Combining array tomography with electron tomography provides insights into leakiness of the blood-brain barrier in mouse cortex.

Like other volume electron microscopy approaches, automated tape-collecting ultramicrotomy (ATUM) enables imaging of serial sections deposited on thick plastic tapes by scanning electron microscopy (SEM). ATUM is unique in enabling hierarchical imaging and thus efficient screening for target structures, as needed for correlative light and electron microscopy. However, SEM of sections on tape can only access the section surface, thereby limiting the axial resolution to the typical size of cellular vesicles with an order of magnitude lower than the acquired xy resolution.

Fluorogenic Chemical Probes for Wash-free Imaging of Cell Membrane Damage in Ferroptosis, Necrosis, and Axon Injury.

Selectively labeling cells with damaged membranes is needed not only for identifying dead cells in culture, but also for imaging membrane barrier dysfunction in pathologies . Most membrane permeability stains are permanently colored or fluorescent dyes that need washing to remove their non-uptaken extracellular background and reach good image contrast. Others are DNA-binding environment-dependent fluorophores, which lack design modularity, have potential toxicity, and can only detect permeabilization of cell volumes containing a nucleus (i.

Validating a minipig model of reversible cerebral demyelination using human diagnostic modalities and electron microscopy.

Background: Inflammatory demyelinating diseases of the central nervous system, such as multiple sclerosis, are significant sources of morbidity in young adults despite therapeutic advances. Current murine models of remyelination have limited applicability due to the low white matter content of their brains, which restricts the spatial resolution of diagnostic imaging. Large animal models might be more suitable but pose significant technological, ethical and logistical challenges.

Immune-mediated denervation of the pineal gland underlies sleep disturbance in cardiac disease.

Disruption of the physiologic sleep-wake cycle and low melatonin levels frequently accompany cardiac disease, yet the underlying mechanism has remained enigmatic. Immunostaining of sympathetic axons in optically cleared pineal glands from humans and mice with cardiac disease revealed their substantial denervation compared with controls. Spatial, single-cell, nuclear, and bulk RNA sequencing traced this defect back to the superior cervical ganglia (SCG), which responded to cardiac disease with accumulation of inflammatory macrophages, fibrosis, and the selective loss of pineal gland-innervating neurons.

Neurons on tape: Automated Tape Collecting Ultramicrotomy-mediated volume EM for targeting neuropathology.

In this chapter, we review Automated Tape Collecting Ultramicrotomy (ATUM), which, among other array tomography methods, substantially simplified large-scale volume electron microscopy (vEM) projects. vEM reveals biological structures at nanometer resolution in three dimensions and resolves ambiguities of two-dimensional representations. However, as the structures of interest-like disease hallmarks emerging from neuropathology-are often rare but the field of view is small, this can easily turn a vEM project into a needle in a haystack problem.

Ex vivo immunocapture and functional characterization of cell-type-specific mitochondria using MitoTag mice.

Mitochondria are key bioenergetic organelles involved in many biosynthetic and signaling pathways. However, their differential contribution to specific functions of cells within complex tissues is difficult to dissect with current methods. The present protocol addresses this need by enabling the ex vivo immunocapture of cell-type-specific mitochondria directly from their tissue context through a MitoTag reporter mouse.

Remyelination by surviving oligodendrocytes is inefficient in the inflamed mammalian cortex.

In multiple sclerosis, an inflammatory attack results in myelin loss, which can be partially reversed by remyelination. Recent studies suggest that mature oligodendrocytes could contribute to remyelination by generating new myelin. Here, we show that in a mouse model of cortical multiple sclerosis pathology, surviving oligodendrocytes can indeed extend new proximal processes but rarely generate new myelin internodes.

Ketogenic diet uncovers differential metabolic plasticity of brain cells.

To maintain homeostasis, the body, including the brain, reprograms its metabolism in response to altered nutrition or disease. However, the consequences of these challenges for the energy metabolism of the different brain cell types remain unknown. Here, we generated a proteome atlas of the major central nervous system (CNS) cell types from young and adult mice, after feeding the therapeutically relevant low-carbohydrate, high-fat ketogenic diet (KD) and during neuroinflammation.

P2R Inhibitors Prevent Antibody-Mediated Complement Activation in an Animal Model of Neuromyelitis Optica : P2R Inhibitors Prevent Autoantibody Injury.

Purinergic 2 receptors (P2Rs) contribute to disease-related immune cell signaling and are upregulated in various pathological settings, including neuroinflammation. P2R inhibitors have been used to treat inflammatory diseases and can protect against complement-mediated cell injury. However, the mechanisms behind these anti-inflammatory properties of P2R inhibitors are not well understood, and their potential in CNS autoimmunity is underexplored.

