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.

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.

Evidence for Concerted and Mosaic Brain Evolution in Dragon Lizards.

The brain plays a critical role in a wide variety of functions including behaviour, perception, motor control, and homeostatic maintenance. Each function can undergo different selective pressures over the course of evolution, and as selection acts on the outputs of brain function, it necessarily alters the structure of the brain. Two models have been proposed to explain the evolutionary patterns observed in brain morphology. The concerted brain evolution model posits that the brain evolves as a single unit and the evolution of different brain regions are coordinated. The mosaic brain evolution model posits that brain regions evolve independently of each other. It is now understood that both models are responsible for driving changes in brain morphology; however, which factors favour concerted or mosaic brain evolution is unclear. Here, we examined the volumes of the 6 major neural subdivisions across 14 species of the agamid lizard genus Ctenophorus (dragons). These species have diverged multiple times in behaviour, ecology, and body morphology, affording a unique opportunity to test neuroevolutionary models across species. We assigned each species to an ecomorph based on habitat use and refuge type, then used MRI to measure total and regional brain volume. We found evidence for both mosaic and concerted brain evolution in dragons: concerted brain evolution with respect to body size, and mosaic brain evolution with respect to ecomorph. Specifically, all brain subdivisions increase in volume relative to body size, yet the tectum and rhombencephalon also show opposite patterns of evolution with respect to ecomorph. Therefore, we find that both models of evolution are occurring simultaneously in the same structures in dragons, but are only detectable when examining particular drivers of selection. We show that the answer to the question of whether concerted or mosaic brain evolution is detected in a system can depend more on the type of selection measured than on the clade of animals studied.

Robust methods to create ex vivo minimum deformation atlases for brain mapping.

Highly detailed ex vivo 3D atlases of average structure are of critical importance to neuroscience and its current push to understanding the global microstructure of the brain. Multiple single slice histology sections can no longer provide sufficient detail of inter-slice microstructure and lack out of plane resolution. Two ex vivo methods have emerged that can create such detailed models. High-field micro MRI with the addition of contrast media has allowed intact whole brain microstructure imaging with an isotropic resolution of 15 µm in mouse. Blockface imaging has similarly evolved to a point where it is now possible to image an entire brain in a rigorous fashion with an out of plane resolution of 10 µm. Despite the destruction of the tissue as part of this process it allows a reconstructed model that is free from cutting artifacts. Both of these methods have been utilised to create minimum deformation atlases that are representative of the respective populations. The MDA atlases allow us unprecedented insight into the commonality and differences in microstructure in cortical structures in specific taxa. In this paper we provide an overview of how to create such MDA models from ex vivo data.

Brain tissue compartment density estimated using diffusion-weighted MRI yields tissue parameters consistent with histology.

We examined whether quantitative density measures of cerebral tissue consistent with histology can be obtained from diffusion magnetic resonance imaging (MRI). By incorporating prior knowledge of myelin and cell membrane densities, absolute tissue density values were estimated from relative intracellular and intraneurite density values obtained from diffusion MRI. The NODDI (neurite orientation distribution and density imaging) technique, which can be applied clinically, was used. Myelin density estimates were compared with the results of electron and light microscopy in ex vivo mouse brain and with published density estimates in a healthy human brain. In ex vivo mouse brain, estimated myelin densities in different subregions of the mouse corpus callosum were almost identical to values obtained from electron microscopy (diffusion MRI: 42 ± 6%, 36 ± 4%, and 43 ± 5%; electron microscopy: 41 ± 10%, 36 ± 8%, and 44 ± 12% in genu, body and splenium, respectively). In the human brain, good agreement was observed between estimated fiber density measurements and previously reported values based on electron microscopy. Estimated density values were unaffected by crossing fibers.

Visualization of mouse barrel cortex using ex-vivo track density imaging.

