Mapping brain function in adults and young children during naturalistic viewing with high-density diffuse optical tomography.

Human studies of early brain development have been limited by extant neuroimaging methods. MRI scanners present logistical challenges for imaging young children, while alternative modalities like functional near-infrared spectroscopy have traditionally been limited by image quality due to sparse sampling. In addition, conventional tasks for brain mapping elicit low task engagement, high head motion, and considerable participant attrition in pediatric populations. As a result, typical and atypical developmental trajectories of processes such as language acquisition remain understudied during sensitive periods over the first years of life. We evaluate high-density diffuse optical tomography (HD-DOT) imaging combined with movie stimuli for high resolution optical neuroimaging in awake children ranging from 1 to 7 years of age. We built an HD-DOT system with design features geared towards enhancing both image quality and child comfort. Furthermore, we characterized a library of animated movie clips as a stimulus set for brain mapping and we optimized associated data analysis pipelines. Together, these tools could map cortical responses to movies and contained features such as speech in both adults and awake young children. This study lays the groundwork for future research to investigate response variability in larger pediatric samples and atypical trajectories of early brain development in clinical populations.

Visual Deprivation during Mouse Critical Period Reorganizes Network-Level Functional Connectivity.

A classic example of experience-dependent plasticity is ocular dominance (OD) shift, in which the responsiveness of neurons in the visual cortex is profoundly altered following monocular deprivation (MD). It has been postulated that OD shifts also modify global neural networks, but such effects have never been demonstrated. Here, we use wide-field fluorescence optical imaging (WFOI) to characterize calcium-based resting-state functional connectivity during acute (3 d) MD in female and male mice with genetically encoded calcium indicators (Thy1-GCaMP6f). We first establish the fundamental performance of WFOI by computing signal to noise properties throughout our data processing pipeline. Following MD, we found that Δ band (0.4-4 Hz) GCaMP6 activity in the deprived visual cortex decreased, suggesting that excitatory activity in this region was reduced by MD. In addition, interhemispheric visual homotopic functional connectivity decreased following MD, which was accompanied by a reduction in parietal and motor homotopic connectivity. Finally, we observed enhanced internetwork connectivity between the visual and parietal cortex that peaked 2 d after MD. Together, these findings support the hypothesis that early MD induces dynamic reorganization of disparate functional networks including the association cortices.

Comprehensive workflow and its validation for simulating diffuse speckle statistics for optical blood flow measurements.

Diffuse optical methods including speckle contrast optical spectroscopy and tomography (SCOS and SCOT), use speckle contrast (κ) to measure deep blood flow. In order to design practical systems, parameters such as signal-to-noise ratio (SNR) and the effects of limited sampling of statistical quantities, should be considered. To that end, we have developed a method for simulating speckle contrast signals including effects of detector noise. The method was validated experimentally, and the simulations were used to study the effects of physical and experimental parameters on the accuracy and precision of κ. These results revealed that systematic detector effects resulted in decreased accuracy and precision of κ in the regime of low detected signals. The method can provide guidelines for the design and usage of SCOS and/or SCOT instruments.

Attention-Based CNN-BiLSTM for Sleep State Classification of Spatiotemporal Wide-Field Calcium Imaging Data.

BACKGROUND: Wide-field calcium imaging (WFCI) with genetically encoded calcium indicators allows for spatiotemporal recordings of neuronal activity in mice. When applied to the study of sleep, WFCI data are manually scored into the sleep states of wakefulness, non-REM (NREM) and REM by use of adjunct EEG and EMG recordings. However, this process is time-consuming, invasive and often suffers from low inter- and intra-rater reliability. Therefore, an automated sleep state classification method that operates on spatiotemporal WFCI data is desired. NEW METHOD: A hybrid network architecture consisting of a convolutional neural network (CNN) to extract spatial features of image frames and a bidirectional long short-term memory network (BiLSTM) with attention mechanism to identify temporal dependencies among different time points was proposed to classify WFCI data into states of wakefulness, NREM and REM sleep. RESULTS: Sleep states were classified with an accuracy of 84% and Cohen's kappa of 0.64. Gradient-weighted class activation maps revealed that the frontal region of the cortex carries more importance when classifying WFCI data into NREM sleep while posterior area contributes most to the identification of wakefulness. The attention scores indicated that the proposed network focuses on short- and long-range temporal dependency in a state-specific manner. COMPARISON WITH EXISTING METHOD: On a 3-hour WFCI recording, the CNN-BiLSTM achieved a kappa of 0.67, comparable to a kappa of 0.65 corresponding to the human EEG/EMG-based scoring. CONCLUSIONS: The CNN-BiLSTM effectively classifies sleep states from spatiotemporal WFCI data and will enable broader application of WFCI in sleep.