Size-Selective Transfer of Lipid Nanoparticle-Based Drug Carriers Across the Blood Brain Barrier Via Vascular Occlusions Following Traumatic Brain Injury.

The current lack of understanding about how nanocarriers cross the blood-brain barrier (BBB) in the healthy and injured brain is hindering the clinical translation of nanoscale brain-targeted drug-delivery systems. Here, the bio-distribution of lipid nano-emulsion droplets (LNDs) of two sizes (30 and 80 nm) in the mouse brain after traumatic brain injury (TBI) is investigated. The highly fluorescent LNDs are prepared by loading them with octadecyl rhodamine B and a bulky hydrophobic counter-ion, tetraphenylborate.

A new form of axonal pathology in a spinal model of neuromyelitis optica.

Neuromyelitis optica is a chronic neuroinflammatory disease, which primarily targets astrocytes and often results in severe axon injury of unknown mechanism. Neuromyelitis optica patients harbour autoantibodies against the astrocytic water channel protein, aquaporin-4 (AQP4-IgG), which induce complement-mediated astrocyte lysis and subsequent axon damage. Using spinal in vivo imaging in a mouse model of such astrocytopathic lesions, we explored the mechanism underlying neuromyelitis optica-related axon injury.

A less painful transfer of power.

Transfer between cells is an unexpected addition to the mitochondrial life cycle. In this issue of Neuron, Van der Vlist et al. now provide evidence that M2-macrophages infiltrating sensory ganglia resolve pain by transferring particles containing mitochondria to neurons-thus boosting nociceptors back to normal function.

Neural labeling and manipulation by neonatal intraventricular viral injection in mice.

This step-by-step protocol provides a fast and easy technique to label and/or genetically manipulate neural cells, achieved by intraventricular injection of viral vectors into neonatal mice under ultrasound guidance. Successful injection of adeno-associated viral vectors (AAV) induces neural transduction as fast as 3 days post injection (dpi) in both the central and peripheral nervous systems. Virally driven expression persists until early adulthood.

Transthyretin Promotes Axon Growth Regulation of Microtubule Dynamics and Tubulin Acetylation.

Transthyretin (TTR), a plasma and cerebrospinal fluid protein, increases axon growth and organelle transport in sensory neurons. While neurons extend their axons, the microtubule (MT) cytoskeleton is crucial for the segregation of functional compartments and axonal outgrowth. Herein, we investigated whether TTR promotes axon elongation by modulating MT dynamics.

Niwaki Instead of Random Forests: Targeted Serial Sectioning Scanning Electron Microscopy With Reimaging Capabilities for Exploring Central Nervous System Cell Biology and Pathology.

Ultrastructural analysis of discrete neurobiological structures by volume scanning electron microscopy (SEM) often constitutes a "needle-in-the-haystack" problem and therefore relies on sophisticated search strategies. The appropriate SEM approach for a given relocation task not only depends on the desired final image quality but also on the complexity and required accuracy of the screening process. Block-face SEM techniques like Focused Ion Beam or serial block-face SEM are "one-shot" imaging runs by nature and, thus, require precise relocation prior to acquisition.

Notch-mediated re-specification of neuronal identity during central nervous system development.

Neuronal identity has long been thought of as immutable, so that once a cell acquires a specific fate, it is maintained for life. Studies using the overexpression of potent transcription factors to experimentally reprogram neuronal fate in the mouse neocortex and retina have challenged this notion by revealing that post-mitotic neurons can switch their identity. Whether fate reprogramming is part of normal development in the central nervous system (CNS) is unclear.

Completion of neuronal remodeling prompts myelination along developing motor axon branches.

Neuronal remodeling and myelination are two fundamental processes during neurodevelopment. How they influence each other remains largely unknown, even though their coordinated execution is critical for circuit function and often disrupted in neuropsychiatric disorders. It is unclear whether myelination stabilizes axon branches during remodeling or whether ongoing remodeling delays myelination.

Phagocyte-mediated synapse removal in cortical neuroinflammation is promoted by local calcium accumulation.

Cortical pathology contributes to chronic cognitive impairment of patients suffering from the neuroinflammatory disease multiple sclerosis (MS). How such gray matter inflammation affects neuronal structure and function is not well understood. In the present study, we use functional and structural in vivo imaging in a mouse model of cortical MS to demonstrate that bouts of cortical inflammation disrupt cortical circuit activity coincident with a widespread, but transient, loss of dendritic spines.

ATUM-FIB microscopy for targeting and multiscale imaging of rare events in mouse cortex.

Here, we describe a detailed workflow for ATUM-FIB microscopy, a hybrid method that combines serial-sectioning scanning electron microscopy (SEM) with focused ion beam SEM (FIB-SEM). This detailed protocol is optimized for mouse cortex samples. The main processing steps include the generation of semi-thick sections from sequentially cured resin blocks using a heated microtomy approach.