A Binary Cre Transgenic Approach Dissects Microglia and CNS Border-Associated Macrophages.

The developmental and molecular heterogeneity of tissue macrophages is unravelling, as are their diverse contributions to physiology and pathophysiology. Moreover, also given tissues harbor macrophages in discrete anatomic locations. Functional contributions of specific cell populations can in mice be dissected using Cre recombinase-mediated mutagenesis. However, single promoter-based Cre models show limited specificity for cell types. Focusing on macrophages in the brain, we establish here a binary transgenic system involving complementation-competent NCre and CCre fragments whose expression is driven by distinct promoters: Sall1ncre: Cx3cr1ccre mice specifically target parenchymal microglia and compound transgenic Lyve1ncre: Cx3cr1ccre animals target vasculature-associated macrophages, in the brain, as well as other tissues. We imaged the respective cell populations and retrieved their specific translatomes using the RiboTag in order to define them and analyze their differential responses to a challenge. Collectively, we establish the value of binary transgenesis to dissect tissue macrophage compartments and their functions.

In-Brain Multiphoton Imaging of Vaterite Cargoes Loaded with Carbon Dots.

Biocompatible fluorescent agents are key contributors to the theranostic paradigm by enabling real-time in vivo imaging. This study explores the optical properties of phenylenediamine carbon dots (CDs) and demonstrates their potential for fluorescence imaging in cells and brain blood vessels. The nonlinear absorption cross-section of the CDs was measured and achieved values near 50 Goeppert-Mayer (GM) units with efficient excitation in the 775-895 nm spectral range. Mesoporous vaterite nanoparticles were loaded with CDs to examine the possibility of a biocompatible imaging platform. Efficient one- and two-photon imaging of the CD-vaterite composites uptaken by diverse cells was demonstrated. For an in vivo scenario, CD-vaterite composites were injected into the bloodstream of a mouse, and their flow was monitored within the blood vessels of the brain through a cranial window. These results show the potential of the platform for high-brightness biocompatible imaging with the potential for both sensing and simultaneous drug delivery.

Distinct spatiotemporal features of microglia and monocyte-derived macrophages in glioma.

Gliomas are the most frequent primary tumors of the brain. Glioma progression is regulated by the tumor microenvironment, which is mainly composed of tumor-associated microglia (TA-MG) and monocyte-derived macrophages (MDM). Recent studies have highlighted the distinct properties of these cells in glioma progression. However, their spatiotemporal alteration during tumor progression has not been fully explored. Using a genetic lineage tracing approach, we show that TA-MG and MDMs differ in their spatiotemporal distribution and interaction with other components of the glioma microenvironment. MDM were present only inside the tumor, whereas TA-MG accumulated both outside and inside the tumor. However, TA-MG was eliminated from the tumor mass as the tumor progressed. Depletion of MDM led to enhanced occupancy of TA-MG in the tumor core, indicating that TA-MG elimination was regulated by MDM. TA-MG and MDM are heterogeneous cell populations whose compositions and properties can change during tumor progression. Finally, MG, TA-MG and MDM were enriched in the perivascular area (PVA) compared to more distal blood vessel-associated areas. However, inside the tumor, the MDM enrichment in PVA was higher than that in TA-MG. Collectively, we established that TA-MG and MDM exhibit different spatiotemporal features in glioma, suggesting distinctive roles during tumor progression.

Reduced IGF-1 signaling delays age-associated proteotoxicity in mice.

The insulin/insulin growth factor (IGF) signaling (IIS) pathway is a key regulator of aging of worms, flies, mice, and likely humans. Delayed aging by IIS reduction protects the nematode C. elegans from toxicity associated with the aggregation of the Alzheimer's disease-linked human peptide, Abeta. We reduced IGF signaling in Alzheimer's model mice and discovered that these animals are protected from Alzheimer's-like disease symptoms, including reduced behavioral impairment, neuroinflammation, and neuronal loss. This protection is correlated with the hyperaggregation of Abeta leading to tightly packed, ordered plaques, suggesting that one aspect of the protection conferred by reduced IGF signaling is the sequestration of soluble Abeta oligomers into dense aggregates of lower toxicity. These findings indicate that the IGF signaling-regulated mechanism that protects from Abeta toxicity is conserved from worms to mammals and point to the modulation of this signaling pathway as a promising strategy for the development of Alzheimer's disease therapy.