A high-density diffuse optical tomography dataset of naturalistic viewing.

Traditional laboratory tasks offer tight experimental control but lack the richness of our everyday human experience. As a result many cognitive neuroscientists have been motivated to adopt experimental paradigms that are more natural, such as stories and movies. Here we describe data collected from 58 healthy adult participants (aged 18-76 years) who viewed 10 minutes of a movie (The Good, the Bad, and the Ugly, 1966). Most (36) participants viewed the clip more than once, resulting in 106 sessions of data. Cortical responses were mapped using high-density diffuse optical tomography (first- through fourth nearest neighbor separations of 1.3, 3.0, 3.9, and 4.7 cm), covering large portions of superficial occipital, temporal, parietal, and frontal lobes. Consistency of measured activity across subjects was quantified using intersubject correlation analysis. Data are provided in both channel format (SNIRF) and projected to standard space (NIfTI), using an atlas-based light model. These data are suitable for methods exploration as well as investigating a wide variety of cognitive phenomena.

Identifying Naturalistic Movies from Human Brain Activity with High-Density Diffuse Optical Tomography.

Modern neuroimaging modalities, particularly functional MRI (fMRI), can decode detailed human experiences. Thousands of viewed images can be identified or classified, and sentences can be reconstructed. Decoding paradigms often leverage encoding models that reduce the stimulus space into a smaller yet generalizable feature set. However, the neuroimaging devices used for detailed decoding are non-portable, like fMRI, or invasive, like electrocorticography, excluding application in naturalistic use. Wearable, non-invasive, but lower-resolution devices such as electroencephalography and functional near-infrared spectroscopy (fNIRS) have been limited to decoding between stimuli used during training. Herein we develop and evaluate model-based decoding with high-density diffuse optical tomography (HD-DOT), a higher-resolution expansion of fNIRS with demonstrated promise as a surrogate for fMRI. Using a motion energy model of visual content, we decoded the identities of novel movie clips outside the training set with accuracy far above chance for single-trial decoding. Decoding was robust to modulations of testing time window, different training and test imaging sessions, hemodynamic contrast, and optode array density. Our results suggest that HD-DOT can translate detailed decoding into naturalistic use.

Evaluation of gliovascular functions of AQP4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links the astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, which preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel AQP4 mouse line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wild-type (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to WT and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. An increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two was unchanged. However, at 100% readthrough, AQP4x localization and the formation of higher order complexes were disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for an increased proportion of endothelial cells with budding vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Anatomical Modeling and Optimization of Speckle Contrast Optical Tomography.

Traditional methods for mapping cerebral blood flow (CBF), such as positron emission tomography and magnetic resonance imaging, offer only isolated snapshots of CBF due to scanner logistics. Speckle contrast optical tomography (SCOT) is a promising optical technique for mapping CBF. However, while SCOT has been established in mice, the method has not yet been demonstrated in humans - partly due to a lack of anatomical reconstruction methods and uncertainty over the optimal design parameters. Herein we develop SCOT reconstruction methods that leverage MRI-based anatomical head models and finite-element modeling of the SCOT forward problem (NIRFASTer). We then simulate SCOT for CBF perturbations to evaluate sensitivity of imaging performance to exposure time and SD-distances. We find image resolution comparable to intensity-based diffuse optical tomography at superficial cortical tissue depth (~1.5 cm). Localization errors can be reduced by including longer SD-measurements. With longer exposure times speckle contrast decreases, however, noise decreases faster, resulting in a net increase in SNR. Specifically, extending exposure time from 10µs to 10ms increased SCOT SNR by 1000X. Overall, our modeling methods provide anatomically-based image reconstructions that can be used to evaluate a broad range of tissue conditions, measurement parameters, and noise sources and inform SCOT system design.

Evaluation of gliovascular functions of Aqp4 readthrough isoforms.

