Imaging Approaches to Investigate Pathophysiological Mechanisms of Brain Disease in Zebrafish.

Zebrafish has become an essential model organism in modern biomedical research. Owing to its distinctive features and high grade of genomic homology with humans, it is increasingly employed to model diverse neurological disorders, both through genetic and pharmacological intervention. The use of this vertebrate model has recently enhanced research efforts, both in the optical technology and in the bioengineering fields, aiming at developing novel tools for high spatiotemporal resolution imaging. Indeed, the ever-increasing use of imaging methods, often combined with fluorescent reporters or tags, enable a unique chance for translational neuroscience research at different levels, ranging from behavior (whole-organism) to functional aspects (whole-brain) and down to structural features (cellular and subcellular). In this work, we present a review of the imaging approaches employed to investigate pathophysiological mechanisms underlying functional, structural, and behavioral alterations of human neurological diseases modeled in zebrafish.

Multimodal Characterization of Seizures in Zebrafish Larvae.

Epilepsy accounts for a significant proportion of the world's disease burden. Indeed, many research efforts are produced both to investigate the basic mechanism ruling its genesis and to find more effective therapies. In this framework, the use of zebrafish larvae, owing to their peculiar features, offers a great opportunity. Here, we employ transgenic zebrafish larvae expressing GCaMP6s in all neurons to characterize functional alterations occurring during seizures induced by pentylenetetrazole. Using a custom two-photon light-sheet microscope, we perform fast volumetric functional imaging of the entire larval brain, investigating how different brain regions contribute to seizure onset and propagation. Moreover, employing a custom behavioral tracking system, we outline the progressive alteration of larval swim kinematics, resulting from different grades of seizures. Collectively, our results show that the epileptic larval brain undergoes transitions between diverse neuronal activity regimes. Moreover, we observe that different brain regions are progressively recruited into the generation of seizures of diverse severity. We demonstrate that midbrain regions exhibit highest susceptibility to the convulsant effects and that, during periods preceding abrupt hypersynchronous paroxysmal activity, they show a consistent increase in functional connectivity. These aspects, coupled with the hub-like role that these regions exert, represent important cues in their identification as epileptogenic hubs.

Fast whole-brain imaging of seizures in zebrafish larvae by two-photon light-sheet microscopy.

Light-sheet fluorescence microscopy (LSFM) enables real-time whole-brain functional imaging in zebrafish larvae. Conventional one-photon LSFM can however induce undesirable visual stimulation due to the use of visible excitation light. The use of two-photon (2P) excitation, employing near-infrared invisible light, provides unbiased investigation of neuronal circuit dynamics. However, due to the low efficiency of the 2P absorption process, the imaging speed of this technique is typically limited by the signal-to-noise-ratio. Here, we describe a 2P LSFM setup designed for non-invasive imaging that enables quintuplicating state-of-the-art volumetric acquisition rate of the larval zebrafish brain (5 Hz) while keeping low the laser intensity on the specimen. We applied our system to the study of pharmacologically-induced acute seizures, characterizing the spatial-temporal dynamics of pathological activity and describing for the first time the appearance of caudo-rostral ictal waves (CRIWs).

Power-effective scanning with AODs for 3D optogenetic applications.

Two-photon (2P) excitation is a cornerstone approach widely employed in neuroscience microscopy for deep optical access and sub-micrometric-resolution light targeting into the brain. However, besides structural and functional imaging, 2P optogenetic stimulations are less routinary, especially in 3D. This is because of the adopted scanning systems, often feebly effective, slow and mechanically constricted. Faster illumination can be achieved through acousto-optic deflectors (AODs) although their applicability to large volumes excitation has been limited by large efficiency drop along the optical axis. Here, we present a new AOD-based scheme for 2P 3D scanning that improves the power delivery between different illumination planes. We applied this approach to photostimulate an optogenetic actuator in zebrafish larvae, demonstrating the method efficiency observing increased activity responses and uniform activation probabilities from neuronal clusters addressed in the volume. This novel driving scheme can open to new AOD applications in neuroscience, allowing more effective 3D interrogation in large neuronal networks.

