitude correlated linearly with spike amplitude. Moreover, the CBF response added linearly in time over a large range of spiking rates. CBF responses could thus be predicted by a linear model of the kind currently used for the interpretation of fMRI data, but including also the spikes’ amplitudes as additional information. Predicted and measured CBF responses matched accurately. For high spiking frequencies (above ∼0.2 Hz), the responses saturated but could eventually recover, indicating the presence of multiple neurovascular coupling mechanisms, which might act at different spatiotemporal scales. Spatially, CBF responses peaked at the center of epileptic activity and displayed a spatial specificity at least as good as the millimeter. These results suggest that simultaneous electroencephalographic and blood flow-based fMRI recordings should be suitable for the noninvasive precise localization of hyperexcitable regions in epileptic patients candidate for neurosurgery.

International audience; Interpretation of hemodynamic responses in epilepsy is hampered by an incomplete understanding of the underlying neurovascular coupling, especially the contributions of excitation and inhibition. We made simultaneous multimodal recordings of local field potentials (LFPs), firing of individual neurons, blood flow, and oxygen level in the somatosensory cortex of anesthetized rats. Epileptiform discharges induced by bicuculline injections were used to trigger large local events. LFP and blood flow were robustly coupled, as were LFP and tissue oxygen. In a parametric linear model, LFP and the baseline activities of cerebral blood flow and tissue partial oxygen tension contributed significantly to blood flow and oxygen responses. In an analysis of recordings from 402 neurons, blood flow/tissue oxygen correlated with the discharge of putative interneurons but not of principal cells. Our results show that interneuron activity is important in the vascular and metabolic responses during epileptiform discharges.

Optical imaging based on intrinsic signals has provided a new level of understanding of the principles underlying cortical development, organization, and function, providing a spatial resolution of up to 20 µm for mapping cortical columns in vivo. This introduction briefly reviews the development of this technique, the types of applications that have been pursued, and the general implications of some findings for other neuroimaging techniques based on hemodynamic responses (e.g., functional magnetic resonance imaging).

Neural computations underlying sensory perception, cognition, and motor control are performed by populations of neurons at different anatomical and temporal scales. Few techniques are currently available for exploring the dynamics of local and large range populations. Voltage-sensitive dye imaging (VSDI), based on organic voltage probes, reveals neural population activity in areas ranging from a few tens of micrometers to a couple of centimeters, or two areas up to ~10 cm apart. VSDI provides a submillisecond temporal resolution and a spatial resolution of ~50 µm. The dye signal emphasizes subthreshold synaptic potentials. VSDI has been applied in the mouse, rat, gerbil, ferret, tree shrew, cat, and monkey cortices to explore the lateral spread of retinotopic or somatotopic activation; the dynamic spatiotemporal pattern resulting from sensory activation, including the somatosensory, olfactory, auditory, and visual modalities; and motor preparation and the properties of spontaneously occurring population activity. In this introduction, we focus on VSDI in vivo and review results obtained mostly in the visual system in our laboratory.

The ultimate goal of high-resolution functional brain mapping is single-condition (stimulus versus no-stimulus maps) rather than differential imaging (comparing two "stimulus maps"), because the appropriate ("orthogonal") stimuli are rarely available. This requires some component(s) of activity-dependent hemodynamic signals to closely colocalize with electrical activity, like the early increase in deoxyhemoglobin, shown previously to yield high-quality functional single-condition maps. Conversely, nonlocal vascular responses dominate in cerebral blood volume (CBV)-based single-condition maps. Differential CBV maps are largely restricted to the parenchyma, implying that part of the CBV response does colocalize with electrical activity at fine spatial scale. By removing surface vascular activation from optical imaging data, we document the existence of a capillary CBV response component, regulated at fine spatial scale and yielding single-condition maps exhibiting approximately 100 microm resolution. Blood volume and -flow based single-condition functional mapping at columnar level should thus be feasible, provided that the capillary response component is selectively imaged.