Patterns of fatty acid usage in two nocturnal insectivores: the Mediterranean house gecko (Hemidactylus turcicus) and the Etruscan pygmy shrew (Suncus etruscus).

Dietary fatty acids (FAs) have been demonstrated to be differentially stored or used as a metabolic fuel, depending on carbon chain length or saturation level. However, intestinal absorption also differs among FAs, potentially biasing conclusions on functional differences and their subsequent implications. We tested dietary FA usage in a nocturnal insectivorous reptile and a nocturnal insectivorous mammal of similar size: the gecko Hemidactylus turcicus and the shrew Suncus etruscus. We compared the relative presence of 13C isotopes in breath and feces following ingestion of three isotopically enriched fatty acids: linoleic acid (a polyunsaturated FA), oleic acid (monounsaturated) and palmitic acid (saturated). Both species oxidized linoleic and oleic acids at much higher levels than palmitic acid. Egestion of palmitic acid in feces was much higher than that of linoleic and oleic acids. The major difference between geckos and shrews was that the latter digested fatty acids much faster, which was best explained by the difference in the metabolic rates of the species. Circadian differences were evident for gecko metabolic and FA oxidation rates, peaking at night; for shrews, peak oxidation was achieved faster at night but rates did not differ. Our study is among the first to integrate oxidation and absorption patterns, as well as metabolic rates and their rhythms, providing important insights into the utilization of different dietary FAs in different species.

Prophylactic TLR9 stimulation reduces brain metastasis through microglia activation.

Brain metastases are prevalent in various types of cancer and are often terminal, given the low efficacy of available therapies. Therefore, preventing them is of utmost clinical relevance, and prophylactic treatments are perhaps the most efficient strategy. Here, we show that systemic prophylactic administration of a toll-like receptor (TLR) 9 agonist, CpG-C, is effective against brain metastases. Acute and chronic systemic administration of CpG-C reduced tumor cell seeding and growth in the brain in three tumor models in mice, including metastasis of human and mouse lung cancer, and spontaneous melanoma-derived brain metastasis. Studying mechanisms underlying the therapeutic effects of CpG-C, we found that in the brain, unlike in the periphery, natural killer (NK) cells and monocytes are not involved in controlling metastasis. Next, we demonstrated that the systemically administered CpG-C is taken up by endothelial cells, astrocytes, and microglia, without affecting blood-brain barrier (BBB) integrity and tumor brain extravasation. In vitro assays pointed to microglia, but not astrocytes, as mediators of CpG- C effects through increased tumor killing and phagocytosis, mediated by direct microglia-tumor contact. In vivo, CpG-C-activated microglia displayed elevated mRNA expression levels of apoptosis-inducing and phagocytosis-related genes. Intravital imaging showed that CpG-C-activated microglia cells contact, kill, and phagocytize tumor cells in the early stages of tumor brain invasion more than nonactivated microglia. Blocking in vivo activation of microglia with minocycline, and depletion of microglia with a colony-stimulating factor 1 inhibitor, indicated that microglia mediate the antitumor effects of CpG-C. Overall, the results suggest prophylactic CpG-C treatment as a new intervention against brain metastasis, through an essential activation of microglia.

Single Cortical Microinfarcts Lead to Widespread Microglia/Macrophage Migration Along the White Matter.

Loss of cognitive function with aging is a complex and poorly understood process. Recently, clinical research has linked the occurrence of cortical microinfarcts to cognitive decline. Cortical microinfarcts form following the occlusion of penetrating vessels and are considered to be restricted to the proximity of the occluded vessel. Whether and how such local events propagate and affect remote brain regions remain unknown. To this end, we combined histological analysis and longitudinal diffusion tensor imaging (DTI), following the targeted-photothrombotic occlusion of single cortical penetrating vessels. Occlusions resulted in distant tissue reorganization across the mouse brain. This remodeling co-occurred with the formation of a microglia/macrophage migratory path along subcortical white matter tracts, reaching the contralateral hemisphere through the corpus callosum and leaving a microstructural signature detected by DTI-tractography. CX3CR1-deficient mice exhibited shorter trail lengths, differential remodeling, and only ipsilateral white matter tract changes. We concluded that microinfarcts lead to brain-wide remodeling in a microglial CX3CR1-dependent manner.

