Seeing the Forest and Its Trees Together: Implementing 3D Light Microscopy Pipelines for Cell Type Mapping in the Mouse Brain

The brain is composed of diverse neuronal and non-neuronal cell types with complex regional connectivity patterns that create the anatomical infrastructure underlying cognition. Remarkable advances in neuroscience techniques enable labeling and imaging of these individual cell types and their interactions throughout intact mammalian brains at a cellular resolution allowing neuroscientists to examine microscopic details in macroscopic brain circuits. Nevertheless, implementing these tools is fraught with many technical and analytical challenges with a need for high-level data analysis. Here we review key technical considerations for implementing a brain mapping pipeline using the mouse brain as a primary model system. Specifically, we provide practical details for choosing methods including cell type specific labeling, sample preparation (e.g., tissue clearing), microscopy modalities, image processing, and data analysis (e.g., image registration to standard atlases). We also highlight the need to develop better 3D atlases with standardized anatomical labels and nomenclature across species and developmental time points to extend the mapping to other species including humans and to facilitate data sharing, confederation, and integrative analysis. In summary, this review provides key elements and currently available resources to consider while developing and implementing high-resolution mapping methods.

Beyond pulmonary vein isolation for persistent atrial fibrillation: sequential high-resolution mapping to guide ablation

Purpose We aimed to evaluate whether outcomes with ablation in persistent (PsAF) and long-standing persistent (LsPsAF) AF can be improved beyond what can be achieved with pulmonary vein isolation (PVI) alone, using individualized mapping to guide ablation. Methods We studied 20 pts (15 M, 68 ± 11y) with PsAF (14) or LsPsAF (6) referred for first-time AF ablation. Following antral PVI, individualized mapping (IM) was performed using a high-density mapping catheter stably and fully deployed for 30 s at each of 23 ± 9 sites per patient. Activation data were reviewed, and an ablation strategy designed to intersect areas of focal and rotational activity. Mean follow-up was 429 ± 131 days. The study population was compared to a matched contemporary control cohort (CC) of 20 consecutive patients undergoing conventional ablation. Results Despite the IM group having a higher median comorbidities score, 3.5 vs. 2.5 in the CC group, indicating potentially more complex patients and more advanced substrate, cumulative freedom from AF after a single procedure was achieved in 94% of patients in the IM group vs. 75% in the CC group at 1 year and remained the same in both groups at the conclusion of the study (p = 0.02). There was a similar trend in atrial arrhythmia-free survival between both groups (84% vs. 67% at 1 year) that did not reach statistical significance. The procedure duration was longer in the IM group by a median of 31.5 min (p = 0.004). Conclusions Individualized mapping to guide AF ablation appears to achieve significantly greater AF-free survival compared to conventional PVI when applied as a primary ablation treatment. The results of this pilot study need to be confirmed in a larger, randomized trial.

Atrial fibrillation driver identification through regional mutual information networks: a modeling perspective

Purpose Effective identification of electrical drivers within remodeled tissue is a key for improving ablation treatment for atrial fibrillation. We have developed a mutual information, graph-based approach to identify and propose fault tolerance metric of local efficiency as a distinguishing feature of rotational activation and remodeled atrial tissue. Methods Voltage data were extracted from atrial tissue simulations (2D Karma, 3D physiological, and the Multiscale Cardiac Simulation Framework (MSCSF)) using multi-spline open and parallel regional mapping catheter geometries. Graphs were generated based on varied mutual information thresholds between electrode pairs and the local efficiency for each graph was calculated. Results High-resolution mapping catheter geometries can distinguish between rotational and irregular activation patterns using the derivative of local efficiency as a function of increasing mutual information threshold. The derivative is decreased for rotational activation patterns comparing to irregular activations in both a simplified 2D model (0.0017 ± 1 × 10−4 vs. 0.0032 ± 1 × 10−4, p < 0.01) and a more realistic 3D model (0.00092 ± 5 × 10−5 vs. 0.0014 ± 4 × 10−5, p < 0.01). Average local efficiency derivative can also distinguish between degrees of remodeling. Simulations using the MSCSF model, with 10 vs. 90% remodeling, display distinct derivatives in the grid design parallel spline catheter configuration (0.0015 ± 5 × 10−5 vs. 0.0019 ± 6 × 10−5, p < 0.01) and the flower shaped open spline configuration (0.0011 ± 5 × 10−5 vs. 0.0016 ± 4 × 10−5, p < 0.01). Conclusion A decreased derivative of local efficiency characterizes rotational activation and varies with atrial remodeling. This suggests a distinct communication pattern in cardiac rotational activation detectable via high-resolution regional mapping and could enable identification of electrical drivers for targeted ablation.

