Neuromodulation of striatal D1 cells shapes BOLD fluctuations in anatomically connected thalamic and cortical regions.

Understanding how the brain's macroscale dynamics are shaped by underlying microscale mechanisms is a key problem in neuroscience. In animal models, we can now investigate this relationship in unprecedented detail by directly manipulating cellular-level properties while measuring the whole-brain response using resting-state fMRI. Here, we focused on understanding how blood-oxygen-level-dependent (BOLD) dynamics, measured within a structurally well-defined striato-thalamo-cortical circuit in mice, are shaped by chemogenetically exciting or inhibiting D1 medium spiny neurons (MSNs) of the right dorsomedial caudate putamen (CPdm). We characterize changes in both the BOLD dynamics of individual cortical and subcortical brain areas, and patterns of inter-regional coupling (functional connectivity) between pairs of areas. Using a classification approach based on a large and diverse set of time-series properties, we found that CPdm neuromodulation alters BOLD dynamics within thalamic subregions that project back to dorsomedial striatum. In the cortex, changes in local dynamics were strongest in unimodal regions (which process information from a single sensory modality) and weakened along a hierarchical gradient towards transmodal regions. In contrast, a decrease in functional connectivity was observed only for cortico-striatal connections after D1 excitation. Our results show that targeted cellular-level manipulations affect local BOLD dynamics at the macroscale, such as by making BOLD dynamics more predictable over time by increasing its self-correlation structure. This contributes to ongoing attempts to understand the influence of structure-function relationships in shaping inter-regional communication at subcortical and cortical levels.

Imaging crossing fibers in mouse, pig, monkey, and human brain using small-angle X-ray scattering.

Myelinated axons (nerve fibers) efficiently transmit signals throughout the brain via action potentials. Multiple methods that are sensitive to axon orientations, from microscopy to magnetic resonance imaging, aim to reconstruct the brain's structural connectome. As billions of nerve fibers traverse the brain with various possible geometries at each point, resolving fiber crossings is necessary to generate accurate structural connectivity maps. However, doing so with specificity is a challenging task because signals originating from oriented fibers can be influenced by brain (micro)structures unrelated to myelinated axons. X-ray scattering can specifically probe myelinated axons due to the periodicity of the myelin sheath, which yields distinct peaks in the scattering pattern. Here, we show that small-angle X-ray scattering (SAXS) can be used to detect myelinated, axon-specific fiber crossings. We first demonstrate the capability using strips of human corpus callosum to create artificial double- and triple-crossing fiber geometries, and we then apply the method in mouse, pig, vervet monkey, and human brains. We compare results to polarized light imaging (3D-PLI), tracer experiments, and to outputs from diffusion MRI that sometimes fails to detect crossings. Given its specificity, capability of 3-dimensional sampling and high resolution, SAXS could serve as a ground truth for validating fiber orientations derived using diffusion MRI as well as microscopy-based methods. STATEMENT OF SIGNIFICANCE: To study how the nerve fibers in our brain are interconnected, scientists need to visualize their trajectories, which often cross one another. Here, we show the unique capacity of small-angle X-ray scattering (SAXS) to study these fiber crossings without use of labeling, taking advantage of SAXS's specificity to myelin - the insulating sheath that is wrapped around nerve fibers. We use SAXS to detect double and triple crossing fibers and unveil intricate crossings in mouse, pig, vervet monkey, and human brains. This non-destructive method can uncover complex fiber trajectories and validate other less specific imaging methods (e.g., MRI or microscopy), towards accurate mapping of neuronal connectivity in the animal and human brain.

Non-invasive visualization of amyloid-beta deposits in Alzheimer amyloidosis mice using magnetic resonance imaging and fluorescence molecular tomography.

