Behavioral as well as hippocampal transcriptomic and microglial responses differ across sexes in adult mouse offspring exposed to a dual genetic and environmental challenge.

INTRODUCTION: A wide range of positive, negative, and cognitive symptoms compose the clinical presentation of schizophrenia. Schizophrenia is a multifactorial disorder in which genetic and environmental risk factors interact for a full emergence of the disorder. Infectious challenges during pregnancy are a well-known environmental risk factor for schizophrenia. Also, genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia. Translational animal models recapitulating these complex gene-environment associations have a great potential to untangle schizophrenia neurobiology and propose new therapeutic strategies. METHODS: Given that genetic variants affecting the function of fractalkine signaling between neurons and microglia were linked to schizophrenia, we compared the outcomes of a well-characterized model of maternal immune activation induced using the viral mimetic polyinosinic:polycytidylic acid (Poly I:C) in wild-type versus fractalkine receptor knockout mice. Possible behavioral and immune alterations were assessed in male and female offspring during adulthood. Considering the role of the hippocampus in schizophrenia, microglial analyses and bulk RNA sequencing were performed within this region to assess the neuroimmune dynamics at play. Males and females were examined separately. RESULTS: Offspring exposed to the dual challenge paradigm exhibited symptoms relevant to schizophrenia and unpredictably to mood disorders. Males displayed social and cognitive deficits related to schizophrenia, while females mainly presented anxiety-like behaviors related to mood disorders. Hippocampal microglia in females exposed to the dual challenge were hypertrophic, indicative of an increased surveillance, whereas those in males showed on the other end of the spectrum blunted morphologies with a reduced phagocytosis. Hippocampal bulk-RNA sequencing further revealed a downregulation in females of genes related to GABAergic transmission, which represents one of the main proposed causes of mood disorders. CONCLUSIONS: Building on previous results, we identified in the current study distinctive behavioral phenotypes in female mice exposed to a dual genetic and environmental challenge, thus proposing a new model of neurodevelopmentally-associated mood and affective symptoms. This paves the way to future sex-specific investigations into the susceptibility to developmental challenges using animal models based on genetic and immune vulnerability as presented here.

The implication of a diversity of non-neuronal cells in disorders affecting brain networks.

In the central nervous system (CNS) neurons are classically considered the functional unit of the brain. Analysis of the physical connections and co-activation of neurons, referred to as structural and functional connectivity, respectively, is a metric used to understand their interplay at a higher level. A myriad of glial cell types throughout the brain composed of microglia, astrocytes and oligodendrocytes are key players in the maintenance and regulation of neuronal network dynamics. Microglia are the central immune cells of the CNS, able to affect neuronal populations in number and connectivity, allowing for maturation and plasticity of the CNS. Microglia and astrocytes are part of the neurovascular unit, and together they are essential to protect and supply nutrients to the CNS. Oligodendrocytes are known for their canonical role in axonal myelination, but also contribute, with microglia and astrocytes, to CNS energy metabolism. Glial cells can achieve this variety of roles because of their heterogeneous populations comprised of different states. The neuroglial relationship can be compromised in various manners in case of pathologies affecting development and plasticity of the CNS, but also consciousness and mood. This review covers structural and functional connectivity alterations in schizophrenia, major depressive disorder, and disorder of consciousness, as well as their correlation with vascular connectivity. These networks are further explored at the cellular scale by integrating the role of glial cell diversity across the CNS to explain how these networks are affected in pathology.

Baseline oxygen consumption decreases with cortical depth.

The cerebral cortex is organized in cortical layers that differ in their cellular density, composition, and wiring. Cortical laminar architecture is also readily revealed by staining for cytochrome oxidase-the last enzyme in the respiratory electron transport chain located in the inner mitochondrial membrane. It has been hypothesized that a high-density band of cytochrome oxidase in cortical layer IV reflects higher oxygen consumption under baseline (unstimulated) conditions. Here, we tested the above hypothesis using direct measurements of the partial pressure of O2 (pO2) in cortical tissue by means of 2-photon phosphorescence lifetime microscopy (2PLM). We revisited our previously developed method for extraction of the cerebral metabolic rate of O2 (CMRO2) based on 2-photon pO2 measurements around diving arterioles and applied this method to estimate baseline CMRO2 in awake mice across cortical layers. To our surprise, our results revealed a decrease in baseline CMRO2 from layer I to layer IV. This decrease of CMRO2 with cortical depth was paralleled by an increase in tissue oxygenation. Higher baseline oxygenation and cytochrome density in layer IV may serve as an O2 reserve during surges of neuronal activity or certain metabolically active brain states rather than reflecting baseline energy needs. Our study provides to our knowledge the first quantification of microscopically resolved CMRO2 across cortical layers as a step towards better understanding of brain energy metabolism.

