Analysis of resting-state neurovascular coupling and locomotion-associated neural dynamics using wide-field optical mapping

Understanding the relationship between neural activity and cortical hemodynamics, or neurovascular coupling is the foundation to interpret neuroimaging signals such as functional magnetic resonance imaging (fMRI) which measure local changes in hemodynamics as a proxy for underlying neural activity. Even though the stereotypical stimulus-evoked hemodynamic response pattern with increased concentration of oxy- and total-hemoglobin and decrease in concentration of deoxy-hemoglobin has been well-recognized, the linearity of neurovascular coupling and its variances depending on brain state and tasks haven’t been thoroughly evaluated. To directly assess the cortical neurovascular coupling, simultaneous recordings of neural and hemodynamic activity were imaged by wide-field optical mapping (WFOM) over the bilateral dorsal surface of the mouse brain through a bilateral thinned-skull cranial window. Neural imaging is achieved through wide-field fluorescence imaging in animals expressing genetically encoded calcium sensor (Thy1-GCaMP). Hemodynamics are recorded via simultaneous imaging of multi-spectral reflectance. Significant hemodynamic crosstalk was found in the detected fluorescence signal and the physical model of the contamination, methods of correction as well as electrophysiological verification are presented. A linear model between neural and hemodynamic signals was used to fit spatiotemporal hemodynamics can be predicted by convolving local fluorescence changes with hemodynamic response functions derived through both deconvolution and gamma-variate fitting. Beyond confirming that the resting-state hemodynamics in the awake and anesthetized brain are coupled to underlying neural activity, the patterns of bilaterally symmetric spontaneous neural activity observed by WFOM emulate the functionally connected networks detected by fMRI. This result provides reassurance that resting-state functional connectivity has neural origins. With the access to cortical neural activity at mesoscopic level, we further explore the cortical neural representations preceding and during spontaneous locomotion

Maternal fluoxetine exposure alters cortical hemodynamic and calcium response of offspring to somatosensory stimuli

Epidemiological studies have found an increased incidence of neurodevelopmental disorders in populations prenatally exposed to selective serotonin reuptake inhibitors (SSRIs). Optical imaging provides a minimally invasive way to determine if perinatal SSRI exposure has long-term effects on cortical function. Herein we probed the functional neuroimaging effects of perinatal SSRI exposure in a fluoxetine (FLX)-exposed mouse model. While resting-state homotopic contralateral functional connectivity was unperturbed, the evoked cortical response to forepaw stimulation was altered in FLX mice. The stimulated cortex showed decreased activity for FLX versus controls, by both hemodynamic responses [oxyhemoglobin (Hb

Interrogating spatiotemporal patterns of resting state neuronal and hemodynamic activity in the awake mouse model

Since the advent of functional magnetic resonance imaging (fMRI) and the rise in popularity of its use for resting state functional connectivity mapping (rs-FCM) to non-invasively detect correlated networks of brain activity in human and animal models, many resting state FCM studies have reported differences in these networks under pathologies such as Alzheimer’s or schizophrenia, highlighting the potential for the method’s diagnostic relevance. A common underlying assumption of this analysis, however, is that the blood oxygen level dependent (BOLD) signal of fMRI is a direct measurement of local neural activity. The BOLD signal is in fact a measurement of the local changes in concentration of deoxy-hemoglobin (HbR). Thus, it is imperative that neurovascular coupling—the relationship between neuronal activity and subsequent hemodynamic activity—be better characterized to enable accurate interpretation of resting state fMRI in the context of clinical usage. This dissertation first describes the development and utility of WFOM paradigm for the robust and easily adaptable imaging of simultaneous neuronal and hemodynamic activity in awake mouse models of health or disease in strains with genetically encoded fluorescent calcium reporters. Subsequent exploration of resting state WFOM data collected in Thy1-GCaMP3 and Thy1-GCaMP6f mouse strains is then presented, namely the characterization of spatiotemporal patterns of neuronal and hemodynamic activity and different modulatory depths of neuronal activity via a toolbox of unsupervised blind source separation (e.g. k-means clustering) and supervised (e.g. non-negative least squares, Pearson correlation) analysis tools. The presence of these different modulatory depths of neuronal activity were then confirmed in another Thy1-jRGECO1a mouse strain using the same imaging scheme. Finally, the dissertation documents the application of the WFOM paradigm and select analysis tools to a novel mouse model of diffusely infiltrating glioma, through which neuronal and hemodynamic activity changes during diffusely infiltrating glioma development which impact temporal coherence of the tumor region activity relative to non-tumor regions activity were recorded and analyzed. The paradigm also allowed for recording of numerous spontaneous occurrences of interictal neuronal activity during which neurovascular coupling is modified in the tumor, as well as occurrences of non-convulsive generalized seizure activity (during which neurovascular is non-linear and cortex eventually suffers hypoxia). The detection of spatiotemporal patterns and different modulatory depths of activity in the awake mouse cortex, as well as observation of changes in functional activity in the context of diffusely infiltrating glioma, provide us with new insights into the possible mechanisms underlying variations in resting state connectivity networks found in resting state fMRI studies comparing health and disease states