Abnormal cerebral accumulation of amyloid-beta peptide (Aß) is a major hallmark of Alzheimer's disease. Non-invasive monitoring of Aß deposits enables assessing the disease burden in patients and animal models mimicking aspects of the human disease as well as evaluating the efficacy of Aß-modulating therapies. Previous in vivo assessments of plaque load have been predominantly based on macroscopic fluorescence reflectance imaging (FRI) and confocal or two-photon microscopy using Aß-specific imaging agents. However, the former method lacks depth resolution, whereas the latter is restricted by the limited field of view preventing a full coverage of the large brain region. Here, we utilized a fluorescence molecular tomography (FMT)-magnetic resonance imaging (MRI) pipeline with the curcumin derivative fluorescent probe CRANAD-2 to achieve full 3D brain coverage for detecting Aß accumulation in the arcAß mouse model of cerebral amyloidosis. A homebuilt FMT system was used for data acquisition, whereas a customized software platform enabled the integration of MRI-derived anatomical information as prior information for FMT image reconstruction. The results obtained from the FMT-MRI study were compared to those from conventional planar FRI recorded under similar physiological conditions, yielding comparable time courses of the fluorescence intensity following intravenous injection of CRANAD-2 in a region-of-interest comprising the brain. In conclusion, we have demonstrated the feasibility of visualizing Aß deposition in 3D using a multimodal FMT-MRI strategy. This hybrid imaging method provides complementary anatomical, physiological and molecular information, thereby enabling the detailed characterization of the disease status in arcAß mouse models, which can also facilitate monitoring the efficacy of putative treatments targeting Aß.

Hybrid fiber optic-fMRI for multimodal cell-specific recording and manipulation of neural activity in rodents.

Significance: Multiscale imaging holds particular relevance to neuroscience, where it helps integrate the cellular and molecular biological scale, which is most accessible to interventions, with holistic organ-level evaluations, most relevant with respect to function. Being inextricably interdisciplinary, multiscale imaging benefits substantially from incremental technology adoption, and a detailed overview of the state-of-the-art is vital to an informed application of imaging methods. Aim: In this article, we lay out the background and methodological aspects of multimodal approaches combining functional magnetic resonance imaging (fMRI) with simultaneous optical measurement or stimulation. Approach: We focus on optical techniques as these allow, in conjunction with genetically encoded proteins (e.g. calcium indicators or optical signal transducers), unprecedented read-out and control specificity for individual cell-types during fMRI experiments, while leveraging non-interfering modalities. Results: A variety of different solutions for optical/fMRI methods has been reported ranging from bulk fluorescence recordings via fiber photometry to high resolution microscopy. In particular, the plethora of optogenetic tools has enabled the transformation of stimulus-evoked fMRI into a cell biological interrogation method. We discuss the capabilities and limitations of these genetically encoded molecular tools in the study of brain phenomena of great methodological and neuropsychiatric interest-such as neurovascular coupling (NVC) and neuronal network mapping. We provide a methodological description of this interdisciplinary field of study, and focus in particular on the limitations of the widely used blood oxygen level dependent (BOLD) signal and how multimodal readouts can shed light on the contributions arising from neurons, astrocytes, or the vasculature. Conclusion: We conclude that information from multiple signaling pathways must be incorporated in future forward models of the BOLD response to prevent erroneous conclusions when using fMRI as a surrogate measure for neural activity. Further, we highlight the potential of direct neuronal stimulation via genetically defined brain networks towards advancing neurophysiological understanding and better estimating effective connectivity.

Whole-brain opto-fMRI map of mouse VTA dopaminergic activation reflects structural projections with small but significant deviations.

Ascending dopaminergic projections from neurons located in the Ventral Tegmental Area (VTA) are key to the etiology, dysfunction, and control of motivation, learning, and addiction. Due to the evolutionary conservation of this nucleus and the extensive use of mice as disease models, establishing an assay for VTA dopaminergic signaling in the mouse brain is crucial for the translational investigation of motivational control as well as of neuronal function phenotypes for diseases and interventions. In this article we use optogenetic stimulation directed at VTA dopaminergic neurons in combination with functional Magnetic Resonance Imaging (fMRI), a method widely used in human deep brain imaging. We present a comprehensive assay producing the first whole-brain opto-fMRI map of dopaminergic activation in the mouse, and show that VTA dopaminergic system function is consistent with its structural VTA projections, diverging only in a few key aspects. While the activation map predominantly highlights target areas according to their relative projection densities (e.g., strong activation of the nucleus accumbens and low activation of the hippocampus), it also includes areas for which a structural connection is not well established (such as the dorsomedial striatum). We further detail the variability of the assay with regard to multiple experimental parameters, including stimulation protocol and implant position, and provide evidence-based recommendations for assay reuse, publishing both reference results and a reference analysis workflow implementation.

