Distinct molecular profiles of skull bone marrow in health and neurological disorders.

The bone marrow in the skull is important for shaping immune responses in the brain and meninges, but its molecular makeup among bones and relevance in human diseases remain unclear. Here, we show that the mouse skull has the most distinct transcriptomic profile compared with other bones in states of health and injury, characterized by a late-stage neutrophil phenotype. In humans, proteome analysis reveals that the skull marrow is the most distinct, with differentially expressed neutrophil-related pathways and a unique synaptic protein signature. 3D imaging demonstrates the structural and cellular details of human skull-meninges connections (SMCs) compared with veins. Last, using translocator protein positron emission tomography (TSPO-PET) imaging, we show that the skull bone marrow reflects inflammatory brain responses with a disease-specific spatial distribution in patients with various neurological disorders. The unique molecular profile and anatomical and functional connections of the skull show its potential as a site for diagnosing, monitoring, and treating brain diseases.

Whole-mouse clearing and imaging at the cellular level with vDISCO.

Homeostatic and pathological phenomena often affect multiple organs across the whole organism. Tissue clearing methods, together with recent advances in microscopy, have made holistic examinations of biological samples feasible. Here, we report the detailed protocol for nanobody(VHH)-boosted 3D imaging of solvent-cleared organs (vDISCO), a pressure-driven, nanobody-based whole-body immunolabeling and clearing method that renders whole mice transparent in 3 weeks, consistently enhancing the signal of fluorescent proteins, stabilizing them for years. This allows the reliable detection and quantification of fluorescent signal in intact rodents enabling the analysis of an entire body at cellular resolution. Here, we show the high versatility of vDISCO applied to boost the fluorescence signal of genetically expressed reporters and clear multiple dissected organs and tissues, as well as how to image processed samples using multiple fluorescence microscopy systems. The entire protocol is accessible to laboratories with limited expertise in tissue clearing. In addition to its applications in obtaining a whole-mouse neuronal projection map, detecting single-cell metastases in whole mice and identifying previously undescribed anatomical structures, we further show the visualization of the entire mouse lymphatic system, the application for virus tracing and the visualization of all pericytes in the brain. Taken together, our vDISCO pipeline allows systematic and comprehensive studies of cellular phenomena and connectivity in whole bodies.

Retino-cortical stimulus frequency-dependent gamma coupling: evidence and functional implications of oscillatory potentials.

Long-range gamma band EEG oscillations mediate information transmission between distant brain regions. Gamma band-based coupling may not be restricted to cortex-to-cortex communication but may include extracortical parts of the visual system. The retinogram and visual event-related evoked potentials exhibit time-locked, forward propagating oscillations that are candidates of gamma oscillatory coupling between the retina and the visual cortex. In this study, we tested if this gamma coupling is present as indicated by the coherence of gamma-range (70-200 Hz) oscillatory potentials (OPs) recorded simultaneously from the retina and the primary visual cortex in freely moving, adult rats. We found significant retino-cortical OP coherence in a wide range of stimulus duration (0.01-1000 msec), stimulus intensity (800-5000 mcd/mm2), interstimulus interval (10-400 msec), and stimulus frequency (0.25-25 Hz). However, at low stimulus frequencies, the OPs were time-locked, flickering light at 25 Hz entrained continuous OP coherence (steady-state response, SSR). Our results suggest that the retina and the visual cortex exhibit oscillatory coupling at high-gamma frequency with precise time locking and synchronization of information transfer from the retina to the visual cortex, similar to cortico-cortical gamma coupling. The temporal fusion of retino-cortical gamma coherence at stimulus rates of theater movies may explain the mechanism of the visual illusion of continuity. How visual perception depends on early transformations of ascending sensory information is incompletely understood. By simultaneous measurement of flash-evoked potentials in the retina and the visual cortex in awake, freely moving rats, we demonstrate for the first time that time-locked gamma oscillatory potentials exhibit stable retino-cortical synchrony across a wide range of stimulus parameters and that the temporal continuity of coherence changes with stimulus frequency according to the expected change in the visual illusion of continuity.