Entrainment of Arteriole Vasomotor Fluctuations by Neural Activity Is a Basis of Blood-Oxygenation-Level-Dependent "Resting-State" Connectivity.

Resting-state signals in blood-oxygenation-level-dependent (BOLD) imaging are used to parcellate brain regions and define "functional connections" between regions. Yet a physiological link between fluctuations in blood oxygenation with those in neuronal signaling pathways is missing. We present evidence from studies on mouse cortex that modulation of vasomotion, i.e., intrinsic ultra-slow (0.1 Hz) fluctuations in arteriole diameter, provides this link. First, ultra-slow fluctuations in neuronal signaling, which occur as an envelope over γ-band activity, entrains vasomotion. Second, optogenetic manipulations confirm that entrainment is unidirectional. Third, co-fluctuations in the diameter of pairs of arterioles within the same hemisphere diminish to chance for separations >1.4 mm. Yet the diameters of arterioles in distant (>5 mm), mirrored transhemispheric sites strongly co-fluctuate; these correlations are diminished in acallosal mice. Fourth, fluctuations in arteriole diameter coherently drive fluctuations in blood oxygenation. Thus, entrainment of vasomotion links neuronal pathways to functional connections.

The impact of vessel size, orientation and intravascular contribution on the neurovascular fingerprint of BOLD bSSFP fMRI.

Monte Carlo simulations have been used to analyze oxygenation-related signal changes in pass-band balanced steady state free precession (bSSFP) as well as in gradient echo (GE) and spin echo (SE) sequences. Signal changes were calculated for artificial cylinders and neurovascular networks acquired from the mouse parietal cortex by two-photon laser scanning microscopy at 1 µm isotropic resolution. Signal changes as a function of vessel size, blood volume, vessel orientation to the main magnetic field B0 as well as relations of intra- and extravascular and of micro- and macrovascular contributions have been analyzed. The results show that bSSFP is highly sensitive to extravascular and microvascular components. Furthermore, GE and bSSFP, and to a lesser extent SE, exhibit a strong dependence of their signal change on the orientation of the vessel network to B0.

Depth-dependent flow and pressure characteristics in cortical microvascular networks.

A better knowledge of the flow and pressure distribution in realistic microvascular networks is needed for improving our understanding of neurovascular coupling mechanisms and the related measurement techniques. Here, numerical simulations with discrete tracking of red blood cells (RBCs) are performed in three realistic microvascular networks from the mouse cerebral cortex. Our analysis is based on trajectories of individual RBCs and focuses on layer-specific flow phenomena until a cortical depth of 1 mm. The individual RBC trajectories reveal that in the capillary bed RBCs preferentially move in plane. Hence, the capillary flow field shows laminar patterns and a layer-specific analysis is valid. We demonstrate that for RBCs entering the capillary bed close to the cortical surface (< 400 µm) the largest pressure drop takes place in the capillaries (37%), while for deeper regions arterioles are responsible for 61% of the total pressure drop. Further flow characteristics, such as capillary transit time or RBC velocity, also vary significantly over cortical depth. Comparison of purely topological characteristics with flow-based ones shows that a combined interpretation of topology and flow is indispensable. Our results provide evidence that it is crucial to consider layer-specific differences for all investigations related to the flow and pressure distribution in the cortical vasculature. These findings support the hypothesis that for an efficient oxygen up-regulation at least two regulation mechanisms must be playing hand in hand, namely cerebral blood flow increase and microvascular flow homogenization. However, the contribution of both regulation mechanisms to oxygen up-regulation likely varies over depth.

Pericyte degeneration leads to neurovascular uncoupling and limits oxygen supply to brain.

Pericytes are perivascular mural cells of brain capillaries. They are positioned centrally in the neurovascular unit between endothelial cells, astrocytes and neurons. This position allows them to regulate key neurovascular functions of the brain. The role of pericytes in the regulation of cerebral blood flow (CBF) and neurovascular coupling remains, however, under debate. Using loss-of-function pericyte-deficient mice, here we show that pericyte degeneration diminishes global and individual capillary CBF responses to neuronal stimuli, resulting in neurovascular uncoupling, reduced oxygen supply to the brain and metabolic stress. Neurovascular deficits lead over time to impaired neuronal excitability and neurodegenerative changes. Thus, pericyte degeneration as seen in neurological disorders such as Alzheimer's disease may contribute to neurovascular dysfunction and neurodegeneration associated with human disease.