An optimized registration workflow and standard geometric space for small animal brain imaging.

The reliability of scientific results critically depends on reproducible and transparent data processing. Cross-subject and cross-study comparability of imaging data in general, and magnetic resonance imaging (MRI) data in particular, is contingent on the quality of registration to a standard reference space. In small animal MRI this is not adequately provided by currently used processing workflows, which utilize high-level scripts optimized for human data, and adapt animal data to fit the scripts, rather than vice-versa. In this fully reproducible article we showcase a generic workflow optimized for the mouse brain, alongside a standard reference space suited to harmonize data between analysis and operation. We introduce four separate metrics for automated quality control (QC), and a visualization method to aid operator inspection. Benchmarking this workflow against common legacy practices reveals that it performs more consistently, better preserves variance across subjects while minimizing variance across sessions, and improves both volume and smoothness conservation RMSE approximately 2-fold. We propose this open source workflow and the QC metrics as a new standard for small animal MRI registration, ensuring workflow robustness, data comparability, and region assignment validity, all of which are indispensable prerequisites for the comparability of scientific results across experiments and centers.

Nanostructure-specific X-ray tomography reveals myelin levels, integrity and axon orientations in mouse and human nervous tissue.

Myelin insulates neuronal axons and enables fast signal transmission, constituting a key component of brain development, aging and disease. Yet, myelin-specific imaging of macroscopic samples remains a challenge. Here, we exploit myelin's nanostructural periodicity, and use small-angle X-ray scattering tensor tomography (SAXS-TT) to simultaneously quantify myelin levels, nanostructural integrity and axon orientations in nervous tissue. Proof-of-principle is demonstrated in whole mouse brain, mouse spinal cord and human white and gray matter samples. Outcomes are validated by 2D/3D histology and compared to MRI measurements sensitive to myelin and axon orientations. Specificity to nanostructure is exemplified by concomitantly imaging different myelin types with distinct periodicities. Finally, we illustrate the method's sensitivity towards myelin-related diseases by quantifying myelin alterations in dysmyelinated mouse brain. This non-destructive, stain-free molecular imaging approach enables quantitative studies of myelination within and across samples during development, aging, disease and treatment, and is applicable to other ordered biomolecules or nanostructures.

Pericyte hypoxia-inducible factor-1 (HIF-1) drives blood-brain barrier disruption and impacts acute ischemic stroke outcome.

Pericytes play essential roles in blood-brain barrier integrity and their dysfunction is implicated in neurological disorders such as stroke although the underlying mechanisms remain unknown. Hypoxia-inducible factor-1 (HIF-1), a master regulator of injury responses, has divergent roles in different cells especially during stress scenarios. On one hand HIF-1 is neuroprotective but on the other it induces vascular permeability. Since pericytes are critical for barrier stability, we asked if pericyte HIF-1 signaling impacts barrier integrity and injury severity in a mouse model of ischemic stroke. We show that pericyte HIF-1 loss of function (LoF) diminishes ischemic damage and barrier permeability at 3 days reperfusion. HIF-1 deficiency preserved barrier integrity by reducing pericyte death thereby maintaining vessel coverage and junctional protein organization, and suppressing vascular remodeling. Importantly, considerable improvements in sensorimotor function were observed in HIF-1 LoF mice indicating that better vascular functionality post stroke improves outcome. Thus, boosting vascular integrity by inhibiting pericytic HIF-1 activation and/or increasing pericyte survival may be a lucrative option to accelerate recovery after severe brain injury.

Psilocybin exerts distinct effects on resting state networks associated with serotonin and dopamine in mice.