Toward an integrative neurovascular framework for studying brain networks.

Brain functional connectivity based on the measure of blood oxygen level-dependent (BOLD) functional magnetic resonance imaging (fMRI) signals has become one of the most widely used measurements in human neuroimaging. However, the nature of the functional networks revealed by BOLD fMRI can be ambiguous, as highlighted by a recent series of experiments that have suggested that typical resting-state networks can be replicated from purely vascular or physiologically driven BOLD signals. After going through a brief review of the key concepts of brain network analysis, we explore how the vascular and neuronal systems interact to give rise to the brain functional networks measured with BOLD fMRI. This leads us to emphasize a view of the vascular network not only as a confounding element in fMRI but also as a functionally relevant system that is entangled with the neuronal network. To study the vascular and neuronal underpinnings of BOLD functional connectivity, we consider a combination of methodological avenues based on multiscale and multimodal optical imaging in mice, used in combination with computational models that allow the integration of vascular information to explain functional connectivity.

Optical measurement of microvascular oxygenation and blood flow responses in awake mouse cortex during functional activation.

The cerebral cortex has a number of conserved morphological and functional characteristics across brain regions and species. Among them, the laminar differences in microvascular density and mitochondrial cytochrome c oxidase staining suggest potential laminar variability in the baseline O2 metabolism and/or laminar variability in both O2 demand and hemodynamic response. Here, we investigate the laminar profile of stimulus-induced intravascular partial pressure of O2 (pO2) transients to stimulus-induced neuronal activation in fully awake mice using two-photon phosphorescence lifetime microscopy. Our results demonstrate that stimulus-induced changes in intravascular pO2 are conserved across cortical layers I-IV, suggesting a tightly controlled neurovascular response to provide adequate O2 supply across cortical depth. In addition, we observed a larger change in venular O2 saturation (ΔsO2) compared to arterioles, a gradual increase in venular ΔsO2 response towards the cortical surface, and absence of the intravascular "initial dip" previously reported under anesthesia. This study paves the way for quantification of layer-specific cerebral O2 metabolic responses, facilitating investigation of brain energetics in health and disease and informed interpretation of laminar blood oxygen level dependent functional magnetic resonance imaging signals.

Movement correction method for laser speckle contrast imaging of cerebral blood flow in cranial windows in rodents.

Laser speckle contrast imaging (LSCI) is used in clinical research to dynamically image blood flow. One drawback is its susceptibility to movement artifacts. We demonstrate a new, simple method to correct motion artifacts in LSCI signals measured in awake mice with cranial windows during sensory stimulation. The principle is to identify a region in the image in which speckle contrast (SC) is independent of blood flow and only varies with animal movement, then to regress out this signal from the data. We show that (1) the regressed signal correlates well with mouse head movement, (2) the corrected signal correlates better with independently measured blood volume and (3) it has a (59 ± 6)% higher signal-to-noise ratio. Compared to three alternative correction methods, ours has the best performance. Regressing out flow-independent global variations in SC is a simple and accessible way to improve the quality of LSCI measurements.

A Suite of Neurophotonic Tools to Underpin the Contribution of Internal Brain States in fMRI.

Recent developments in optical microscopy, applicable for large-scale and longitudinal imaging of cortical activity in behaving animals, open unprecedented opportunities to gain a deeper understanding of neurovascular and neurometabolic coupling during different brain states. Future studies will leverage these tools to deliver foundational knowledge about brain state-dependent regulation of cerebral blood flow and metabolism as well as regulation as a function of brain maturation and aging. This knowledge is of critical importance to interpret hemodynamic signals observed with functional magnetic resonance imaging (fMRI).

Modeling of vascular space occupancy and BOLD functional MRI from first principles using real microvascular angiograms.

PURPOSE: The vascular space occupancy (VASO) is a functional MRI technique for probing cerebral blood volume changes noninvasively, including during neuronal activation in humans. An important consideration when implementing VASO is the BOLD effect in the signal. Assessing the physical origin of this BOLD contamination and the capabilities of correction methods could improve the quantification of cerebral blood volume changes with VASO. METHODS: Given the heterogeneity of cerebral microvascular architecture, the vascular geometry within an MRI voxel can influence both BOLD and VASO signals. To investigate this effect, 3D high-resolution images of mouse cerebral vasculature measured with two-photon microscopy were used to model BOLD and VASO signals from first principles using Monte Carlo diffusion of water protons. Quantitative plots of VASO together with intravascular and extravascular BOLD signals as a function of TE at B0 fields 1.5 T to 14 T were obtained. RESULTS: The BOLD contamination of the VASO response was on the order of 50% for gradient echo and 5% for spin echo at 7 T and TE = 6 ms and significantly increased with TE and B0 . Two currently used correction schemes were shown to account for most of this contamination and recover accurate relative signal changes, with optimal correction obtained using TEs as short as possible. CONCLUSION: These results may provide useful information for optimizing sequence parameters in VASO and BOLD functional MRI, leading the way to a wider application of these techniques in healthy and diseased brain.