Ultra-large field-of-view two-photon microscopy.

We present a two-photon microscope that images the full extent of murine cortex with an objective-limited spatial resolution across an 8 mm by 10 mm field. The lateral resolution is approximately 1 µm and the maximum scan speed is 5 mm/ms. The scan pathway employs large diameter compound lenses to minimize aberrations and performs near theoretical limits. We demonstrate the special utility of the microscope by recording resting-state vasomotion across both hemispheres of the murine brain through a transcranial window and by imaging histological sections without the need to stitch.

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.

All-optical osteotomy to create windows for transcranial imaging in mice.

Surgical procedures as a prelude to optical imaging are a rate-limiting step in experimental neuroscience. Towards automation of these procedures, we describe the use of nonlinear optical techniques to create a thinned skull window for transcranial imaging. Metrology by second harmonic generation was used to map the surfaces of the skull and define a cutting path. Plasma-mediated laser ablation was utilized to cut bone. Mice prepared with these techniques were used to image subsurface cortical vasculature and blood flow. The viability of the brain tissue was confirmed via histological analysis and supports the utility of solely optical techniques for osteotomy and potentially other surgical procedures.

The cortical angiome: an interconnected vascular network with noncolumnar patterns of blood flow.

What is the nature of the vascular architecture in the cortex that allows the brain to meet the energy demands of neuronal computations? We used high-throughput histology to reconstruct the complete angioarchitecture and the positions of all neuronal somata of multiple cubic millimeter regions of vibrissa primary sensory cortex in mouse. Vascular networks were derived from the reconstruction. In contrast with the standard model of cortical columns that are tightly linked with the vascular network, graph-theoretical analyses revealed that the subsurface microvasculature formed interconnected loops with a topology that was invariant to the position and boundary of columns. Furthermore, the calculated patterns of blood flow in the networks were unrelated to location of columns. Rather, blood sourced by penetrating arterioles was effectively drained by the penetrating venules to limit lateral perfusion. This analysis provides the underpinning to understand functional imaging and the effect of penetrating vessels strokes on brain viability.

All-optical in situ histology of brain tissue with femtosecond laser pulses.

This protocol describes the application of laser pulses to image and ablate neuronal tissue for the purpose of automated histology. The histology is accomplished in situ using serial two-photon imaging of labeled tissue and removal of the imaged tissue with amplified, femtosecond pulses. Together with the use of endogenous fluorescent indicators and/or deep penetration of antibody labels and organic dyes, this method may be used to automatically image, reconstruct, and vectorize structures of interest across millimeter to centimeter regions of brain with micrometer resolution.

The smallest stroke: occlusion of one penetrating vessel leads to infarction and a cognitive deficit.

Microinfarctions are present in the aged and injured human brain. Their clinical relevance is controversial, with postulated sequelae ranging from cognitive sparing to vascular dementia. To address the consequences of microinfarcts, we used controlled optical methods to create occlusions of individual penetrating arterioles or venules in rat cortex. Single microinfarcts, targeted to encompass all or part of a cortical column, impaired performance in a macrovibrissa-based behavioral task. Furthermore, the targeting of multiple vessels resulted in tissue damage that coalesced across cortex, even though the intervening penetrating vessels were acutely patent. Post-occlusion administration of memantine, a glutamate receptor antagonist that reduces cognitive decline in Alzheimer's disease, ameliorated tissue damage and perceptual deficits. Collectively, these data imply that microinfarcts likely contribute to cognitive decline. Strategies that have received limited success in the treatment of ischemic injury, which include therapeutics against excitotoxicity, may be successful against the progressive nature of vascular dementia.

Vectorization of optically sectioned brain microvasculature: learning aids completion of vascular graphs by connecting gaps and deleting open-ended segments.