Hallucinogenic agents have been proposed as potent antidepressants; this includes the serotonin (5-HT) receptor 2A agonist psilocybin. In human subjects, psilocybin alters functional connectivity (FC) within the default-mode network (DMN), a constellation of inter-connected regions that displays altered FC in depressive disorders. In this study, we investigated the effects of psilocybin on FC across the entire brain with a view to investigate underlying mechanisms. Psilocybin effects were investigated in lightly-anaesthetized mice using resting-state fMRI. Dual-regression analysis identified reduced FC within the ventral striatum in psilocybin- relative to vehicle-treated mice. Refinement of the analysis using spatial references derived from both gene expression maps and viral tracer projection fields revealed two distinct effects of psilocybin: it increased FC between 5-HT-associated networks and cortical areas, including elements of the murine DMN, thalamus, and midbrain; it decreased FC within dopamine (DA)-associated striatal networks. These results suggest that interactions between 5-HT- and DA-regulated neural networks contribute to the neural and therefore psychological effects of psilocybin. Furthermore, they highlight how information on molecular expression patterns and structural connectivity can assist in the interpretation of pharmaco-fMRI findings.

Cortical Excitation:Inhibition Imbalance Causes Abnormal Brain Network Dynamics as Observed in Neurodevelopmental Disorders.

Abnormal brain development manifests itself at different spatial scales. However, whether abnormalities at the cellular level can be diagnosed from network activity measured with functional magnetic resonance imaging (fMRI) is largely unknown, yet of high clinical relevance. Here a putative mechanism reported in neurodevelopmental disorders, that is, excitation-to-inhibition ratio (E:I), was chemogenetically increased within cortical microcircuits of the mouse brain and measured via fMRI. Increased E:I caused a significant "reduction" of long-range connectivity, irrespective of whether excitatory neurons were facilitated or inhibitory Parvalbumin (PV) interneurons were suppressed. Training a classifier on fMRI signals, we were able to accurately classify cortical areas exhibiting increased E:I. This classifier was validated in an independent cohort of Fmr1y/- knockout mice, a model for autism with well-documented loss of parvalbumin neurons and chronic alterations of E:I. Our findings demonstrate a promising novel approach towards inferring microcircuit abnormalities from macroscopic fMRI measurements.

Multimodal imaging combining time-domain near-infrared optical tomography and continuous-wave fluorescence molecular tomography.

Fluorescence molecular tomography (FMT) emerges as a powerful non-invasive imaging tool with the ability to resolve fluorescence signals from sources located deep in living tissues. Yet, the accuracy of FMT reconstruction depends on the deviation of the assumed optical properties from the actual values. In this work, we improved the accuracy of the initial optical properties required for FMT using a new-generation time-domain (TD) near-infrared optical tomography (NIROT) system, which effectively decouples scattering and absorption coefficients. We proposed a multimodal paradigm combining TD-NIROT and continuous-wave (CW) FMT. Both numerical simulation and experiments were performed on a heterogeneous phantom containing a fluorescent inclusion. The results demonstrate significant improvement in the FMT reconstruction by taking the NIROT-derived optical properties as prior information. The multimodal method is attractive for preclinical studies and tumor diagnostics since both functional and molecular information can be obtained.

An Automated Open-Source Workflow for Standards-Compliant Integration of Small Animal Magnetic Resonance Imaging Data.

Large-scale research integration is contingent on seamless access to data in standardized formats. Standards enable researchers to understand external experiment structures, pool results, and apply homogeneous preprocessing and analysis workflows. Particularly, they facilitate these features without the need for numerous potentially confounding compatibility add-ons. In small animal magnetic resonance imaging, an overwhelming proportion of data is acquired via the ParaVision software of the Bruker Corporation. The original data structure is predominantly transparent, but fundamentally incompatible with modern pipelines. Additionally, it sources metadata from free-field operator input, which diverges strongly between laboratories and researchers. In this article we present an open-source workflow which automatically converts and reposits data from the ParaVision structure into the widely supported and openly documented Brain Imaging Data Structure (BIDS). Complementing this workflow we also present operator guidelines for appropriate ParaVision data input, and a programmatic walk-through detailing how preexisting scans with uninterpretable metadata records can easily be made compliant after the acquisition.

Validation and Comparison of Monte Carlo and Finite Element Method in Forward Modeling for Near Infrared Optical Tomography.

