Enhanced characterization of the zebrafish brain as revealed by super-resolution track-density imaging

In this study, we explored the use of super-resolution track-density imaging (TDI) for neuroanatomical characterization of the adult zebrafish brain. We compared the quality of image contrast and resolution obtained with T-2* magnetic resonance imaging (MRI), diffusion tensor-based imaging (DTI), TDI, and histology. The anatomical structures visualized in 5 mu m TDI maps corresponded with histology. Moreover, the super-resolution property and the local-directional information provided by directionally encoded color TDI facilitated delineation of a larger number of brain regions, commissures and small white matter tracks when compared to conventional MRI and DTI. In total, we were able to visualize 17 structures that were previously unidentifiable using MR microimaging, such as the four layers of the optic tectum. This study demonstrates the use of TDI for characterization of the adult zebrafish brain as a pivotal tool for future phenotypic examination of transgenic models of neurological diseases

Optic-nerve-transmitted eyeshine, a new type of light emission from fish eyes

Background: Most animal eyes feature an opaque pigmented eyecup to assure that light can enter from one direction only. We challenge this dogma by describing a previously unknown form of eyeshine resulting from light that enters the eye through the top of the head and optic nerve, eventually emanating through the pupil as a narrow beam: the Optic-Nerve-Transmitted (ONT) eyeshine. We characterize ONT eyeshine in the triplefin blenny Tripterygion delaisi (Tripterygiidae) in comparison to three other teleost species, using behavioural and anatomical observations, spectrophotometry, histology, and magnetic resonance imaging. The study’s aim is to identify the factors that determine ONT eyeshine occurrence and intensity, and whether these are specifically adapted for that purpose. Results: ONT eyeshine intensity benefits from locally reduced head pigmentation, a thin skull, the gap between eyes and forebrain, the potential light-guiding properties of the optic nerve, and, most importantly, a short distance between the head surface and the optic nerves. Conclusions: The generality of these factors and the lack of specifically adapted features implies that ONT eyeshine is widespread among small fish species. Nevertheless, its intensity varies considerably, depending on the specific combination and varying expression of common anatomical features. We discuss whether ONT eyeshine might affect visual performance, and speculate about possible functions such as predator detection, camouflage, and intraspecific communication. Electronic supplementary material The online version of this article (doi:10.1186/s12983-017-0198-9) contains supplementary material, which is available to authorized users

An ontologically consistent MRI-based atlas of the mouse diencephalon

In topological terms, the diencephalon lies between the hypothalamus and the midbrain. It is made up of three segments, prosomere 1 (pretectum), prosomere 2 (thalamus), and prosomere 3 (the prethalamus). A number of MRI-based atlases of different parts of the mouse brain have already been published, but none of them displays the segments the diencephalon and their component nuclei. In this study we present a new volumetric atlas identifying 89 structures in the diencephalon of the male C57BL/6J 12 week mouse. This atlas is based on an average of MR scans of 18 mouse brains imaged with a 16.4T scanner. This atlas is available for download at www.imaging.org.au/AMBMC. Additionally, we have created an FSL package to enable nonlinear registration of novel data sets to the AMBMC model and subsequent automatic segmentation

Acute multi-sgRNA knockdown of KEOPS complex genes reproduces the microcephaly phenotype of the stable knockout zebrafish model

Until recently, morpholino oligonucleotides have been widely employed in zebrafish as an acute and efficient loss-of-function assay. However, off-target effects and reproducibility issues when compared to stable knockout lines have compromised their further use. Here we employed an acute CRISPR/Cas approach using multiple single guide RNAs targeting simultaneously different positions in two exemplar genes (osgep or tprkb) to increase the likelihood of generating mutations on both alleles in the injected F0 generation and to achieve a similar effect as morpholinos but with the reproducibility of stable lines. This multi single guide RNA approach resulted in median likelihoods for at least one mutation on each allele of >99% and sgRNA specific insertion/deletion profiles as revealed by deep-sequencing. Immunoblot showed a significant reduction for Osgep and Tprkb proteins. For both genes, the acute multi-sgRNA knockout recapitulated the microcephaly phenotype and reduction in survival that we observed previously in stable knockout lines, though milder in the acute multi-sgRNA knockout. Finally, we quantify the degree of mutagenesis by deep sequencing, and provide a mathematical model to quantitate the chance for a biallelic loss-of-function mutation. Our findings can be generalized to acute and stable CRISPR/Cas targeting for any zebrafish gene of interest