A graph of tissue vasculature is an essential requirement to model the exchange of gasses and nutriments between the blood and cells in the brain. Such a graph is derived from a vectorized representation of anatomical data, provides a map of all vessels as vertices and segments, and may include the location of nonvascular components, such as neuronal and glial somata. Yet vectorized data sets typically contain erroneous gaps, spurious endpoints, and spuriously merged strands. Current methods to correct such defects only address the issue of connecting gaps and further require manual tuning of parameters in a high dimensional algorithm. To address these shortcomings, we introduce a supervised machine learning method that (1) connects vessel gaps by "learned threshold relaxation"; (2) removes spurious segments by "learning to eliminate deletion candidate strands"; and (3) enforces consistency in the joint space of learned vascular graph corrections through "consistency learning." Human operators are only required to label individual objects they recognize in a training set and are not burdened with tuning parameters. The supervised learning procedure examines the geometry and topology of features in the neighborhood of each vessel segment under consideration. We demonstrate the effectiveness of these methods on four sets of microvascular data, each with >800(3) voxels, obtained with all optical histology of mouse tissue and vectorization by state-of-the-art techniques in image segmentation. Through statistically validated sampling and analysis in terms of precision recall curves, we find that learning with bagged boosted decision trees reduces equal-error error rates for threshold relaxation by 5-21% and strand elimination performance by 18-57%. We benchmark generalization performance across datasets; while improvements vary between data sets, learning always leads to a useful reduction in error rates. Overall, learning is shown to more than halve the total error rate, and therefore, human time spent manually correcting such vectorizations.

A polished and reinforced thinned-skull window for long-term imaging of the mouse brain.

In vivo imaging of cortical function requires optical access to the brain without disruption of the intracranial environment. We present a method to form a polished and reinforced thinned skull (PoRTS) window in the mouse skull that spans several millimeters in diameter and is stable for months. The skull is thinned to 10 to 15 µm in thickness with a hand held drill to achieve optical clarity, and is then overlaid with cyanoacrylate glue and a cover glass to: 1) provide rigidity, 2) inhibit bone regrowth and 3) reduce light scattering from irregularities on the bone surface. Since the skull is not breached, any inflammation that could affect the process being studied is greatly reduced. Imaging depths of up to 250 µm below the cortical surface can be achieved using two-photon laser scanning microscopy. This window is well suited to study cerebral blood flow and cellular function in both anesthetized and awake preparations. It further offers the opportunity to manipulate cell activity using optogenetics or to disrupt blood flow in targeted vessels by irradiation of circulating photosensitizers.

Prospect for feedback guided surgery with ultra-short pulsed laser light.

The controlled cutting of tissue with laser light is a natural technology to combine with automated stereotaxic surgery. A central challenge is to cut hard tissue, such as bone, without inducing damage to juxtaposed soft tissue, such as nerve and dura. We review past work that demonstrates the feasibility of such control through the use of ultrafast laser light to both cut and generate optical feedback signals via second harmonic generation and laser induced plasma spectra.

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.

A guide to delineate the logic of neurovascular signaling in the brain.

The neurovascular system may be viewed as a distributed nervous system within the brain. It transforms local neuronal activity into a change in the tone of smooth muscle that lines the walls of arterioles and microvessels. We review the current state of neurovascular coupling, with an emphasis on signaling molecules that convey information from neurons to neighboring vessels. At the level of neocortex, this coupling is mediated by: (i) a likely direct interaction with inhibitory neurons, (ii) indirect interaction, via astrocytes, with excitatory neurons, and (iii) fiber tracts from subcortical layers. Substantial evidence shows that control involves competition between signals that promote vasoconstriction versus vasodilation. Consistent with this picture is evidence that, under certain circumstances, increased neuronal activity can lead to vasoconstriction rather than vasodilation. This confounds naïve interpretations of functional brain images. We discuss experimental approaches to detect signaling molecules in vivo with the goal of formulating an empirical basis for the observed logic of neurovascular control.

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.

Temporally focused femtosecond laser pulses for low numerical aperture micromachining through optically transparent materials.

Temporal focusing of spatially chirped femtosecond laser pulses overcomes previous limitations for ablating high aspect ratio features with low numerical aperture (NA) beams. Simultaneous spatial and temporal focusing reduces nonlinear interactions, such as self-focusing, prior to the focal plane so that deep (approximately 1 mm) features with parallel sidewalls are ablated at high material removal rates (25 microm(3) per 80 microJ pulse) at 0.04-0.05 NA. This technique is applied to the fabrication of microfluidic devices by ablation through the back surface of thick (6 mm) fused silica substrates. It is also used to ablate bone under aqueous immersion to produce craniotomies.