Pax6 regulation of Sox9 in the mouse retinal pigmented epithelium controls its timely differentiation and choroid vasculature development.

The synchronized differentiation of neuronal and vascular tissues is crucial for normal organ development and function, although there is limited information about the mechanisms regulating the coordinated development of these tissues. The choroid vasculature of the eye serves as the main blood supply to the metabolically active photoreceptors, and develops together with the retinal pigmented epithelium (RPE). Here, we describe a novel regulatory relationship between the RPE transcription factors Pax6 and Sox9 that controls the timing of RPE differentiation and the adjacent choroid maturation. We used a novel machine learning algorithm tool to analyze high resolution imaging of the choroid in Pax6 and Sox9 conditional mutant mice. Additional unbiased transcriptomic analyses in mutant mice and RPE cells generated from human embryonic stem cells, as well as chromatin immunoprecipitation and high-throughput analyses, revealed secreted factors that are regulated by Pax6 and Sox9. These factors might be involved in choroid development and in the pathogenesis of the common blinding disease: age-related macular degeneration (AMD).

Demystifying the extracellular matrix and its proteolytic remodeling in the brain: structural and functional insights.

The extracellular matrix (ECM) plays diverse roles in several physiological and pathological conditions. In the brain, the ECM is unique both in its composition and in functions. Furthermore, almost all the cells in the central nervous system contribute to different aspects of this intricate structure. Brain ECM, enriched with proteoglycans and other small proteins, aggregate into distinct structures around neurons and oligodendrocytes. These special structures have cardinal functions in the normal functioning of the brain, such as learning, memory, and synapse regulation. In this review, we have compiled the current knowledge about the structure and function of important ECM molecules in the brain and their proteolytic remodeling by matrix metalloproteinases and other enzymes, highlighting the special structures they form. In particular, the proteoglycans in brain ECM, which are essential for several vital functions, are emphasized in detail.

Comparing two classes of biological distribution systems using network analysis.

Distribution networks-from vasculature to urban transportation pathways-are spatially embedded networks that must route resources efficiently in the face of pressures induced by the costs of building and maintaining network infrastructure. Such requirements are thought to constrain the topological and spatial organization of these systems, but at the same time, different kinds of distribution networks may exhibit variable architectural features within those general constraints. In this study, we use methods from network science to compare and contrast two classes of biological transport networks: mycelial fungi and vasculature from the surface of rodent brains. These systems differ in terms of their growth and transport mechanisms, as well as the environments in which they typically exist. Though both types of networks have been studied independently, the goal of this study is to quantify similarities and differences in their network designs. We begin by characterizing the structural backbone of these systems with a collection of measures that assess various kinds of network organization across topological and spatial scales, ranging from measures of loop density, to those that quantify connected pathways between different network regions, and hierarchical organization. Most importantly, we next carry out a network analysis that directly considers the spatial embedding and properties especially relevant to the function of distribution systems. We find that although both the vasculature and mycelia are highly constrained planar networks, there are clear distinctions in how they balance tradeoffs in network measures of wiring length, efficiency, and robustness. While the vasculature appears well organized for low cost, but relatively high efficiency, the mycelia tend to form more expensive but in turn more robust networks. As a whole, this work demonstrates the utility of network-based methods to identify both common features and variations in the network structure of different classes of biological transport systems.

Linking brain vascular physiology to hemodynamic response in ultra-high field MRI.