Understanding of the spatio-temporal characteristics of the sensory-evoked cortical blood-volume and oxygenation changes is important from the physiological perspective as well as for the interpretation of results obtained by various neuroimaging techniques, such as optical imaging, PET and f-MRI, and for their improvement. The detailed picture, however, has remained elusive for more than a century. We investigated the blood-volume and oxygenation changes in anesthetized cats and awake monkeys using intrinsic imaging at isosbestic and other wavelengths, laser Doppler, imaging spectroscopy, phosphorescence quenching and fluorescence imaging of activity-dependent responses of intravenously injected extrinsic probes. We found that the onset of blood-volume changes was delayed (>300 ms) with respect to a fast decrease in blood oxygenation. Thus, the blood-volume effects cannot merely explain the “initial dip”. 570-nm measurements and high-resolution imaging (80 ms, 7 μm) of a fluorescent tracer injected into the blood circulation facilitated the resolution of the responses of different microvascular compartments. Preliminary results show that the arterioles led the blood volume increase, rapidly spreading towards the other microvascular compartments. Veins lagged behind. Functional maps of stimulus vs. blank (single condition maps) in a conscious macaque and an anesthetized cat were obtained only during the early deoxygenation phase at 605 nm. At later times or at 570 nm, the vessel artifacts dominated. These results indicated a stronger co-localization of oxygen consumption and electrical activity as compared to the subsequent volume and flow increase, which are not well regulated at the cortical column level.

Increasing evidence suggests that sensory stimulation not only changes the level of cortical activity with respect to baseline but also its structure. Despite having been reported in a multitude of conditions and preparations (for instance, as a quenching of intertrial variability, Churchland et al., 2010), such changes remain relatively poorly characterized. Here, we used optical imaging of voltage-sensitive dyes to explore, in V4 of an awake macaque, the spatiotemporal characteristics of both visually evoked and spontaneously ongoing neuronal activity and their difference. With respect to the spontaneous case, we detected a reduction in large-scale activity (cortical extent>1 mm) in the alpha range (5 to 12.5 Hz) during sensory inflow accompanied by a decrease in pairwise correlations. Moreover, the spatial patterns of correlation obtained during the different visual stimuli were on the average more similar one to another than they were to that obtained in the absence of stimulation. Finally, these observed changes in activity dynamics approached saturation already at very low stimulus contrasts, unlike the progressive, near-linear increase of the mean raw evoked responses over a wide range of contrast values, which could indicate a specific switching in the presence of a sensory inflow. This work was supported by the European Union (Integrated Project BrainScales), Award No. IST-FET-2010-269921; a French-Hungarian International ANR grant MULTISCALEFUNIM; and recurrent funding by CNRS and Aix-Marseille Université.

Author(s): Devor, A; Sakadžic, S; Yaseen, MA; Roussakis, E; Tian, P; Slovin, H; Vanzetta, I; Teng, I; Saisan, PA; Sinks, LE; Dale, AM; Vinogradov, SA; Boas, DA | Abstract: Microscopic in vivo measurements of cerebral oxygenation are of key importance for understanding normal cerebral energy metabolism and its dysregulation in a wide range of clinical conditions. Relevant cerebral pathologies include compromised blood perfusion following stroke and a decrease in efficiency of single-cell respiratory processes that occurs in neurodegenerative diseases such as Alzheimer's and Parkinson's disease. In this chapter we review a number of quantitative optical approaches to measuring oxygenation of blood and cerebral tissue. These methods can be applied to map the hemodynamic response and study neurovascular and neurometabolic coupling, and can provide microscopic imaging of biomarkers in animal models of human disease, which would be useful for screening potential therapeutic approaches. © 2014 Springer Science+Business Media New York.

9 pages; International audience; It is shown that using elliptically polarized light permits selecting well-defined subsurface volumes in a turbid medium. This suggests the possibility of probing biological tissues at specific depths. First, we present the method and preliminary results obtained on an Intralipid phantom. We next report on the method's performance on a biological phantom (chicken breast) and, finally, on the exposed cortex of an anesthetized rat.