We describe the visualization of the barrel cortex of the primary somatosensory area (S1) of ex vivo adult mouse brain with short-tracks track density imaging (stTDI). stTDI produced much higher definition of barrel structures than conventional fractional anisotropy (FA), directionally-encoded color FA maps, spin-echo T1- and T2-weighted imaging and gradient echo T1/T2*-weighted imaging. 3D high angular resolution diffusion imaging (HARDI) data were acquired at 48 micron isotropic resolution for a (3mm)(3) block of cortex containing the barrel field and reconstructed using stTDI at 10 micron isotropic resolution. HARDI data were also acquired at 100 micron isotropic resolution to image the whole brain and reconstructed using stTDI at 20 micron isotropic resolution. The 10 micron resolution stTDI maps showed exceptionally clear delineation of barrel structures. Individual barrels could also be distinguished in the 20 micron stTDI maps but the septa separating the individual barrels appeared thicker compared to the 10 micron maps, indicating that the ability of stTDI to produce high quality structural delineation is dependent upon acquisition resolution. Close homology was observed between the barrel structure delineated using stTDI and reconstructed histological data from the same samples. stTDI also detects barrel deletions in the posterior medial barrel sub-field in mice with infraorbital nerve cuts. The results demonstrate that stTDI is a novel imaging technique that enables three-dimensional characterization of complex structures such as the barrels in S1 and provides an important complementary non-invasive imaging tool for studying synaptic connectivity, development and plasticity of the sensory system.

Zebrafish models of candidate human epilepsy-associated genes provide evidence of hyperexcitability.

Hundreds of novel candidate human epilepsy-associated genes have been identified thanks to advancements in next-generation sequencing and large genome-wide association studies, but establishing genetic etiology requires functional validation. We generated a list of >2200 candidate epilepsy-associated genes, of which 81 were determined suitable for the generation of loss-of-function zebrafish models via CRISPR/Cas9 gene editing. Of those 81 crispants, 48 were successfully established as stable mutant lines and assessed for seizure-like swim patterns in a primary F2 screen. Evidence of seizure-like behavior was present in 5 (arfgef1, kcnd2, kcnv1, ubr5, wnt8b) of the 48 mutant lines assessed. Further characterization of those 5 lines provided evidence for epileptiform activity via electrophysiology in kcnd2 and wnt8b mutants. Additionally, arfgef1 and wnt8b mutants showed a decrease in the number of inhibitory interneurons in the optic tectum of larval animals. Furthermore, RNAseq revealed convergent transcriptional abnormalities between mutant lines, consistent with their developmental defects and hyperexcitable phenotypes. These zebrafish models provide strongest experimental evidence supporting the role of ARFGEF1, KCND2, and WNT8B in human epilepsy and further demonstrate the utility of this model system for evaluating candidate human epilepsy genes.

A segmentation protocol and MRI atlas of the C57BL/6J mouse neocortex.

The neocortex is the largest component of the mammalian cerebral cortex. It integrates sensory inputs with experiences and memory to produce sophisticated responses to an organism's internal and external environment. While areal patterning of the mouse neocortex has been mapped using histological techniques, the neocortex has not been comprehensively segmented in magnetic resonance images. This study presents a method for systematic segmentation of the C57BL/6J mouse neocortex. We created a minimum deformation atlas, which was hierarchically segmented into 74 neocortical and cortical-related regions, making it the most detailed atlas of the mouse neocortex currently available. In addition, we provide mean volumes and relative intensities for each structure as well as a nomenclature comparison between the two most cited histological atlases of the mouse brain. This MR atlas is available for download, and it should enable researchers to perform automated segmentation in genetic models of cortical disorders.

A method for detecting molecular transport within the cerebral ventricles of live zebrafish (Danio rerio) larvae.

The production and flow of cerebrospinal fluid performs an important role in the development and homeostasis of the central nervous system.However, these processes are difficult to study in the mammalian brain because the ventricles are situated deep within the parenchyma.In this communication we introduce the zebrafish larva as an in vivo model for studying cerebral ventricle and blood­brain barrier function. Using confocal microscopy we show that zebrafish ventricles are topologically similar to those of the mammalian brain.We describe a new method for measuring the dynamics of molecular transport within the ventricles of live zebrafish by means of the uncaging of a fluorescein derivative. Furthermore, we determine that in 5­6 days post-fertilization zebrafish, the dispersal of molecules in the ventricles is driven by a combination of ciliary motion and diffusion. The zebrafish presents a tractable system with the advantage of genetics, size and transparency for exploring ventricular physiology and for mounting large-scale high throughput experiments.