Aquaporin-4 (AQP4) is a water channel protein that links astrocytic endfeet to the blood-brain barrier (BBB) and regulates water and potassium homeostasis in the brain, as well as the glymphatic clearance of waste products that would otherwise potentiate neurological diseases. Recently, translational readthrough was shown to generate a C-terminally extended variant of AQP4, known as AQP4x, that preferentially localizes around the BBB through interaction with the scaffolding protein α-syntrophin, and loss of AQP4x disrupts waste clearance from the brain. To investigate the function of AQP4x, we generated a novel mouse AQP4 line (AllX) to increase relative levels of the readthrough variant above the ~15% of AQP4 in the brain of wildtype (WT) mice. We validated the line and assessed characteristics that are affected by the presence of AQP4x, including AQP4 and α-syntrophin localization, integrity of the BBB, and neurovascular coupling. We compared AllXHom and AllXHet mice to wildtype, and to previously characterized AQP4 NoXHet and NoXHom mice, which cannot produce AQP4x. Increased dose of AQP4x enhanced perivascular localization of α-syntrophin and AQP4, while total protein expression of the two were unchanged. However, at 100% readthrough, AQP4x localization and formation of higher-order complexes was disrupted. Electron microscopy showed that overall blood vessel morphology was unchanged except for increased endothelial cell vesicles in NoXHom mice, which may correspond to a leakier BBB or altered efflux that was identified in NoX mice using MRI. These data demonstrate that AQP4x plays a small but measurable role in maintaining BBB integrity as well as recruiting structural and functional support proteins to the blood vessel. This also establishes a new set of genetic tools for quantitatively modulating AQP4x levels.

Ultra-high density imaging arrays for diffuse optical tomography of human brain improve resolution, signal-to-noise, and information decoding.

Functional magnetic resonance imaging (fMRI) has dramatically advanced non-invasive human brain mapping and decoding. Functional near-infrared spectroscopy (fNIRS) and high-density diffuse optical tomography (HD-DOT) non-invasively measure blood oxygen fluctuations related to brain activity, like fMRI, at the brain surface, using more-lightweight equipment that circumvents ergonomic and logistical limitations of fMRI. HD-DOT grids have smaller inter-optode spacing (∼13 mm) than sparse fNIRS (∼30 mm) and therefore provide higher image quality, with spatial resolution ∼1/2 that of fMRI. Herein, simulations indicated reducing inter-optode spacing to 6.5 mm would further improve image quality and noise-resolution tradeoff, with diminishing returns below 6.5 mm. We then constructed an ultra-high-density DOT system (6.5-mm spacing) with 140 dB dynamic range that imaged stimulus-evoked activations with 30-50% higher spatial resolution and repeatable multi-focal activity with excellent agreement with participant-matched fMRI. Further, this system decoded visual stimulus position with 19-35% lower error than previous HD-DOT, throughout occipital cortex.

A comprehensive workflow and its validation for simulating diffuse speckle statistics for optical blood flow measurements.

Diffuse optical methods including speckle contrast optical spectroscopy and tomography (SCOS and SCOT), use speckle contrast (κ) to measure deep blood flow. In order to design practical systems, parameters such as signal-to-noise ratio (SNR) and the effects of limited sampling of statistical quantities, should be considered. To that end, we have developed a method for simulating speckle contrast signals including effects of detector noise. The method was validated experimentally, and the simulations were used to study the effects of physical and experimental parameters on the accuracy and precision of κ. These results revealed that systematic detector effects resulted in decreased accuracy and precision of κ in the regime of low detected signals. The method can provide guidelines for the design and usage of SCOS and/or SCOT instruments.

Visual deprivation during mouse critical period reorganizes network-level functional connectivity.

A classic example of experience-dependent plasticity is ocular dominance (OD) shift, in which the responsiveness of neurons in the visual cortex is profoundly altered following monocular deprivation (MD). It has been postulated that OD shifts also modify global neural networks, but such effects have never been demonstrated. Here, we used longitudinal wide-field optical calcium imaging to measure resting-state functional connectivity during acute (3-day) MD in mice. First, delta GCaMP6 power in the deprived visual cortex decreased, suggesting that excitatory activity was reduced in the region. In parallel, interhemispheric visual homotopic functional connectivity was rapidly reduced by the disruption of visual drive through MD and was sustained significantly below baseline state. This reduction of visual homotopic connectivity was accompanied by a reduction in parietal and motor homotopic connectivity. Finally, we observed enhanced internetwork connectivity between visual and parietal cortex that peaked at MD2. Together, these findings support the hypothesis that early MD induces dynamic reorganization of disparate functional networks including association cortices.

Myeloid cell activation during Zika virus encephalitis predicts recovery of functional cortical connectivity.