Imaging the Neuroimmune Dynamics Across Space and Time.

The immune system is essential for maintaining homeostasis, as well as promoting growth and healing throughout the brain and body. Considering that immune cells respond rapidly to changes in their microenvironment, they are very difficult to study without affecting their structure and function. The advancement of non-invasive imaging methods greatly contributed to elucidating the physiological roles performed by immune cells in the brain across stages of the lifespan and contexts of health and disease. For instance, techniques like two-photon in vivo microscopy were pivotal for studying microglial functional dynamics in the healthy brain. Through these observations, their interactions with neurons, astrocytes, blood vessels and synapses were uncovered. High-resolution electron microscopy with immunostaining and 3D-reconstruction, as well as super-resolution fluorescence microscopy, provided complementary insights by revealing microglial interventions at synapses (phagocytosis, trogocytosis, synaptic stripping, etc.). In addition, serial block-face scanning electron microscopy has provided the first 3D reconstruction of a microglial cell at nanoscale resolution. This review will discuss the technical toolbox that currently allows to study microglia and other immune cells in the brain, as well as introduce emerging methods that were developed and could be used to increase the spatial and temporal resolution of neuroimmune imaging. A special attention will also be placed on positron emission tomography and the development of selective functional radiotracers for microglia and peripheral macrophages, considering their strong potential for research translation between animals and humans, notably when paired with other imaging modalities such as magnetic resonance imaging.

Structural and Functional Features of Developing Brain Capillaries, and Their Alteration in Schizophrenia.

Schizophrenia affects more than 1% of the world's population and shows very high heterogeneity in the positive, negative, and cognitive symptoms experienced by patients. The pathogenic mechanisms underlying this neurodevelopmental disorder are largely unknown, although it is proposed to emerge from multiple genetic and environmental risk factors. In this work, we explore the potential alterations in the developing blood vessel network which could contribute to the development of schizophrenia. Specifically, we discuss how the vascular network evolves during early postnatal life and how genetic and environmental risk factors can lead to detrimental changes. Blood vessels, capillaries in particular, constitute a dynamic and complex infrastructure distributing oxygen and nutrients to the brain. During postnatal development, capillaries undergo many structural and anatomical changes in order to form a fully functional, mature vascular network. Advanced technologies like magnetic resonance imaging and near infrared spectroscopy are now enabling to study how the brain vasculature and its supporting features are established in humans from birth until adulthood. Furthermore, the contribution of the different neurovascular unit elements, including pericytes, endothelial cells, astrocytes and microglia, to proper brain function and behavior, can be dissected. This investigation conducted among different brain regions altered in schizophrenia, such as the prefrontal cortex, may provide further evidence that schizophrenia can be considered a neurovascular disorder.

Chronic Cranial Windows for Long Term Multimodal Neurovascular Imaging in Mice.

Chronic cranial windows allow for longitudinal brain imaging experiments in awake, behaving mice. Different imaging technologies have their unique advantages and combining multiple imaging modalities offers measurements of a wide spectrum of neuronal, glial, vascular, and metabolic parameters needed for comprehensive investigation of physiological and pathophysiological mechanisms. Here, we detail a suite of surgical techniques for installation of different cranial windows targeted for specific imaging technologies and their combination. Following these techniques and practices will yield higher experimental success and reproducibility of results.

More homogeneous capillary flow and oxygenation in deeper cortical layers correlate with increased oxygen extraction.

Our understanding of how capillary blood flow and oxygen distribute across cortical layers to meet the local metabolic demand is incomplete. We addressed this question by using two-photon imaging of resting-state microvascular oxygen partial pressure (PO2) and flow in the whisker barrel cortex in awake mice. Our measurements in layers I-V show that the capillary red-blood-cell flux and oxygenation heterogeneity, and the intracapillary resistance to oxygen delivery, all decrease with depth, reaching a minimum around layer IV, while the depth-dependent oxygen extraction fraction is increased in layer IV, where oxygen demand is presumably the highest. Our findings suggest that more homogeneous distribution of the physiological observables relevant to oxygen transport to tissue is an important part of the microvascular network adaptation to local brain metabolism. These results will inform the biophysical models of layer-specific cerebral oxygen delivery and consumption and improve our understanding of the diseases that affect cerebral microcirculation.