Evaluating endothelial function during neurovascular coupling in awake behaving mice using advanced imaging technologies

Local neuronal activity in the brain results in increased blood flow and is called neurovascular coupling. Such blood flow changes result in the blood-oxygen level dependent (BOLD) fluctuations detectable by functional magnetic resonance imaging (fMRI). The hemodynamic response is also an essential component of brain health and is impaired in various models of cognitive dysfunction. However, we still do not understand why functional hyperemia in the brain is important. To understand this question, various groups have studied brain metabolic activity as well as the mechanisms underlying neurovascular coupling. Over the years, several cell types have been proposed to contribute to functional hyperemia in the brain, including neurons, astrocytes and pericytes. However, the picture remains incomplete – controversies abound regarding the exact role of astrocytes, and pericytes in neurovascular coupling. Our lab has studies the mechanisms of neurovascular coupling from a mesoscopic perspective, as vasodilation in the rodent cortex involves capillaries and diving arterioles in the brain parenchyma as well as surface vasculature in the brain. We proposed that the vascular endothelium itself might provide a continuous conduit for transmitting vasodilatory signals initiated at the capillary level due to local neuronal activity. Given that systemic endothelial dysfunction could contribute to decreased neurovascular function, this hypothesis raised important concerns regarding endothelial vulnerabilities in common diseases like hypertension and diabetes and its role in diminished cognitive function and neurodegeneration. Based on findings from vascular research in other organ systems, we hypothesized that two distinct mechanisms of endothelium-derived vasodilation significantly contribute to neurovascular coupling the brain. These two mechanisms were expected to consist of fast long-range endothelium-derived hyperpolarization (EDH) dependent vasodilation (conducted vasodilation) and slower, more localized endothelium calcium-wave dependent vasodilation (propagated vasodilation). Together, we expected these mechanisms to shape the spatio-temporal evolution of hemodynamic responses in the brain. This dual mechanism of endothelial control of the hyperemic response in the brain might explain the complex spatiotemporal properties and non-linearities of the fMRI blood oxygen level dependent (BOLD) signal. My initial experiments were conducted in anesthetized rats, where I pharmacologically inhibited endothelial dependent vasodilation during functional hyperemia in the somatosensory cortex under a hind-paw electrical stimulus paradigm. While the results gleaned from these experiments were very revealing, it was important to consider the effect of the pharmacological manipulations on neuronal activity in the brain. In addition, neurovascular coupling and overall brain blood flow in anesthetized animals is dramatically altered when compared to awake animals. In order to accomplish these goals, I built a wide-field optical imaging system that could simultaneously measure fluorescence-based neuronal activity and reflectance-based hemodynamic activity in awake head-restrained mice. I then used non-blood brain barrier permeable pharmacology to study endothelial mechanisms of neurovascular coupling in awake Thy1-GCaMP6f mice, which express the calcium fluorophore in a subset of excitatory neurons in the cortex. I found that using this pharmacology I could dissect out the hypothesized two spatiotemporally distinct components of whisker-stimulus evoked neurovascular coupling in awake mice. With simultaneous recording of the neuronal activity driving this blood flow, I was able to build a mathematical model for neurovascular coupling that accounted for these two mechanisms by allowing for the superposition of a time-invariant, constant hemodynamic response with a hemodynamic response obtained by convolving the underlying neuronal response with a hemodynamic response function (HRF). I was able to linearize these apparent non-linearities in the hemodynamic response by studying the properties of deconvolved HRFs for stimuli of different durations before and after pharmacological manipulation of endothelial activity. Two important considerations remain. Firstly, our wide-field, mesoscopic view of the brain prevents observations of endothelial function (hyperpolarization and calcium activity) and the propagation dynamics of dilation best observed at the microscopic level. To accomplish this task, ongoing experiments currently use our high-speed volumetric imaging technology (SCAPE – Swept Confocally Aligned Planar Excitation microscopy) to study stimulus-evoked vascular dynamics in mouse lines expressing GFP and GCaMP8 in endothelial cells. Secondly, our longitudinal imaging of these animals is ideal for studying the acute and long-term effects of endothelial dysfunction on cognitive function. This requires adequate study of changes in mouse behavior during manipulations of endothelial function longitudinally in awake mice. Future experiments should involve the development and implementation of appropriate task-based behavior experiments, and analysis methods for more carefully exploring changes in neuronal activity in the mouse brain during stimulus and non-stimulus dependent activity