Correlations of neuronal and microvascular densities in murine cortex revealed by direct counting and colocalization of nuclei and vessels.

It is well known that the density of neurons varies within the adult brain. In neocortex, this includes variations in neuronal density between different lamina as well as between different regions. Yet the concomitant variation of the microvessels is largely uncharted. Here, we present automated histological, imaging, and analysis tools to simultaneously map the locations of all neuronal and non-neuronal nuclei and the centerlines and diameters of all blood vessels within thick slabs of neocortex from mice. Based on total inventory measurements of different cortical regions ( approximately 10(7) cells vectorized across brains), these methods revealed: (1) In three dimensions, the mean distance of the center of neuronal somata to the closest microvessel was 15 mum. (2) Volume samples within lamina of a given region show that the density of microvessels does not match the strong laminar variation in neuronal density. This holds for both agranular and granular cortex. (3) Volume samples in successive radii from the midline to the ventral-lateral edge, where each volume summed the number of cells and microvessels from the pia to the white matter, show a significant correlation between neuronal and microvessel densities. These data show that while neuronal and vascular densities do not track each other on the 100 mum scale of cortical lamina, they do track each other on the 1-10 mm scale of the cortical mantle. The absence of a disproportionate density of blood vessels in granular lamina is argued to be consistent with the initial locus of functional brain imaging signals.

Acute vascular disruption and aquaporin 4 loss after stroke.

BACKGROUND AND PURPOSE: Ischemic protection has been demonstrated by a decrease in stroke-infarct size in transgenic mice with deficient Aquaporin 4 (AQP4) expression. However, it is not known whether AQP4 is rapidly reduced during acute stroke in animals with normal AQP4 phenotype, which may provide a potential self-protective mechanism. METHODS: Adult male rats underwent transient occlusion of the middle cerebral artery (tMCAo) for 1 to 8 hours followed by reperfusion for 30 minutes. Protein and mRNA expression of AQP4 and glial fibrillary acidic protein (GFAP) were determined by Western blot and rtPCR. Fluorescence quantitation was obtained with laser scanning cytometry (LSC) for Cy5-tagged immunoreactivity along with fluorescein signals from pathological uptake of plasma-borne high-molecular-weight fluorescein-dextran. Cell death was assessed with in vivo Propidium Iodide (PI) nucleus labeling. RESULTS: In the ischemic hemisphere in tissue sections, patches of fluorescein-dextran uptake were overlapped with sites of focal loss of AQP4 immunoreactivity after tMCAo of 1 to 8 hours duration. However, the average levels of AQP4 protein and mRNA, determined in homogenates of whole striatum, were not significantly reduced after 8 hours of tMCAo. Tissue section cytometry (LSC) of immunoreactivity in scan areas with high densities of fluorescein-dextran uptake demonstrated reductions in AQP4, but not in IgG or GFAP, after tMCAo of 2 hours or longer. Scan areas with low densities of fluorescein-dextran did not lose AQP4. There was sparse astrocyte cell death as only 1.7+/-0.85% (mean, SD) of DAPI labeled cells were PI- and GFAP-labeled after 8 hours of tMCAo. CONCLUSIONS: During acute tMCAo, a rapid loss of AQP4 immunoreactivity from viable astrocytes can occur. However, AQP4 loss is spatially selective and occurs primarily in regions of vascular damage.

Plasma-mediated ablation: an optical tool for submicrometer surgery on neuronal and vascular systems.

Plasma-mediated ablation makes use of high energy laser pulses to ionize molecules within the first few femtoseconds of the pulse. This process leads to a submicrometer-sized bubble of plasma that can ablate tissue with negligible heat transfer and collateral damage to neighboring tissue. We review the physics of plasma-mediated ablation and its use as a tool to generate targeted insults at the subcellular level to neurons and blood vessels deep within nervous tissue. Illustrative examples from axon regeneration and microvascular research highlight the utility of this tool. We further discuss the use of ablation as an integral part of automated histology.