Segmentation of the C57BL/6J mouse cerebellum in magnetic resonance images.

The C57BL mouse is the centerpiece of efforts to use gene-targeting technology to understand cerebellar pathology, thus creating a need for a detailed magnetic resonance imaging (MRI) atlas of the cerebellum of this strain. In this study we present a methodology for systematic delineation of the vermal and hemispheric lobules of the C57BL/6J mouse cerebellum in magnetic resonance images. We have successfully delineated 38 cerebellar and cerebellar-related structures. The higher signal-to-noise ratio achieved by group averaging facilitated the identification of anatomical structures. In addition, we have calculated average region volumes and created probabilistic maps for each structure. The segmentation method and the probabilistic maps we have created will provide a foundation for future studies of cerebellar disorders using transgenic mouse models.

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.

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 T2-weighted magnetic resonance histology (MRH) images acquired on a 16.4-T superconducting magnet. We achieved an isotropic resolution of 10 microm, 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 microm, 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.

Magnetic resonance histology of the adult zebrafish brain: optimization of fixation and gadolinium contrast enhancement.

Magnetic resonance histology (MRH) has become a widespread tool to examine brain morphology in situ or ex vivo. Samples are routinely fixed and stained to allow for longer scan times with increased contrast and resolution. Although the zebrafish is an important model for neuroscience, to date most MRH studies have focused almost exclusively on mice. In this paper, we examined, for the first time, the zebrafish brain using MRH. We compared a range of fixatives, contrast agents, and fixation/staining durations to determine optimal imaging of the zebrafish brain. By quantifying the T(1), T(2), and T(2)* relaxation values, we demonstrated that ethanol and potassium permanganate are unviable for imaging and significant differences exist between mono and di-aldehydes. Furthermore, we compared two commercially available gadolinium-based contrast agents, Magnevist® and Optimark®, at five different concentrations. For both contrast agents, a concentration of 0.5% was determined to be ideal as it significantly shortened the T(1) but maintained a relatively long T(2) and T(2)*. Subsequently, we analyzed the duration of fixation/staining and established a period of 12 h, which best minimized T(1) values but maintained T(2) and T(2)* values. Finally, using this optimized fixation and staining protocol, we performed a gradient-echo T(2)*-weighted imaging to obtain an image set of the adult zebrafish brain at an isotropic resolution of 10 µm.

Quantitative assessment of brain volumes in fish: comparison of methodologies.

When correlating brain areas with behavioral and environmental characteristics, a variety of techniques are employed. In fishes (elasmobranchs and teleosts), 2 methods, histology and the idealized ellipsoid and/or half-ellipsoid technique, are primarily used to calculate the volume of a brain area and therefore its relationship to social or ecological complexity. In this study on a perciform teleost, we have quantitatively compared brain volumes obtained using the conventional techniques of histology and approximating brain volume to an idealized ellipsoid (or half ellipsoid) and magnetic resonance imaging, an established clinical tool typically used for assessing brain volume in other vertebrates. Our results indicate that, when compared to brain volumes measured using magnetic resonance imaging of brain regions in situ, variations in brain shape and histological artifacts can lead to significant differences in brain volume, especially in the telencephalon and optic tecta. Consequently, in comparative studies of brain volumes, we advise caution when using the histological and/or ellipsoid methods to make correlations between brain area size and environmental, behavioral and social characteristics and, when possible, we propose the use of magnetic resonance imaging.

Precision Calcium Imaging of Dense Neural Populations via a Cell-Body-Targeted Calcium Indicator.