The Correlation between Astrocytic Calcium and fMRI Signals is Related to the Thalamic Regulation of Cortical States

BOLD fMRI has been wildly used for mapping brain activity, but the cellular contribution of BOLD signals is still controversial. In this study, we investigated the correlation between neuronal/astrocytic calcium and the BOLD signal using simultaneous GCaMP-mediated calcium and BOLD signal recording, in the event-related state and in resting state, in anesthetized and in free-moving rats. To our knowledge, the results provide the first demonstration that evoked and intrinsic astrocytic calcium signals could occur concurrently accompanied by opposite BOLD signals which are associated with vasodilation and vasoconstriction. We show that the intrinsic astrocytic calcium is involved in brain state changes and is related to the activation of central thalamus. First, by simultaneous LFP and fiber optic calcium recording, the results show that the coupling between LFP and calcium indicates that neuronal activity is the basis of the calcium signal in both neurons and astrocytes. Second, we found that evoked neuronal and astrocytic calcium signals are always positively correlated with BOLD responses. However, intrinsic astrocytic calcium signals are accompanied by the activation of the central thalamus followed by a striking negative BOLD signal in cortex, which suggests that central thalamus may be involved in the initiation of the intrinsic astrocytic calcium signal. Third, we confirmed that the intrinsic astrocytic calcium signal is preserved in free moving rats. Moreover, the occurrences of intrinsic astrocytic calcium spikes are coincident with the transition between different sleep stages, which suggests intrinsic astrocytic calcium spikes reflect brain state transitions. These results demonstrate that the correlation between astrocytic calcium and fMRI signals is related to the thalamic regulation of cortical states. On the other hand, by studying the relationship between vessel–specific BOLD signals and spontaneous calcium activity from adjacent neurons, we show that low frequency spontaneous neuronal activity is the cellular mechanism of the BOLD signal during resting state

The impact of cortical perturbations on neurovascular dynamics

Neurons and the underlying vascular structure that maintains the nutrients necessary for their normal function are intrinsically linked. The relationship between neural activity and its accompanying blood flow is called neurovascular coupling. Our understanding of the intricacies of this relationship has evolved over the years from one of pure supply and demand to one that is highly complex and involves various cell types. While the exact mechanisms underlying neurovascular coupling still remains unresolved, altered coupling has been implicated in a variety of pathological conditions. The overall motivation of this thesis was to uncover how specific perturbations to either the neural or vascular system affect the resulting interplay between them. Our hope is that the results could act as a framework for guiding more specific mechanistic dissections in the future.Until recently, technological constraints have precluded the ability to comprehensively characterize neurovascular coupling on a large scale. Much of our understanding of the coupling relationship on a circuit level has been inferred from individual measurements of either neuronal firing or blood flow dynamics. Our lab has the ability to study coupling more directly through simultaneous imaging of both neural and hemodynamic activity. In this thesis, I set out to characterize how coupling could be differentially altered at a mesoscopic level by specifically perturbing either blood flow or cortical circuit organization. Thus, this work is split into two projects. The first investigates the downstream effects of an acute ischemic injury and the second focuses on how a developmental change in neuronal circuit structure alters function. My work in the acute ischemia model allowed us to capture a curious phenomenon called cortical spreading depolarization (CSD). CSDs have been implicated in a range of acute brain injuries, including ischemia. Despite being a neural event, CSDs have a profound impact on the cerebrovascular. Unfortunately, existing work in this field has been discordant and the results have been difficult to interpret. We used wide-field optical mapping to characterize the dynamics and impact of ischemia-triggered CSDs. Our imaging technique revealed that CSDs had a spatially heterogeneous impact on tissue depending on factors such as baseline metabolic condition and spatiotemporal properties of the CSDs themselves. Furthermore, we observed that CSDs were not isolated events and that multiple could occur in succession in a short period of time. By tracking each and every CSD, we were able to characterize the cumulative effects of CSDs on tissue oxygenation. Our results provide a contextual framework that reconciles some of the observed experimental variabilities. We conclude that an ischemic insult triggers a CSD and consequently, a combination of CSD dynamics and the tissue’s metabolic condition begets more CSDs. This pushes the brain deeper into a feedback loop of exacerbating damage. The second study was done in collaboration with Dr. Ewoud Schmidt and Dr. Franck Polleux, and looks at the functional changes mediated by expression of a human-specific gene duplication, SRGAP2C. The human brain exhibits unique features that enable its enhanced cognitive abilities. The Polleux lab found that humanized SRGAP2C mice showed similar features that characterize the human brain, such as increased synaptic density and delayed synaptic maturation. This ultimately led to increased local and long-range cortico-cortical connectivity and even improved the behavioral performance in a texture discrimination task. Thus, we were motivated to investigate the functional underpinnings that may explain and link these structural and behavioral differences. We used two-photon imaging to determine whether SRGAP2C expression changed neuronal firing dynamics and found that it increased response reliability and selectivity to whisker inputs, thus improving accuracy of sensory coding. This improvement may help to explain why SRGAP2C mice performed better in a cortex-dependent task that actively relies on engagement of multiple cortical regions. Moreover, by using a humanized SRGAP2C mouse model, our results provide a small step towards better understanding how experimental studies can be interpreted for and translated to humans