Near infrared optical tomography (NIROT) is a non-invasive imaging technique to provide physiological information e.g. the oxygenation of tissue. For image reconstruction in clinical and preclinical scenarios, models to accurately describe light propagation are needed. This work aims to assess the accuracy and efficiency of different models, which paves the way for an optimal design of model-based image reconstruction algorithms in NIROT for realistic tissue geometries and heterogeneities. Two popular simulators were evaluated: the Monte Carlo (MC) method based MCX and the finite element method (FEM) based Toast++. We compared simulated results with experimental data measured on a homogeneous silicone phantom with well-calibrated parameters. The laser light was focused on the center of the phantom surface and images were captured by a CCD camera in both reflection and transmission modes. For transmittance measurements, the two models showed good agreement. Both achieve a cosine similarity of ~99%. In contrast, for reflectance measurements, FEM results deviated more from the measured values than MC, yielding similarity values of 86% and 94%, respectively. This study recommends the use of MC for NIROT in reflection mode and both MC and FEM yield excellent results for transmission mode.

Retrieving neuronal orientations using 3D scanning SAXS and comparison with diffusion MRI.

While diffusion MRI (dMRI) is currently the method of choice to non-invasively probe tissue microstructure and study structural connectivity in the brain, its spatial resolution is limited and its results need structural validation. Current ex vivo methods employed to provide 3D fiber orientations have limitations, including tissue-distorting sample preparation, small field of view or inability to quantify 3D fiber orientation distributions. 3D fiber orientation in tissue sections can be obtained from 3D scanning small-angle X-ray scattering (3D sSAXS) by analyzing the anisotropy of scattering signals. Here we adapt the 3D sSAXS method for use in brain tissue, exploiting the high sensitivity of the SAXS signal to the ordered molecular structure of myelin. We extend the characterization of anisotropy from vectors to tensors, employ the Funk-Radon-Transform for converting scattering information to real space fiber orientations, and demonstrate the feasibility of the method in thin sections of mouse brain with minimal sample preparation. We obtain a second rank tensor representing the fiber orientation distribution function (fODF) for every voxel, thereby generating fODF maps. Finally, we illustrate the potential of 3D sSAXS by comparing the result with diffusion MRI fiber orientations in the same mouse brain. We show a remarkably good correspondence, considering the orthogonality of the two methods, i.e. the different physical processes underlying the two signals. 3D sSAXS can serve as validation method for microstructural MRI, and can provide novel microstructural insights for the nervous system, given the method's orthogonality to dMRI, high sensitivity to myelin sheath's orientation and abundance, and the possibility to extract myelin-specific signal and to perform micrometer-resolution scanning.

Smart Toolkit for Fluorescence Tomography: Simulation, Reconstruction, and Validation.

OBJECTIVE: Fluorescence molecular tomography (FMT) can provide valuable molecular information by mapping the bio-distribution of fluorescent reporter molecules in the intact organism. Various prototype FMT systems have been introduced during the past decade. However, none of them has evolved as a standard tool for routine biomedical research. The goal of this paper is to develop a software package that can automate the complete FMT reconstruction procedure. METHODS: We present smart toolkit for fluorescence tomography (STIFT), a comprehensive platform comprising three major protocols: 1) virtual FMT, i.e., forward modeling and reconstruction of simulated data; 2) control of actual FMT data acquisition; and 3) reconstruction of experimental FMT data. RESULTS: Both simulation and phantom experiments have shown robust reconstruction results for homogeneous and heterogeneous tissue-mimicking phantoms containing fluorescent inclusions. CONCLUSION: STIFT can be used for optimization of FMT experiments, in particular for optimizing illumination patterns. SIGNIFICANCE: This paper facilitates FMT experiments by bridging the gaps between simulation, actual experiments, and data reconstruction.

Common functional networks in the mouse brain revealed by multi-centre resting-state fMRI analysis.