Mutations in KEOPS-Complex Genes Cause Nephrotic Syndrome with Primary Microcephaly

Galloway-Mowat syndrome (GAMOS) is an autosomal-recessive disease characterized by the combination of early-onset nephrotic syndrome (SRNS) and microcephaly with brain anomalies. Here we identified recessive mutations in OSGEP, TP53RK, TPRKB, and LAGE3, genes encoding the four subunits of the KEOPS complex, in 37 individuals from 32 families with GAMOS. CRISPR-Cas9 knockout in zebrafish and mice recapitulated the human phenotype of primary microcephaly and resulted in early lethality. Knockdown of OSGEP, TP53RK, or TPRKB inhibited cell proliferation, which human mutations did not rescue. Furthermore, knockdown of these genes impaired protein translation, caused endoplasmic reticulum stress, activated DNA-damage-response signaling, and ultimately induced apoptosis. Knockdown of OSGEP or TP53RK induced defects in the actin cytoskeleton and decreased the migration rate of human podocytes, an established intermediate phenotype of SRNS. We thus identified four new monogenic causes of GAMOS, describe a link between KEOPS function and human disease, and delineate potential pathogenic mechanisms

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

A three-dimensional digital atlas of the zebrafish brain

In the past three decades, the zebrafish has become a vital animal model in a range of biological sciences. To augment current neurobiological research, we have developed the first three-dimensional digital atlas of the zebrafish brain from T(2)*-weighted magnetic resonance histology (MRH) images acquired on a 16.4-T superconducting magnet. We achieved an isotropic resolution of 10 mu m, which is the highest resolution achieved in a vertebrate brain and, for the first time, is comparable in slice thickness to conventional histology. By using manual segmentation, 53 anatomical structures, including fiber tracts as small as 40 pm, were delineated. Using Amira software, structures were also individually segmented and reconstructed to create three-dimensional animations. Additional quantitative information including, volume, surface areas, and mean gray scale intensities were also determined. Finally, we established a stereotaxic coordinate system as a framework in which maps created from other modalities can be incorporated into the atlas. (C) 2010 Elsevier Inc. All rights reserved

Laplace mixture autoregressive models

Autoregressive (AR) models are an important tool in the study of time series data. However, the standard AR model only allows for unimodal marginal and conditional densities, and cannot capture conditional heteroscedasticity. Previously, the Gaussian mixture AR (GMAR) model was considered to remedy these shortcomings by using a Gaussian mixture conditional model. We introduce the Laplace mixture (LMAR) model that utilizes a Laplace mixture conditional model, as an alternative to the GMAR model. We characterize the LMAR model and provide conditions for stationarity. An MM (minorization–maximization) algorithm is then proposed for maximum pseudolikelihood (MPL) estimation of an LMAR model. Conditions for asymptotic inference and a rule for model selection for the MPL estimator are considered. An example analysis of data arising from the calcium imaging of a zebrafish brain is performed

The retinal wholemount technique: A window to understanding the brain and behaviour

The accessibility of the vertebrate retina has provided the opportunity to assess various parameters of the visual abilities of a range of species. This thin but complex extension of the brain achieves a large proportion of the necessary visual processing of an optical image before information is delivered to the brain as neural impulses. Studies of the retina as a wholemount or a flattened sheet of neural tissue are abundant due to the large amount of information that can be analysed, as follows: the level of summation or convergence; the coverage, stratification and potential sites of synaptic connections; the spatial resolving power; the arrangement of neuronal arrays or mosaics; electrophysiological access for the recording of responses to visual stimuli; the spatial arrangement of cell dendritic fields; location of retinal ‘blind spots’ (optic nerve, falciform process and pecten); topographic differences in retinal cell sampling; spectral filters, and reflective structures. The present study examines all aspects of the wholemount technique, including enucleation, fixation, retinal extraction, flattening, staining, visualization of labelled cells and stereological mapping of cell density. Uniquely, it highlights the crucial technical and often species-specific differences encountered when examining a range of vertebrate taxa (fishes, reptiles, birds and mammals). This broad comparative approach will enable future studies to overcome technical difficulties, thus permitting larger conceptual questions to be posed regarding the diversity of visual tasks across phylogenetic boundaries