International audience; Choosing an appropriate set of stimuli is essential in order to characterize the response of a sensory system to a particular functional dimension, such as the eye movement following the motion of a visual scene. Here, we describe a framework to generate random texture movies with controlled information content, i.e., Motion Clouds. These stimuli are defined using a generative model which is based on controlled experimental parametrization. We show that Motion Clouds correspond to dense mixing of localized moving gratings with random positions. Their global envelope is similar to natural-like stimulation with an approximate full-field translation corresponding to a retinal slip. We describe the construction of these stimuli mathematically and propose an open-source python-based implementation. Examples of the use of this framework are shown. We also propose extensions to other modalities such as color vision, touch and audition.

Comprehensive information on the spatio-temporal dynamics of the vascular response is needed to underpin the signals used in hemodynamics-based functional imaging. It has recently been shown that red blood cells (RBCs) velocity and its changes can be extracted from wide-field optical imaging recordings of intrinsic absorption changes in cortex. Here, we describe a complete processing work-flow for reliable RBC velocity estimation in cortical networks. Several pre-processing steps are implemented: image co-registration, necessary to correct for small movements of the vasculature, semi-automatic image segmentation for fast and reproducible vessel selection, reconstruction of RBC trajectories patterns for each micro-vessel, and spatio-temporal filtering to enhance the desired data characteristics. The main analysis step is composed of two robust algorithms for estimating the RBCs' velocity field. Vessel diameter and its changes are also estimated, as well as local changes in backscattered light intensity. This full processing chain is implemented with a software suite that is freely distributed. The software uses efficient data management for handling the very large data sets obtained with in vivo optical imaging. It offers a complete and user-friendly graphical user interface with visualization tools for displaying and exploring data and results. A full data simulation framework is also provided in order to optimize the performances of the algorithm with respect to several characteristics of the data. We illustrate the performance of our method in three different cases of in vivo data. We first document the massive RBC speed response evoked by a spreading depression in anesthetized rat somato-sensory cortex. Second, we show the velocity response elicited by a visual stimulation in anesthetized cat visual cortex. Finally, we report, for the first time, visually-evoked RBC speed responses in an extended vascular network in awake monkey extrastriate cortex.

Over the last 20 years, neuroscientists have become increasingly interested in two-photon microscopy. One of the reasons for this interest is that two-photon fl uorescence excitation allows counterbalancing the deterioration of the optical signals due to light scattering, and this opens the door for high-resolution imaging at considerable depth in living tissue. Due to progress in fl uorescent marking techniques, to date, two-photon microscopy allows the functional exploration of neuronal activity at multiple scales, from the subprocesses of a single cell (dendrites, single spines, etc.), through single cells or small networks of a few neurons, up to large neuronal populations in the order of a cortical column. Here, we provide some information on the practical aspects of two-photon microscopy applied to imaging neurons in living tissue. We fi rst discuss the advantages, shortcomings, and possible developments of the technique. We provide some practical considerations on the choice of the microscope itself, as well as on its principal elements. Because of recent progress in tackling high-speed imaging of 3D objects, we devote particular attention to the discussion of z -axis scanning techniques. Next, we illustrate some common applications, such as calcium imaging of neuronal activity, in vitro and in vivo. We also brie fl y illustrate how two-photon microscopy can be used for the imaging of erythrocyte fl ow in individual capillaries. Some practical considerations on speci fi c protocols are provided in the form of self-consistent text boxes.

International audience; Depth selectivity is crucial for accurate depth volume probing in vivo in a large number of medical applications such as brain monitoring. Polarization gating has been widely used to analyze biological tissues. It is shown that using polarized light allows probing tissues on a specific depth depending on the polarization illumination type (linearly, circularly) and the tissues properties. However, accurate depth investigation of the tissue requires a high selectivity of the probed depth. We propose and simulate the use of different elliptically polarized illuminations for continuous depth examination between linearly and circularly polarized illumination. Monte Carlo simulations verify that circularly polarized illumination penetrates deeper than linearly polarized illumination in biological scattering media. Furthermore, we show that elliptically polarized light can be tuned in its penetration depth continuously between the penetration depth of linearly polarized light and circularly polarized light. Experimental results obtained on phantoms mimicking in vivo situations are presented.