Awake Mouse Imaging: From Two-Photon Microscopy to Blood Oxygen Level-Dependent Functional Magnetic Resonance Imaging.

BACKGROUND: Functional magnetic resonance imaging (fMRI) in awake behaving mice is well positioned to bridge the detailed cellular-level view of brain activity, which has become available owing to recent advances in microscopic optical imaging and genetics, to the macroscopic scale of human noninvasive observables. However, though microscopic (e.g., two-photon imaging) studies in behaving mice have become a reality in many laboratories, awake mouse fMRI remains a challenge. Owing to variability in behavior among animals, performing all types of measurements within the same subject is highly desirable and can lead to higher scientific rigor. METHODS: We demonstrated blood oxygenation level-dependent fMRI in awake mice implanted with long-term cranial windows that allowed optical access for microscopic imaging modalities and optogenetic stimulation. We started with two-photon imaging of single-vessel diameter changes (n = 1). Next, we implemented intrinsic optical imaging of blood oxygenation and flow combined with laser speckle imaging of blood flow obtaining a mesoscopic picture of the hemodynamic response (n = 16). Then we obtained corresponding blood oxygenation level-dependent fMRI data (n = 5). All measurements could be performed in the same mice in response to identical sensory and optogenetic stimuli. RESULTS: The cranial window did not deteriorate the quality of fMRI and allowed alternation between imaging modalities in each subject. CONCLUSIONS: This report provides a proof of feasibility for multiscale imaging approaches in awake mice. In the future, this protocol could be extended to include complex cognitive behaviors translatable to humans, such as sensory discrimination or attention.

Deep 2-photon imaging and artifact-free optogenetics through transparent graphene microelectrode arrays.

Recent advances in optical technologies such as multi-photon microscopy and optogenetics have revolutionized our ability to record and manipulate neuronal activity. Combining optical techniques with electrical recordings is of critical importance to connect the large body of neuroscience knowledge obtained from animal models to human studies mainly relying on electrophysiological recordings of brain-scale activity. However, integration of optical modalities with electrical recordings is challenging due to generation of light-induced artifacts. Here we report a transparent graphene microelectrode technology that eliminates light-induced artifacts to enable crosstalk-free integration of 2-photon microscopy, optogenetic stimulation, and cortical recordings in the same in vivo experiment. We achieve fabrication of crack- and residue-free graphene electrode surfaces yielding high optical transmittance for 2-photon imaging down to ~ 1 mm below the cortical surface. Transparent graphene microelectrode technology offers a practical pathway to investigate neuronal activity over multiple spatial scales extending from single neurons to large neuronal populations.

Magnetic resonance fingerprinting based on realistic vasculature in mice.

Magnetic resonance fingerprinting (MRF) was recently proposed as a novel strategy for MR data acquisition and analysis. A variant of MRF called vascular MRF (vMRF) followed, that extracted maps of three parameters of physiological importance: cerebral oxygen saturation (SatO2), mean vessel radius and cerebral blood volume (CBV). However, this estimation was based on idealized 2-dimensional simulations of vascular networks using random cylinders and the empirical Bloch equations convolved with a diffusion kernel. Here we focus on studying the vascular MR fingerprint using real mouse angiograms and physiological values as the substrate for the MR simulations. The MR signal is calculated ab initio with a Monte Carlo approximation, by tracking the accumulated phase from a large number of protons diffusing within the angiogram. We first study the identifiability of parameters in simulations, showing that parameters are fully estimable at realistically high signal-to-noise ratios (SNR) when the same angiogram is used for dictionary generation and parameter estimation, but that large biases in the estimates persist when the angiograms are different. Despite these biases, simulations show that differences in parameters remain estimable. We then applied this methodology to data acquired using the GESFIDE sequence with SPIONs injected into 9 young wild type and 9 old atherosclerotic mice. Both the pre injection signal and the ratio of post-to-pre injection signals were modeled, using 5-dimensional dictionaries. The vMRF methodology extracted significant differences in SatO2, mean vessel radius and CBV between the two groups, consistent across brain regions and dictionaries. Further validation work is essential before vMRF can gain wider application.

The roadmap for estimation of cell-type-specific neuronal activity from non-invasive measurements.