Neurologic complications of Zika virus (ZIKV) infection across the lifespan have been described during outbreaks in Southeast Asia, South America, and Central America since 2016. In the adult CNS ZIKV tropism for neurons is tightly linked to its effects, with neuronal loss within the hippocampus during acute infection and protracted synapse loss during recovery, which is associated with cognitive deficits. The effects of ZIKV on cortical networks have not been evaluated. Although animal behavior assays have been used previously to model cognitive impairment, in vivo brain imaging can provide orthogonal information regarding the health of brain networks in real time, providing a tool to translate findings in animal models to humans. In this study, we use widefield optical imaging to measure cortical functional connectivity (FC) in mice during acute infection with, and recovery from, intracranial infection with a mouse-adapted strain of ZIKV. Acute ZIKV infection leads to high levels of myeloid cell activation, with loss of neurons and presynaptic termini in the cerebral cortex and associated loss of FC primarily within the somatosensory cortex. During recovery, neuron numbers, synapses and FC recover to levels near those of healthy mice. However, hippocampal injury and impaired spatial cognition persist. The magnitude of activated myeloid cells during acute infection predicted both recovery of synapses and the degree of FC recovery after recovery from ZIKV infection. These findings suggest that a robust inflammatory response may contribute to the health of functional brain networks after recovery from infection.

Large-scale encoding of emotion concepts becomes increasingly similar between individuals from childhood to adolescence.

Humans require a shared conceptualization of others' emotions for adaptive social functioning. A concept is a mental blueprint that gives our brains parameters for predicting what will happen next. Emotion concepts undergo refinement with development, but it is not known whether their neural representations change in parallel. Here, in a sample of 5-15-year-old children (n = 823), we show that the brain represents different emotion concepts distinctly throughout the cortex, cerebellum and caudate. Patterns of activation to each emotion changed little across development. Using a model-free approach, we show that activation patterns were more similar between older children than between younger children. Moreover, scenes that required inferring negative emotional states elicited higher default mode network activation similarity in older children than younger children. These results suggest that representations of emotion concepts are relatively stable by mid to late childhood and synchronize between individuals during adolescence.

Mapping cortical activations underlying covert and overt language production using high-density diffuse optical tomography.

Gold standard neuroimaging modalities such as functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and more recently electrocorticography (ECoG) have provided profound insights regarding the neural mechanisms underlying the processing of language, but they are limited in applications involving naturalistic language production especially in developing brains, during face-to-face dialogues, or as a brain-computer interface. High-density diffuse optical tomography (HD-DOT) provides high-fidelity mapping of human brain function with comparable spatial resolution to that of fMRI but in a silent and open scanning environment similar to real-life social scenarios. Therefore, HD-DOT has potential to be used in naturalistic settings where other neuroimaging modalities are limited. While HD-DOT has been previously validated against fMRI for mapping the neural correlates underlying language comprehension and covert (i.e., "silent") language production, HD-DOT has not yet been established for mapping the cortical responses to overt (i.e., "out loud") language production. In this study, we assessed the brain regions supporting a simple hierarchy of language tasks: silent reading of single words, covert production of verbs, and overt production of verbs in normal hearing right-handed native English speakers (n = 33). First, we found that HD-DOT brain mapping is resilient to movement associated with overt speaking. Second, we observed that HD-DOT is sensitive to key activations and deactivations in brain function underlying the perception and naturalistic production of language. Specifically, statistically significant results were observed that show recruitment of regions in occipital, temporal, motor, and prefrontal cortices across all three tasks after performing stringent cluster-extent based thresholding. Our findings lay the foundation for future HD-DOT studies of imaging naturalistic language comprehension and production during real-life social interactions and for broader applications such as presurgical language assessment and brain-machine interfaces.

Open-source statistical and data processing tools for wide-field optical imaging data in mice.

Significance: Wide-field optical imaging (WOI) can produce concurrent hemodynamic and cell-specific calcium recordings across the entire cerebral cortex in animal models. There have been multiple studies using WOI to image mouse models with various environmental or genetic manipulations to understand various diseases. Despite the utility of pursuing mouse WOI alongside human functional magnetic resonance imaging (fMRI), and the multitude of analysis toolboxes in the fMRI literature, there is not an available open-source, user-friendly data processing and statistical analysis toolbox for WOI data. Aim: To assemble a MATLAB toolbox for processing WOI data, as described and adapted to combine techniques from multiple WOI groups and fMRI. Approach: We outline our MATLAB toolbox on GitHub with multiple data analysis packages and translate a commonly used statistical approach from the fMRI literature to the WOI data. To illustrate the utility of our MATLAB toolbox, we demonstrate the ability of the processing and analysis framework to detect a well-established deficit in a mouse model of stroke and plot activation areas during an electrical paw stimulus experiment. Results: Our processing toolbox and statistical methods isolate a somatosensory-based deficit 3 days following photothrombotic stroke and cleanly localize sensory stimulus activations. Conclusions: The toolbox presented here details an open-source, user-friendly compilation of WOI processing tools with statistical methods to apply to any biological question investigated with WOI techniques.