The method proposed here allows to perform a selection of a well defined subsurface volume in a turbid medium allowing SNR enhancement for functional imaging of the cortex. The principle consists in sequentially probing the biological tissue with light polarized linearly or circularly. The method and preliminary results obtained on phantoms are presented.

International audience; Measuring erythrocyte velocity in individual microvessels has important applications for biomedical and functional imaging. Recent multiphoton fluorescence microscopy approaches require injecting fluorescent tracers; moreover, only one or few vessels can be imaged at a time. To overcome these shortcomings, we used CCD-based optical imaging of intrinsic absorption changes in macroscopic vascular networks to record erythrocytes' trajectories over several mm2 of cortical surface. We then demonstrate the feasibility of erythrocyte velocity estimation from such wide-field data, using two robust, independent, algorithms. The first one is a recently published Radon transform-based algorithm that estimates erythrocyte velocity locally. We adapt it to data obtained in wide-field imaging and show, for the first time, its performance on such datasets. The second ("fasttrack") algorithm is novel. It is based on global energy minimization techniques to estimate the full spatiotemporal erythrocytes' trajectories inside vessels. We test the two algorithms on both simulated and biological data, obtained in rat cerebral cortex in a spreading depression experiment. On vessels with medium-slow erythrocyte velocities both algorithms performed well, allowing their usage as benchmark one for another. However, our novel fasttrack algorithm outperformed the other one for higher velocities, as encountered in the arterial network.

Interpreting fMRI data relies on the assumption that hemodynamic responses reflect neuronal activity. Some recently reported results seem to suggest that this assumption might be less robust than what has been thought so far. Data by Schummers et al. (2008) suggest that hemodynamic responses depend on functional properties of astrocytes as mediators of neuronal activity to blood vessels, and therefore reflect neuronal tuning properties only indirectly. The question is how much the final outcome differs from a linear integration of the local neuronal responses.

Functional magnetic resonance imaging can now resolve individual cortical columns, which should provide insights into sensory perception and higher cognitive functions.

International audience; Several functional and biomedical imaging techniques rely on determining hemodynamic variables and their changes in large vascular networks. To do so at micro-vascular resolution requires taking into account the - usually small but often non-rigid - mechanical deformations of the imaged vasculature induced by the cardiac pulsation and/or the subjects'body movements. Here, we present two new algorithmic approaches, allowing (i) to efficiently and accurately co-register large sets of such images in a non-rigid manner using Scale-Invariant Feature Transform (SIFT) keypoints, and (ii) to extract blood vessels and their diameters based on blood-flow information using a fast marching algorithm. These methods were applied to optical imaging data of intrinsic signals from awake monkey visual cortex at high spatiotemporal resolution (30μm, 5ms). The movement of red blood cells in the sequences could be enhanced by a Beer-Lambert-based image preprocessing. Our SIFT-based registration could be directly compared to a rigid registration, whereas the vessel extraction algorithm was tested by verifying flow conservation in vascular branching points. Finally, both methods together proved to improve a lot the estimation of the blood velocity in the vessels.

In the neocortex, neurons with similar response properties are often clustered together in column-like structures, giving rise to what has become known as functional architecture-the mapping of various stimulus feature dimensions onto the cortical sheet. At least partially, we owe this finding to the availability of several functional brain imaging techniques, both post-mortem and in-vivo, which have become available over the last two generations, revolutionizing neuroscience by yielding information about the spatial organization of active neurons in the brain. Here, we focus on how our understanding of such functional architecture is linked to the development of those functional imaging methodologies, especially to those that image neuronal activity indirectly, through metabolic or haemodynamic signals, rather than directly through measurement of electrical activity. Some of those approaches allow exploring functional architecture at higher spatial resolution than others. In particular, optical imaging of intrinsic signals reaches the striking detail of approximately 50 mum, and, together with other methodologies, it has allowed characterizing the metabolic and haemodynamic responses induced by sensory-evoked neuronal activity. Here, we review those findings about the spatio-temporal characteristics of neurovascular coupling and discuss their implications for functional brain imaging, including position emission tomography, and non-invasive neuroimaging techniques, such as funtional magnetic resonance imaging, applicable also to the human brain.