Functional MRI using blood oxygenation level-dependent (BOLD) contrast indirectly probes neuronal activity via evoked cerebral blood volume (CBV) and oxygenation changes. Thus, its spatio-temporal characteristics are determined by vascular physiology and MRI parameters. In this paper, we focus on the spatial distribution and time course of the fMRI signal and their magnetic field strength dependence. Even though much is still unknown, the following consistent picture is emerging: a) For high spatial resolution imaging, fMRI contrast-to-noise increases supra-linearly with field strength. b) The location and spacing of penetrating arteries and ascending veins in the cortical tissue are not correlated to cortical columns, imposing limitations on achievable point-spread function (PSF) in fMRI. c) Baseline CBV distribution may vary over cortical layers biasing fMRI signal to layers with high CBV values. d) The largest CBV change is in the tissue microvasculature, less in surface arteries and even less in pial veins. e) Venous CBV changes are only relevant for longer stimuli, and oxygenation changes are largest in post-capillary blood vessels. f) The balloon effect (i.e. slow recovery of CBV to baseline) is located in the tissue, consistent with the fact that the post-stimulus undershoot has narrower spatial PSF than the positive BOLD response. g) The onset time following stimulation has been found to be shortest in middle/lower layers, both in optical imaging and high-resolution fMRI, but we argue and demonstrate with simulations that varying signal latencies can also be caused by vascular properties and, therefore, may potentially not be interpreted as neural latencies. With simulations, we illustrate the field strength dependency of fMRI signal transients, such as the adaptation during stimulation, initial dip and the post-stimulus undershoot. In sum, vascular structure and function impose limitations on the achievable PSF of fMRI and give rise to complex fMRI transients, which contain time-varying amount of excitatory and inhibitory neuronal information. Nevertheless, non-invasive fMRI at ultra-high magnetic fields not only provides high contrast-to-noise but also an unprecedented detailed view on cognitive processes in the human brain.

Maintaining unperturbed cerebral blood flow is key in the study of brain metastasis and its interactions with stress and inflammatory responses.

Blood-borne brain metastases are associated with poor prognosis, but little is known about the interplay between cerebral blood flow, surgical stress responses, and the metastatic process. The intra-carotid inoculation approach, traditionally used in animal studies, involves permanent occlusion of the common carotid artery (CCA). Herein we introduced a novel intra-carotid inoculation approach that avoids CCA ligation, namely - assisted external carotid artery inoculation (aECAi) - and compared it to the traditional approach in C57/BL6 mice, assessing cerebral blood flow; particle distribution; blood-brain barrier (BBB) integrity; stress, inflammatory and immune responses; and brain tumor retention and growth. Doppler flowmetry and two-photon imaging confirmed that only in the traditional approach regional and capillary cerebral blood flux were significantly reduced. Corticosterone and plasma IL-6 levels were higher in the traditional approach, splenic numbers of NK, CD3+, granulocytes, and dendritic cells were lower, and many of these indices were more profoundly affected by surgical stress in the traditional approach. BBB integrity was unaffected. Administration of spherical beads indicated that CCA ligation significantly limited brain distribution of injected particles, and inoculation of D122-LLC syngeneic tumor cells resulted in 10-fold lower brain tumor-cell retention in the traditional approach. Last, while most of the injected tumor cells were arrested in extra-cranial head areas, our method improved targeting of brain-tissue by 7-fold. This head versus brain distribution difference, commonly overlooked, cannot be detected using in vivo bioluminescent imaging. Overall, it is crucial to maintain unperturbed cerebral blood flow while studying brain metastasis and interactions with stress and inflammatory responses.

Astrocytes from old Alzheimer's disease mice are impaired in Aß uptake and in neuroprotection.