High-resolution screening for marine prokaryotic and eukaryotic taxa with selective preference for PE and PET surfaces

Marine plastic debris serve as substrates for the colonization of a variety of prokaryote and eukaryote organisms. Of particular interest are the microorganisms that have adapted to thrive on plastic as they may contain genes, enzymes or pathways involved in the colonization or metabolism of plastics. We implemented DNA metabarcoding with nanopore MinION sequencing to compare the one-month-old biomes of hydrolysable (polyethylene terephthalate) and non-hydrolysable (polyethylene) plastics surfaces vs. those of glass and the surrounding water in a Mediterranean Sea marina. We sequenced longer 16S rRNA, 18S rRNA and ITS barcode loci for a more comprehensive taxonomic profiling of the bacterial, protist and fungal communities respectively. Long read sequencing enabled high-resolution mapping to genera and species. Using differential abundance screening we identified 32 bacteria and five eukaryotes, that were significantly differentially abundant on PE or PET compared to glass. This approach may be used in the future to characterize the plastisphere communities and to screen for microorganisms with a plastic-metabolism potential.

3D mapping of the submerged Crowie barge using electrical resistivity tomography

This study explores the applicability and effectiveness of electrical resistivity tomography (ERT) as a tool for the high-resolution mapping of submerged and buried shipwrecks in 3D. This approach was trialled through modelling and field studies of Crowie, a paddle steamer barge which sunk at anchor in the Murray River at Morgan, South Australia, in the late 1950s. The mainly metallic structure of the ship is easily recognisable in the ERT data and was mapped in 3D both subaqueously and beneath the sedimentwater interface.The innovative and successful use of ERT in this case study demonstrates that 3D ERT can be used for the detailed mapping of submerged cultural material. It will be particularly useful where other geophysical and diver based mapping techniques may be inappropriate due to shallow water depths, poor visibility, or other constraints.

5-Hydroxymethylcytosine-mediated active demethylation is required for mammalian neuronal differentiation and function

Although high levels of 5-hydroxymethylcytosine (5hmC) accumulate in mammalian neurons, our knowledge of its roles in terminal differentiation or as an intermediate in active DNA demethylation is incomplete. We report high-resolution mapping of DNA methylation and hydroxymethylation, chromatin accessibility, and histone marks in developing postmitotic Purkinje cells (PCs) in Mus musculus. Our data reveal new relationships between PC transcriptional and epigenetic programs, and identify a class of genes that lose both 5-methylcytosine (5mC) and 5hmC during terminal differentiation. Deletion of the 5hmC writers Tet1, Tet2, and Tet3 from postmitotic PCs prevents loss of 5mC and 5hmC in regulatory domains and gene bodies, and hinders transcriptional and epigenetic developmental transitions. Our data demonstrate that Tet-mediated active DNA demethylation occurs in vivo, and that acquisition of the precise molecular properties of adult PCs require continued oxidation of 5mC to 5hmC during the final phases of differentiation.

High-resolution relaxometry-based calibrated fMRI in murine brain: Metabolic differences between awake and anesthetized states

Functional magnetic resonance imaging (fMRI) techniques using the blood-oxygen level-dependent (BOLD) signal have shown great potential as clinical biomarkers of disease. Thus, using these techniques in preclinical rodent models is an urgent need. Calibrated fMRI is a promising technique that can provide high-resolution mapping of cerebral oxygen metabolism (CMRO2). However, calibrated fMRI is difficult to use in rodent models for several reasons: rodents are anesthetized, stimulation-induced changes are small, and gas challenges induce noisy CMRO2 predictions. We used, in mice, a relaxometry-based calibrated fMRI method which uses cerebral blood flow (CBF) and the BOLD-sensitive magnetic relaxation component, R2′, the same parameter derived in the deoxyhemoglobin-dilution model of calibrated fMRI. This method does not use any gas challenges, which we tested on mice in both awake and anesthetized states. As anesthesia induces a whole-brain change, our protocol allowed us to overcome the former limitations of rodent studies using calibrated fMRI. We revealed 1.5-2 times higher CMRO2, dependent upon brain region, in the awake state versus the anesthetized state. Our results agree with alternative measurements of whole-brain CMRO2 in the same mice and previous human anesthesia studies. The use of calibrated fMRI in rodents has much potential for preclinical fMRI.

iMUT-seq: high-resolution mapping of DSB mutational landscapes reveals new insights into the mutagenic mechanisms of DSB repair

DNA double-strand breaks (DSBs) are the most mutagenic form of DNA damage, and play a significant role in cancer biology, neurodegeneration and aging. However, studying DSB-induced mutagenesis is currently limited by the tools available for mapping these mutations. Here, we describe iMUT-seq, a technique that profiles DSB-induced mutations at high-sensitivity and single-nucleotide resolution around endogenous DSBs spread across the genome. By depleting 20 different DSB-repair factors we defined their mutational signatures in detail, revealing remarkable insights into the mechanisms of DSB-induced mutagenesis. We find that homologous-recombination (HR) is mutagenic in nature, displaying high levels of base substitutions and mononucleotide deletions due to DNA-polymerase errors, but simultaneously reduced translocation events, suggesting the primary role of HR is the specific suppression of genomic rearrangements. The results presented here offer new fundamental insights into DSB-induced mutagenesis and have significant implications for our understanding of cancer biology and the development of DDR-targeting chemotherapeutics.