Three-dimensional Ca2+ imaging advances understanding of astrocyte biology.

Astrocyte communication is typically studied by two-dimensional calcium ion (Ca2+) imaging, but this method has not yielded conclusive data on the role of astrocytes in synaptic and vascular function. We developed a three-dimensional two-photon imaging approach and studied Ca2+ dynamics in entire astrocyte volumes, including during axon-astrocyte interactions. In both awake mice and brain slices, we found that Ca2+ activity in an individual astrocyte is scattered throughout the cell, largely compartmented between regions, preponderantly local within regions, and heterogeneously distributed regionally and locally. Processes and endfeet displayed frequent fast activity, whereas the soma was infrequently active. In awake mice, activity was higher than in brain slices, particularly in endfeet and processes, and displayed occasional multifocal cellwide events. Astrocytes responded locally to minimal axonal firing with time-correlated Ca2+ spots

Neuronal and Hemodynamic Functional Connectivity in the Awake Mouse

Resting State functional Magnetic Resonance Imaging (rs-fMRI) has revealed brain-wide correlation patterns throughout the human brain, interpreted as Functional Connectivity. Dynamic Functional Connectivity (DFC) has recently expanded on this technique via sliding window correlation analysis, revealing moment-to-moment changes in functional connectivity across an imaging session. However, the meaning of these transitions in terms of neural activity and behavior are not well understood.In this work, I utilized Dynamic Functional Connectivity analytical techniques in conjunction with Wide Field Optical Mapping (WFOM) in the awake, freely behaving mouse. I hypothesized that neural and hemodynamic activity observed with WFOM would exhibit similar transitions between functional connectivity states as reported by fMRI DFC studies. I also explored whether changes in functional connectivity would correspond to changes in behavior. Simultaneous neural and hemodynamic activity was collected using WFOM from five freely behaving head-fixed Thy1-jRGECO1a mice. Behavioral metrics of movement, whisking and pupillometry were acquired simultaneously. Raw neuroimaging data were dimensionally reduced to representative time courses across the dorsal surface of the cortex for each subject utilizing a semi-supervised clustering technique. Functional Connectivity analysis revealed rich spatiotemporal structures within neural and hemodynamic activity, which were consistent across imaging sessions and subjects. I observed broad changes in Functional Connectivity metrics during rest, locomotion, and transitional epochs between the two by directly comparing windows captured during these epochs. It was also observed that Functional Connectivity metrics immediately following locomotion offset could be distinguished from periods of sustained rest. Similar to human fMRI studies, a distinct increase in bilateral connectivity of anterior lateral prefrontal cortex was observed, which became significantly less synchronized with posterior brain regions during sustained periods of rest. I next used an unsupervised clustering technique on the same data to test if these properties could be observed in an indirect manner. This approach has been previously used in numerous human fMRI studies, and contextualized this work to human fMRI studies. A sliding window was used to calculate moment-to-moment Functional Connectivity maps across each imaging session. These dynamic correlation maps were clustered into multiple states, which could then be used to calculate the most representative state for any given epoch. Unsupervised clustering revealed strikingly similar dynamic states to our previous observations. These dynamic states also exhibited independent distributions of behavioral activity both in neural and hemodynamic models, leading us to conclude that there is not only a meaningful link between Functional Connectivity in neural and hemodynamic activity, but that behavioral shifts largely drive these changes. My findings provide strong evidence that Dynamic Functional Connectivity has neural origins, and hemodynamic responses are able to depict correlation patterns that tracks rapid changes in behavior and internal brain states such as the level of arousal or alertness. Future studies are necessary to further investigate this speculation, but this offers an excellent framework to better understand the rich, dynamic properties of brain activity