Preclinical applications of resting-state functional magnetic resonance imaging (rsfMRI) offer the possibility to non-invasively probe whole-brain network dynamics and to investigate the determinants of altered network signatures observed in human studies. Mouse rsfMRI has been increasingly adopted by numerous laboratories worldwide. Here we describe a multi-centre comparison of 17 mouse rsfMRI datasets via a common image processing and analysis pipeline. Despite prominent cross-laboratory differences in equipment and imaging procedures, we report the reproducible identification of several large-scale resting-state networks (RSN), including a mouse default-mode network, in the majority of datasets. A combination of factors was associated with enhanced reproducibility in functional connectivity parameter estimation, including animal handling procedures and equipment performance. RSN spatial specificity was enhanced in datasets acquired at higher field strength, with cryoprobes, in ventilated animals, and under medetomidine-isoflurane combination sedation. Our work describes a set of representative RSNs in the mouse brain and highlights key experimental parameters that can critically guide the design and analysis of future rodent rsfMRI investigations.

Lymphatic outflow of cerebrospinal fluid is reduced in glioma.

Glioblastoma is a malignant brain tumor with mean overall survival of less than 15 months. Blood vessel leakage and peritumoral edema lead to increased intracranial pressure and augment neurological deficits which profoundly decrease the quality of life of glioblastoma patients. It is unknown how the dynamics of cerebrospinal fluid (CSF) turnover are affected during this process. By monitoring the transport of CSF tracers to the systemic blood circulation after infusion into the cisterna magna, we demonstrate that the outflow of CSF is dramatically reduced in glioma-bearing mice. Using a combination of magnetic resonance imaging (MRI) and near-infrared (NIR) imaging, we found that the circulation of CSF tracers was hindered after cisterna magna injection with reduced signals along the exiting cranial nerves and downstream lymph nodes, which represent the major CSF outflow route in mice. Due to blockage of the normal routes of CSF bulk flow within and from the cranial cavity, CSF tracers were redirected into the spinal space. In some mice, impaired CSF clearance from the cranium was compensated by a lymphatic outflow from the sacral spine.

Correction to: Retinal pathology in experimental optic neuritis is characterized by retrograde degeneration and gliosis.

In the original publication of this article [1], Fig. 10 contained two panels "C" as panel "F" was accidentally omitted. The incorrect (Fig. 1) and correct (Fig. 2) versions are published in this correction article.

Retinal pathology in experimental optic neuritis is characterized by retrograde degeneration and gliosis.

The exact mechanisms and temporal sequence of neurodegeneration in multiple sclerosis are still unresolved. The visual pathway including its unmyelinated retinal axons, can serve as a prototypic model of neurodegeneration in experimental optic neuritis. We conducted a longitudinal study combining retinal imaging through optical coherence tomography (OCT) with immunohistochemical analyses of retinal and optic nerve tissue at various time points in experimental autoimmune encephalomyelitis (EAE).Inner retinal layer (IRL) thickness was measured in 30 EAE and 14 healthy control C57BL/6 J mice using OCT. Distribution of marker proteins was assessed by immunofluorescence staining and retinal mRNA levels were assayed using real-time PCR. Histological morphology was evaluated on light and electron microscopy images.Signs of inflammatory edema 11 days post immunisation coincided with IRL thickening, while neuro-axonal degeneration throughout the disease course contributed to IRL thinning observed after 20 days post immunisation. Retinal pathology, including axonal transport impairment, was observed early, prior to cellular infiltration (i.e. T-cells) in the optic nerve 11 days post immunisation. Yet, the effects of early retinal damage on OCT-derived readouts were outweighed by the initial inflammatory edema. Early microglial activation and astrocytosis was detected in the retina prior to retinal ganglion cell loss and persisted until 33 days post immunisation. Müller cell reactivity (i.e. aquaporin-4 and glutamine synthetase decrease) presented after 11 days post immunisation in the IRL. Severe neuro-axonal degeneration was observed in the optic nerve and retina until 33 days post immunisation.Initial signs of retinal pathology subsequent to early glial activity, suggests a need for prophylactic treatment of optic neuritis. Following early inflammation, Müller cells possibly respond to retinal pathology with compensatory mechanisms. Although the majority of the IRL damage observed is likely due to retrograde degeneration following optic neuritis, initial pathology, possibly due to gliosis, may contribute further to IRL thinning. These results add morphological substrate to our OCT findings. The extent and rapid onset of axonal and neuronal damage in this model appears relevant for testing interventions scaled to human optic neuritis.