During the last 20 years, optical imaging methods - either alone or in combination with other recording techniques - has proven a fruitful approach to explore both the physiological and the functional aspects of activity-evoked hemodynamic responses in cortex. One of the main advantages of optical imaging consists in its high spatio-temporal resolution (in the order of few microns and milliseconds), allowing not only to unambiguously distinguish between activity patterns relating to the underlying functional architecture and those originating from the activation of medium/large blood vessels, but also to investigate the various activity-evoked hemodynamic processes at very fine detail. Here, we briefly review the principal findings obtained by optical imaging about the spatio-temporal properties of the various hemodynamic responses in cortex, i.e., changes in blood-oxygenation, blood-volume, and, to some extent, blood-flow. We also discuss the implications of those findings for non-invasive high-resolution functional brain imaging, in particular for fMRI. Finally, we underscore the importance of novel approaches for high-resolution blood-flow imaging, in the context of the need to gather information at fine spatial detail about the blood-flow response, necessary to constrain the multiple free parameters of hemodynamic response models.

Visual neurons integrate information over a finite part of the visual field with high selectivity. This classical receptive field is modulated by peripheral inputs that play a role in both neuronal response normalization and contextual modulations. However, the consequences of these properties for visuomotor transformations are yet incompletely understood. To explore those, we recorded short-latency ocular following responses in humans to large center-only and center-surround stimuli. We found that eye movements are triggered by a mechanism that integrates motion over a restricted portion of the visual field, the size of which depends on stimulus contrast and increases as a function of time after response onset. We also found evidence for a strong nonisodirectional center-surround organization, responsible for normalizing the central, driving input so that motor responses are set to their most linear contrast dynamics. Such response normalization is delayed about 20 ms relative to tracking onset, gradually builds up over time, and is partly tuned for surround orientation/direction. These results outline the spatiotemporal organization of a behavioral receptive field, which might reflect a linear integration among subpopulations of cortical visual motion detectors.

Knowledge about sensory-evoked blood-flow changes is essential for constraining hemodynamic response models used to interpret functional brain imaging signals, such as fMRI. Here, we extracted 2-dimensional blood-flow and its temporal modulations from high-resolution optical imaging data in the awake monkey. Optical imaging allows to track moving erythrocytes (or small clusters thereof), thus providing, albeit noisy, information about their velocity in individual blood vessels, across the whole imaged area. Here, we illustrate the algorithms that allowed us to extract, at the single micro-vessel level, red blood cell (RBC) motion information from the noisy optical signals. For this purpose, we developed an algorithm that is both robust and computationally efficient, using the structure tensor, known to detect an average direction of image intensity gradient. This structure tensor tool is applied to detect trajectory directions in the spatio-temporal data. Since blood-flow modulation by the cardiac pulsation was clearly detected, our method should be applicable also to study blood-flow modulations by neuronal activity, and their spatio-temporal patterns.

Advancement in the treatment of blindness depends on the development of new technologies that enable early detection, follow-up, and treatment of disease. The authors describe direct, noninvasive imaging of four parameters: blood flow, blood oximetry, metabolic state, and hidden vasculature, particularly capillaries. These are functional parameters of the retina known to be degraded by retinal disease. The new Retinal Function Imager (Optical Imaging, Ltd., Rehovot, Israel) can image all four parameters as intrinsic reflectance intensity differences over the retina's surface. During the past 2 decades, imaging of small optical signals has been a powerful tool for high-resolution functional mapping in the neocortex. In this article, this technology is applied to the retina and demonstrates a general tool for noninvasively probing retinal function in many modalities. Imaging functional changes before anatomic consequences arise holds promise as a powerful tool for early diagnosis and treatment of retinal disease.[Ophthalmic Surg Lasers Imaging 2005;36:57–66.]