In Alzheimer's disease (AD), astrocytes undergo morphological changes ranging from atrophy to hypertrophy, but the effect of such changes at the functional level is still largely unknown. Here, we aimed to investigate whether alterations in astrocyte activity in AD are transient and depend on their microenvironment, or whether they are irreversible. We established and characterized a new protocol for the isolation of adult astrocytes and discovered that astrocytes isolated from old 5xFAD mice have higher GFAP expression than astrocytes derived from WT mice, as observed in vivo. We found high C1q levels in brain sections from old 5xFAD mice in close vicinity to amyloid plaques and astrocyte processes. Interestingly, while old 5xFAD astrocytes are impaired in uptake of soluble Aß42, this effect was reversed upon an addition of exogenous C1q, suggesting a potential role for C1q in astrocyte-mediated Aß clearance. Our results suggest that scavenger receptor B1 plays a role in C1q-facilitated Aß uptake by astrocytes and that expression of scavenger receptor B1 is reduced in adult old 5xFAD astrocytes. Furthermore, old 5xFAD astrocytes show impairment in support of neuronal growth in co-culture and neurotoxicity concomitant with an elevation in IL-6 expression. Further understanding of the impact of astrocyte impairment on AD pathology may provide insights into the etiology of AD.

Robust and fragile aspects of cortical blood flow in relation to the underlying angioarchitecture.

We review the organizational principles of the cortical vasculature and the underlying patterns of blood flow under normal conditions and in response to occlusion of single vessels. The cortex is sourced by a two-dimensional network of pial arterioles that feeds a three-dimensional network of subsurface microvessels in close proximity to neurons and glia. Blood flow within the surface and subsurface networks is largely insensitive to occlusion of a single vessel within either network. However, the penetrating arterioles that connect the pial network to the subsurface network are bottlenecks to flow; occlusion of even a single penetrating arteriole results in the death of a 500 µm diameter cylinder of cortical tissue despite the potential for collateral flow through microvessels. This pattern of flow is consistent with that calculated from a full reconstruction of the angioarchitecture. Conceptually, collateral flow is insufficient to compensate for the occlusion of a penetrating arteriole because penetrating venules act as shunts of blood that flows through collaterals. Future directions that stem from the analysis of the angioarchitecture concern cellular-level issues, in particular the regulation of blood flow within the subsurface microvascular network, and system-level issues, in particular the role of penetrating arteriole occlusions in human cognitive impairment.

Sequentially activated discrete modules appear as traveling waves in neuronal measurements with limited spatiotemporal sampling.

Numerous studies have identified traveling waves in the cortex and suggested they play important roles in brain processing. These waves are most often measured using macroscopic methods that are unable to assess the local spiking activity underlying wave dynamics. Here, we investigated the possibility that waves may not be traveling at the single neuron scale. We first show that sequentially activating two discrete brain areas can appear as traveling waves in EEG simulations. We next reproduce these results using an analytical model of two sequentially activated regions. Using this model, we were able to generate wave-like activity with variable directions, velocities, and spatial patterns, and to map the discriminability limits between traveling waves and modular sequential activations. Finally, we investigated the link between field potentials and single neuron excitability using large-scale measurements from turtle cortex ex vivo. We found that while field potentials exhibit wave-like dynamics, the underlying spiking activity was better described by consecutively activated spatially adjacent groups of neurons. Taken together, this study suggests caution when interpreting phase delay measurements as continuously propagating wavefronts in two different spatial scales. A careful distinction between modular and wave excitability profiles across scales will be critical for understanding the nature of cortical computations.

Chronic optical access through a polished and reinforced thinned skull.

We present a method to form an optical window in the mouse skull that spans millimeters and is stable for months without causing brain inflammation. This enabled us to repeatedly image blood flow in cortical capillaries of awake mice and determine long-range correlations in speed. We also repeatedly imaged dendritic spines, microglia and angioarchitecture, as well as used illumination to drive motor output via optogenetics and induce microstrokes via photosensitizers.

Topological basis for the robust distribution of blood to rodent neocortex.