Influence of Focal Activity on Macroscale Brain Dynamics in Health and Disease

Macroscopic recordings of brain activity (e.g. fMRI, EEG) are a sensitive biomarker of the neural networks supporting neurocognitive function. However, it remains largely unclear what mechanisms mediate changes in macroscale networks after focal brain injuries like stroke, seizure, and TBI. Recently, optical neuroimaging in animal models has emerged as a powerful tool to begin addressing these questions. Using widefield imaging of cortical calcium dynamics in mice, this dissertation investigates the mechanisms by which focal disruptions in activity alter brain-wide functional dynamics. In two chapters, I demonstrate 1) that focal sensory stimulation elicits state-dependent, global slow waves propagating from primary somatosensory cortex (S1). Using a focal ischemic stroke model, I show that bilateral activation of somatosensory cortices is required for initiating global SWs, while spontaneous SWs are generated independent of S1. 2) That regional disruption of cortical excitability induces widespread changes across cortical networks, using chemogenetic manipulation of parvalbumin interneurons to model focal epileptiform activity in S1. We further show that local imbalances in excitability propagate differentially through intra- and interhemispheric connections, and can induce plasticity in large-scale networks. These studies begin to define the mechanisms of macro-scale network disruption after focal injuries, adding to our understanding of how local cortical circuits modulate global brain networks

Novel contrasts in photoacoustic tomography

Photoacoustic tomography (PAT) combines rich optical contrast and high ultrasonic resolution in optically scattering tissue at depths. Taking advantage of its 100% sensitivity to optical absorption, PAT has been widely applied to structural, functional and molecular imaging, with both endogenous and exogenous contrasts, at superior depths than pure optical methods. This dissertation explores novel absorption contrast mechanisms of PAT based on optical/thermal patterns, endogenous cellular chromophores, nanoparticles, small-molecule dyes and genetically-encoded proteins. With these novel contrasts, the proof-of-concept applications of PAT have been extended to include homogenous flow measurements, targeted angiogenesis imaging and therapy, label-free white blood cell imaging, 3D-whole-organ cell nuclei imaging with a subcellular resolution, and in vivo neural activity imaging with voltage/calcium-sensitive indicators. Specifically, Chapter 1 introduces photoacoustic microscopy (PAM) and photoacoustic computed tomography (PACT) systems and discuss the motivation of the dissertation. Chapter 2 describes two photoacoustic (PA) flow measurement methods with optical and thermal patterns, which are applicable to homogenous flowing medium. In the first method, a Doppler frequency shift in PA signals of the flow was detected and used to calculate flow speeds. In the second method, unique features in an externally imposed thermal pattern of the flow, captured by repeated B-scans along the flow direction with a PAM system, revealed different flow speeds. Chapter 3 explores the unique PA contrast of macrophages, an important type of white blood cells. Macrophages were imaged by PAM without any label, and their measured PA spectrum was distinctive from the hemoglobin spectrum, so they can be potentially differentiated from red blood cells in the blood stream. Next, with a microtomy-assisted PAM system, cell nuclei distribution in whole organs, including mouse brain and mouse lung, were imaged with subcellular resolution. Chapter 4 introduces a type of target copper nanoparticles, which are less expensive and more biocompatible than its counterpart gold nanoparticles. The PA signals of neovasculature in the mouse flank were enhanced by the ___3-targeted copper nanoparticles. Moreover, the work shows the first example of a systemically targeted antiangiogenic drug delivery with a photoacoustic contrast nanoparticle in vivo. Chapter 5 demonstrates the voltage imaging capability of PA. A voltage sensitive dye with sufficient signal change was discovered and used as a PA voltage indicator for the first time. The mechanism was characterized through both PA imaging and spectroscopic methods. Its use was explored in a mouse epilepsy model and cortical electrical stimulation model in vivo. Finally, the deep imaging potential of PA was realized by imaging the voltage response of cells under 4.5 mm thick slice of rat brain tissue using a PACT system. Chapter 6 proves the neural calcium imaging capability of PA with a genetically encoded calcium indicator. In a fly model, I ambiguously demonstrated for the first time that PA can be used to imaging neural activities in the fly brain without the interference signals from hemoglobin. In the a live-mouse-brain-slice model, I successfully demonstrated the deep imaging capability of PA for calcium imaging by imaging through a 2-mm-thick scattering medium with a PACT system