Mecp2 deletion results in profound alterations of developmental and adult functional connectivity.

As a regressive neurodevelopmental disorder with a well-established genetic cause, Rett syndrome and its Mecp2 loss-of-function mouse model provide an excellent opportunity to define potentially translatable functional signatures of disease progression, as well as offer insight into the role of Mecp2 in functional circuit development. Thus, we applied widefield optical fluorescence imaging to assess mesoscale calcium functional connectivity (FC) in the Mecp2 cortex both at postnatal day (P)35 in development and during the disease-related decline. We found that FC between numerous cortical regions was disrupted in Mecp2 mutant males both in juvenile development and early adulthood. Female Mecp2 mice displayed an increase in homotopic contralateral FC in the motor cortex at P35 but not in adulthood, where instead more posterior parietal regions were implicated. An increase in the amplitude of connection strength, both with more positive correlations and more negative anticorrelations, was observed across the male cortex in numerous functional regions. Widespread rescue of MeCP2 protein in GABAergic neurons rescued none of these functional deficits, nor, surprisingly, the expected male lifespan. Altogether, the female results identify early signs of disease progression, while the results in males indicate MeCP2 protein is required for typical FC in the brain.

Multi-mode fiber-based speckle contrast optical spectroscopy: analysis of speckle statistics.

Speckle contrast optical spectroscopy/tomography (SCOS/T) provides a real-time, non-invasive, and cost-efficient optical imaging approach to mapping of cerebral blood flow. By measuring many speckles (n>>10), SCOS/T has an increased signal-to-noise ratio relative to diffuse correlation spectroscopy, which measures one or a few speckles. However, the current free-space SCOS/T designs are not ideal for large field-of-view imaging in humans because the curved head contour cannot be readily imaged with a single flat sensor and hair obstructs optical access. Herein, we evaluate the feasibility of using cost-efficient multi-mode fiber (MMF) bundles for use in SCOS/T systems. One challenge with speckle contrast measurements is the potential for confounding noise sources (e.g., shot noise, readout noise) which contribute to the standard deviation measure and corrupt the speckle contrast measure that is central to the SCOS/T systems. However, for true speckle measurements, the histogram of pixel intensities from light interference follows a non-Gaussian distribution, specifically a gamma distribution with non-zero skew, whereas most noise sources have pixel intensity distributions that are Gaussian. By evaluating speckle data from static and dynamic targets imaged through an MMF, we use histograms and statistical analysis of pixel histograms to evaluate whether the statistical properties of the speckles are retained. We show that flow-based speckle can be distinguished from static speckle and from sources of system noise through measures of skew in the pixel intensity histograms. Finally, we illustrate in humans that MMF bundles relay blood flow information.

TRPV1-mediated sonogenetic neuromodulation of motor cortex in freely moving mice.

Background.Noninvasive and cell-type-specific neuromodulation tools are critically needed for probing intact brain function. Sonogenetics for noninvasive activation of neurons engineered to express thermosensitive transient receptor potential vanilloid 1 (TRPV1) by transcranial focused ultrasound (FUS) was recently developed to address this need. However, using TRPV1-mediated sonogenetics to evoke behavior by targeting the cortex is challenged by its proximity to the skull due to high skull absorption of ultrasound and increased risks of thermal-induced tissue damage.Objective.This study evaluated the feasibility and safety of TRPV1-mediated sonogenetics in targeting the motor cortex to modulate the locomotor behavior of freely moving mice.Approach.Adeno-associated viral vectors was delivered to the mouse motor cortex via intracranial injection to express TRPV1 in excitatory neurons. A wearable FUS device was installed on the mouse head after a month to control neuronal activity by activating virally expressed TRPV1 through FUS sonication at different acoustic pressures. Immunohistochemistry staining ofex vivobrain slices was performed to verify neuron activation and evaluate safety.Results.TRPV1-mediated sonogenetic stimulation at 0.7 MPa successfully evoked rotational behavior in the direction contralateral to the stimulation site, activated cortical neurons as indicated by the upregulation of c-Fos, and did not induce significant changes in inflammatory or apoptotic markers (GFAP, Iba1, and Caspase-3). Sonogenetic stimulation of TRPV1 mice at a higher acoustic pressure, 1.1 MPa, induced significant changes in motor behavior and upregulation of c-Fos compared with FUS sonication of naïve mice at 1.1 MPa. However, signs of damage at the meninges were observed at 1.1 MPa.Significance.TRPV1-mediated sonogenetics can achieve effective and safe neuromodulation at the cortex with carefully selected FUS parameters. These findings expand the application of this technique to include superficial brain targets.