The maintenance of robust blood flow to the brain is crucial to the health of brain tissue. We examined the pial network of the middle cerebral artery, which distributes blood from the cerebral arteries to the penetrating arterioles that source neocortical microvasculature, to characterize how vascular topology may support such robustness. For both mice and rats, two features dominate the topology. First, interconnected loops span the entire territory sourced by the middle cerebral artery. Although the loops comprise <10% of all branches, they maintain the overall connectivity of the network after multiple breaks. Second, >80% of offshoots from the loops are stubs that end in a single penetrating arteriole, as opposed to trees with multiple penetrating arterioles. We hypothesize that the loops and stubs protect blood flow to the parenchyma from an occlusion in a surface vessel. To test this, we assayed the viability of tissue that was sourced by an individual penetrating arteriole following occlusion of a proximal branch in the surface loop. We observed that neurons remained healthy, even when occlusion led to a reduction in the local blood flow. In contrast, direct blockage of a single penetrating arteriole invariably led to neuronal death and formation of a cyst. Our results show that the surface vasculature functions as a grid for the robust allocation of blood in the event of vascular dysfunction. The combined results of the present and prior studies imply that the pial network reallocates blood in response to changing metabolic needs.

Longitudinal in vivo imaging of perineuronal nets.

Significance: Perineuronal nets (PNNs) are extracellular matrix structures implicated in learning, memory, information processing, synaptic plasticity, and neuroprotection. However, our understanding of mechanisms governing the evidently important contribution of PNNs to central nervous system function is lacking. A primary cause for this gap of knowledge is the absence of direct experimental tools to study their role in vivo. Aim: We introduce a robust approach for quantitative longitudinal imaging of PNNs in brains of awake mice at subcellular resolution. Approach: We label PNNs in vivo with commercially available compounds and monitor their dynamics with two-photon imaging. Results: Using our approach, we show that it is possible to longitudinally follow the same PNNs in vivo while monitoring degradation and reconstitution of PNNs. We demonstrate the compatibility of our method to simultaneously monitor neuronal calcium dynamics in vivo and compare the activity of neurons with and without PNNs. Conclusion: Our approach is tailored for studying the intricate role of PNNs in vivo, while paving the road for elucidating their role in different neuropathological conditions.

Large-scale automated histology in the pursuit of connectomes.

How does the brain compute? Answering this question necessitates neuronal connectomes, annotated graphs of all synaptic connections within defined brain areas. Further, understanding the energetics of the brain's computations requires vascular graphs. The assembly of a connectome requires sensitive hardware tools to measure neuronal and neurovascular features in all three dimensions, as well as software and machine learning for data analysis and visualization. We present the state of the art on the reconstruction of circuits and vasculature that link brain anatomy and function. Analysis at the scale of tens of nanometers yields connections between identified neurons, while analysis at the micrometer scale yields probabilistic rules of connection between neurons and exact vascular connectivity.

Versatile software and hardware combo enabling photon counting acquisition and real-time display for multiplexing, 2D and continuous 3D two-photon imaging applications.

Significance: rPySight brings a flexible and highly customizable open-software platform built around a powerful multichannel digitizer; combined, it enables performing complex photon counting-based experiments. We exploited advanced programming technology to share the photon counting stream with the graphical processing unit (GPU), making possible real-time display of two-dimensional (2D) and three-dimensional (3D) experiments and paving the road for other real-time applications. Aim: Photon counting improves multiphoton imaging by providing better signal-to-noise ratio in photon-deprived applications and is becoming more widely implemented, as indicated by its increasing presence in many microscopy vendor portfolios. Despite the relatively easy access to this technology offered in commercial systems, these remain limited to one or two channels of data and might not enable highly tailored experiments, forcing most researchers to develop their own electronics and code. We set to develop a flexible and open-source interface to a cutting-edge multichannel fast digitizer that can be easily integrated into existing imaging systems. Approach: We selected an advanced multichannel digitizer capable of generating 70M tags/s and wrote an open software application, based on Rust and Python languages, to share the stream of detected events with the GPU, enabling real-time data processing. Results: rPySight functionality was showcased in real-time monitoring of 2D imaging, improved calcium imaging, multiplexing, and 3D imaging through a varifocal lens. We provide a detailed protocol for implementing out-of-the-box rPySight and its related hardware. Conclusions: Applying photon-counting approaches is becoming a fundamental component in recent technical developments that push well beyond existing acquisition speed limitations of classical multiphoton approaches. Given the performance of rPySight, we foresee its use to capture, among others, the joint dynamics of hundreds (if not thousands) of neuronal and vascular elements across volumes, as is likely required to uncover in a much broader sense the hemodynamic transform function.