Methods for one-photon fluorescent imaging of calcium dynamics can capture the activity of hundreds of neurons across large fields of view at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk from neuropil, resulting in a decreased signal-to-noise ratio and artifactual correlations of neural activity. We address this problem by engineering cell-body-targeted variants of the fluorescent calcium indicators GCaMP6f and GCaMP7f. We screened fusions of GCaMP to natural, as well as artificial, peptides and identified fusions that localized GCaMP to within 50 µm of the cell body of neurons in mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP in dense neural circuits reported fewer artifactual spikes from neuropil, an increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators facilitates usage of simple, powerful, one-photon methods for imaging neural calcium dynamics.

An Open-Source Husbandry Repository.

Electronic databases provide effective and efficient management of zebrafish colony operations, but commercially available options are expensive. In this study we have developed a free zebrafish management repository alternative using free Google applications. Husbandry information is logged into a Google Sheets-based catalog through Google Form (GF) entries. Form autopopulation can be streamlined by barcodes, which can be generated and deciphered through free smartphone applications. The repository is capable of calculating pertinent husbandry dates from GF input and sending e-mail reminders to users for specified tasks. A Google application-based repository allows for a free simple zebrafish husbandry management solution.

Whole-Volume Clustering of Time Series Data from Zebrafish Brain Calcium Images via Mixture Modeling.

Calcium is a ubiquitous messenger in neural signaling events. An increasing number of techniques are enabling visualization of neurological activity in animal models via luminescent proteins that bind to calcium ions. These techniques generate large volumes of spatially correlated time series. A model-based functional data analysis methodology via Gaussian mixtures is suggested for the clustering of data from such visualizations is proposed. The methodology is theoretically justified and a computationally efficient approach to estimation is suggested. An example analysis of a zebrafish imaging experiment is presented.

A 3D MRI-based atlas of a lizard brain.

Magnetic resonance imaging (MRI) is an established technique for neuroanatomical analysis, being particularly useful in the medical sciences. However, the application of MRI to evolutionary neuroscience is still in its infancy. Few magnetic resonance brain atlases exist outside the standard model organisms in neuroscience and no magnetic resonance atlas has been produced for any reptile brain. A detailed understanding of reptilian brain anatomy is necessary to elucidate the evolutionary origin of enigmatic brain structures such as the cerebral cortex. Here, we present a magnetic resonance atlas for the brain of a representative squamate reptile, the Australian tawny dragon (Agamidae: Ctenophorus decresii), which has been the subject of numerous ecological and behavioral studies. We used a high-field 11.74T magnet, a paramagnetic contrasting-enhancing agent and minimum-deformation modeling of the brains of thirteen adult male individuals. From this, we created a high-resolution three-dimensional model of a lizard brain. The 3D-MRI model can be freely downloaded and allows a better comprehension of brain areas, nuclei, and fiber tracts, facilitating comparison with other species and setting the basis for future comparative evolution imaging studies. The MRI model and atlas of a tawny dragon brain (Ctenophorus decresii) can be viewed online and downloaded using the Wiley Biolucida Server at wiley.biolucida.net.

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.

Disease-modifying effect of intravenous immunoglobulin in an experimental model of epilepsy.

Novel therapies that prevent or modify the development of epilepsy following an initiating brain insult could significantly reduce the burden of this disease. In light of evidence that immune mechanisms play an important role in generating and maintaining the epileptic condition, we evaluated the effect of a well-established immunomodulatory treatment, intravenous immunoglobulin (IVIg), on the development of epilepsy in an experimental model of epileptogenesis. In separate experiments, IVIg was administered either before (pre-treatment) or after (post-treatment) the onset of pilocarpine status epilepticus (SE). Our results show that both pre- and post-treatment with IVIg attenuated acute inflammation in the SE model. Specifically, IVIg reduced local activation of glial cells, complement system activation, and blood-brain barrier damage (BBB), which are all thought to play important roles in the development of epilepsy. Importantly, post-treatment with IVIg was also found to reduce the frequency and duration of subsequent spontaneous recurrent seizures as detected by chronic video-electroencephalographic (video-EEG) recordings. This finding supports a novel application for IVIg, specifically its repurposing as a disease-modifying therapy in epilepsy.