Much work remains to be done to establish the clinical usefulness of the RFI for early diagnosis and treatment guidance. The discoveries obtained by functional optical imaging of the neocortex in the last 15 years and the recent RFI studies of the eyes of normal subjects and patients with diabetic retinopathy, glaucoma, and age-related macular degeneration suggest that functional optical imaging of the retina is likely to become a multi-modality powerful clinical tool.

The ultimate goal of high-resolution functional brain mapping is single-condition (stimulus versus no-stimulus maps) rather than differential imaging (comparing two "stimulus maps"), because the appropriate ("orthogonal") stimuli are rarely available. This requires some component(s) of activity-dependent hemodynamic signals to closely colocalize with electrical activity, like the early increase in deoxyhemoglobin, shown previously to yield high-quality functional single-condition maps. Conversely, nonlocal vascular responses dominate in cerebral blood volume (CBV)-based single-condition maps. Differential CBV maps are largely restricted to the parenchyma, implying that part of the CBV response does colocalize with electrical activity at fine spatial scale. By removing surface vascular activation from optical imaging data, we document the existence of a capillary CBV response component, regulated at fine spatial scale and yielding single-condition maps exhibiting approximately 100 microm resolution. Blood volume and -flow based single-condition functional mapping at columnar level should thus be feasible, provided that the capillary response component is selectively imaged.

Explorations of learning and memory, other long-term plastic changes, and additional cognitive functions in the behaving primate brain would greatly benefit from the ability to image the functional architecture within the same patch of cortex, at the columnar level, for a long period of time. We developed methods for long-term optical imaging based on intrinsic signals and repeatedly visualized the same functional domains in behaving macaque cortex for a period extending over 1 year. Using optical imaging and imaging spectroscopy, we first explored the relationship between electrical activity and hemodynamic events in the awake behaving primate and compared it with anesthetized preparations. We found that, whereas the amplitude of the intrinsic signal was much larger in the awake animal, its temporal pattern was similar to that observed in the anesthetized animals. In both groups, deoxyhemoglobin concentration reached a peak 2–3 sec after stimulus onset. Furthermore, the early activity-dependent increase in deoxyhemoglobin concentration (the “initial dip”) was far more tightly colocalized with electrical activity than the delayed increase in oxyhemoglobin concentration, known to be associated with an increase in blood flow. The implications of these results for improvement of the spatial resolution of blood oxygenation level-dependent functional magnetic resonance imaging are discussed. After the characterization of the intrinsic signal in the behaving primate, we used this new imaging method to explore the stability of cortical maps in the macaque primary visual cortex. Functional maps of orientation and ocular dominance columns were found to be stable for a period longer than 1 year.

Optical imaging, PET and fMRI all rely on vascular responses to image neuronal activity. Although these imaging techniques are successfully used for functional brain mapping, the detailed spatio-temporal dynamics of hemodynamic events in the various microvascular compartments have remained unknown. Here, we used high-resolution optical imaging in area 18 of anesthetized cats to explore sensory-evoked blood-volume (CBV) changes in the various cortical microvascular compartments, selectively. To avoid the confounding effects of hematocrit and oximetry changes, we developed and used a new fluorescent blood plasma tracer and combined these measurements with optical imaging of intrinsic signals at a near-isosbestic wavelength for hemoglobin (565 nm). The vascular response began at the arteriolar level, rapidly spreading towards capillaries and venules. Larger veins lagged behind. Capillaries exhibited clear blood-volume changes. Arterioles and arteries had the largest response, whereas the venous response was smallest. Information about compartment-specific oxygen consumption dynamics was obtained in imaging sessions using 605 nm illumination, a wavelength known to reflect primarily oximetric changes, thus being more directly related to electrical activity than CBV changes. Those images were radically different: the response began at the parenchyma level, followed only later by the other microvascular compartments. These results have implications for the modeling of fMRI responses and suggest that the Balloon Model in its original formulation needs to be modified. Furthermore, functional maps obtained by imaging the capillary CBV response were similar but not identical to those obtained using the early oximetric signal, suggesting the presence of different regulatory mechanisms underlying these two hemodynamic processes.