The computational properties of the human brain arise from an intricate interplay between billions of neurons connected in complex networks. However, our ability to study these networks in healthy human brain is limited by the necessity to use non-invasive technologies. This is in contrast to animal models where a rich, detailed view of cellular-level brain function with cell-type-specific molecular identity has become available due to recent advances in microscopic optical imaging and genetics. Thus, a central challenge facing neuroscience today is leveraging these mechanistic insights from animal studies to accurately draw physiological inferences from non-invasive signals in humans. On the essential path towards this goal is the development of a detailed 'bottom-up' forward model bridging neuronal activity at the level of cell-type-specific populations to non-invasive imaging signals. The general idea is that specific neuronal cell types have identifiable signatures in the way they drive changes in cerebral blood flow, cerebral metabolic rate of O2 (measurable with quantitative functional Magnetic Resonance Imaging), and electrical currents/potentials (measurable with magneto/electroencephalography). This forward model would then provide the 'ground truth' for the development of new tools for tackling the inverse problem-estimation of neuronal activity from multimodal non-invasive imaging data.This article is part of the themed issue 'Interpreting BOLD: a dialogue between cognitive and cellular neuroscience'.

Cell type specificity of neurovascular coupling in cerebral cortex.

Identification of the cellular players and molecular messengers that communicate neuronal activity to the vasculature driving cerebral hemodynamics is important for (1) the basic understanding of cerebrovascular regulation and (2) interpretation of functional Magnetic Resonance Imaging (fMRI) signals. Using a combination of optogenetic stimulation and 2-photon imaging in mice, we demonstrate that selective activation of cortical excitation and inhibition elicits distinct vascular responses and identify the vasoconstrictive mechanism as Neuropeptide Y (NPY) acting on Y1 receptors. The latter implies that task-related negative Blood Oxygenation Level Dependent (BOLD) fMRI signals in the cerebral cortex under normal physiological conditions may be mainly driven by the NPY-positive inhibitory neurons. Further, the NPY-Y1 pathway may offer a potential therapeutic target in cerebrovascular disease.

Preliminary investigation of multispectral retinal tissue oximetry mapping using a hyperspectral retinal camera.

Oximetry measurement of principal retinal vessels represents a first step towards understanding retinal metabolism, but the technique could be significantly enhanced by spectral imaging of the fundus outside of main vessels. In this study, a recently developed Hyperspectral Retinal Camera was used to measure relative oximetric (SatO2) and total hemoglobin (HbT) maps of the retina, outside of large vessels, in healthy volunteers at baseline (N = 7) and during systemic hypoxia (N = 11), as well as in patients with glaucoma (N = 2). Images of the retina, on a field of view of ∼30°, were acquired between 500 and 600 nm with 2 and 5 nm steps, in under 3 s. The reflectance spectrum from each pixel was fitted to a model having oxy- and deoxyhemoglobin as the main absorbers and scattering modeled by a power law, yielding estimates of relative SatO2 and HbT over the fundus. Average optic nerve head (ONH) saturation over 8 eyes was 68 ± 5%. During systemic hypoxia, mean ONH saturation decreased by 12.5% on average. Upon further development and validation, the relative SatO2 and HbT maps of microvasculature obtained with this imaging system could ultimately contribute to the diagnostic and management of diseases affecting the ONH and retina.

Shedding light on the effect of priority instructions during dual-task performance in younger and older adults: A fNIRS study.

Age-related differences in the ability to perform two tasks simultaneously (or dual-task) have become a major concern in aging neurosciences and have often been assessed with two distinct paradigms; the Psychological Refractory Period (PRP) and the Dual-Task (DT) paradigms. PRP studies assess participants when they give Priority to one task over the other (complete A then B), whereas in DT studies participants give Equal priority to both tasks (complete A and B). The Equal condition could be viewed as adding an executive control component to the task since the participants must spontaneously monitor attention between tasks. In the current study, we assessed the effect of priority instructions (Priority vs. Equal) on the dual-task performance and brain activity of younger (n = 16) and older adults (n = 19) with functional near infra-red spectroscopy (fNIRS). In younger adults, the Priority condition showed right-sided activation in the prefrontal cortex during DT execution. Older adults showed bilateral frontal activation, yet restrained to specific areas. They showed increased activation in DT vs. single task condition in the left dorsolateral prefrontal cortex (DLPFC) and the bilateral ventrolateral prefrontal cortex (VLPFC). In the Equal condition, the DT condition showed isolated left DLPFC and VLPFC activation in younger adults and widespread bilateral DLPFC activation in older adults. These results suggest that for both older and younger adults, priority effects are associated with distinct patterns of prefrontal activation. Age-related differences also exist in these patterns such that prefrontal activation seems to be more spread out at different sites in older adults when they are instructed to give Equal priority to both tasks.