Reconstruction scheme for excitatory and inhibitory dynamics with quenched disorder: application to zebrafish imaging.

An inverse procedure is developed and tested to recover functional and structural information from global signals of brains activity. The method assumes a leaky-integrate and fire model with excitatory and inhibitory neurons, coupled via a directed network. Neurons are endowed with a heterogenous current value, which sets their associated dynamical regime. By making use of a heterogenous mean-field approximation, the method seeks to reconstructing from global activity patterns the distribution of in-coming degrees, for both excitatory and inhibitory neurons, as well as the distribution of the assigned currents. The proposed inverse scheme is first validated against synthetic data. Then, time-lapse acquisitions of a zebrafish larva recorded with a two-photon light sheet microscope are used as an input to the reconstruction algorithm. A power law distribution of the in-coming connectivity of the excitatory neurons is found. Local degree distributions are also computed by segmenting the whole brain in sub-regions traced from annotated atlas.

Efficient clofilium tosylate-mediated rescue of POLG-related disease phenotypes in zebrafish.

The DNA polymerase gamma (Polg) is a nuclear-encoded enzyme involved in DNA replication in animal mitochondria. In humans, mutations in the POLG gene underlie a set of mitochondrial diseases characterized by mitochondrial DNA (mtDNA) depletion or deletion and multiorgan defects, named POLG disorders, for which an effective therapy is still needed. By applying antisense strategies, ENU- and CRISPR/Cas9-based mutagenesis, we have generated embryonic, larval-lethal and adult-viable zebrafish Polg models. Morphological and functional characterizations detected a set of phenotypes remarkably associated to POLG disorders, including cardiac, skeletal muscle, hepatic and gonadal defects, as well as mitochondrial dysfunctions and, notably, a perturbed mitochondria-to-nucleus retrograde signaling (CREB and Hypoxia pathways). Next, taking advantage of preliminary evidence on the candidate molecule Clofilium tosylate (CLO), we tested CLO toxicity and then its efficacy in our zebrafish lines. Interestingly, at well tolerated doses, the CLO drug could successfully rescue mtDNA and Complex I respiratory activity to normal levels, even in mutant phenotypes worsened by treatment with Ethidium Bromide. In addition, the CLO drug could efficiently restore cardio-skeletal parameters and mitochondrial mass back to normal values. Altogether, these evidences point to zebrafish as a valuable vertebrate organism to faithfully phenocopy multiple defects detected in POLG patients. Moreover, this model represents an excellent platform to screen, at the whole-animal level, candidate molecules with therapeutic effects in POLG disorders.

Colored visual stimuli evoke spectrally tuned neuronal responses across the central nervous system of zebrafish larvae.

BACKGROUND: Visually guided behaviors such as optomotor and optokinetic responses, phototaxis, and prey capture are crucial for survival in zebrafish and become apparent after just a few days of development. Color vision, which in zebrafish is based on a spatially anisotropic tetrachromatic retina, provides an additional important component of world representation driving fundamental larval behaviors. However, little is known about the central nervous system (CNS) circuitry underlying color vision processing downstream of the retina, and its activity correlates with behavior. Here, we used the transparent larva of zebrafish to image CNS neurons and their activity in response to colored visual stimuli. RESULTS: To investigate the processing of chromatic information in the zebrafish larva brain, we mapped with cellular resolution, spectrally responsive neurons in the larva encephalon and spinal cord. We employed the genetically encoded calcium indicator GCaMP6s and two-photon microscopy to image the neuronal activity while performing visual stimulation with spectrally distinct stimuli at wavelengths matching the absorption peaks of the four zebrafish cone types. We observed the presence of a high number of wavelength-selective neurons not only in the optic tectum, but also in all other regions of the CNS, demonstrating that the circuitry involved in processing spectral information and producing color-selective responses extends to the whole CNS. CONCLUSIONS: Our measurements provide a map of neurons involved in color-driven responses, revealing that spectral information spreads in all regions of the CNS. This suggests the underlying complexity of the circuits involved and opens the way to their detailed future investigation.

Effects of excitation light polarization on fluorescence emission in two-photon light-sheet microscopy.

Light-sheet microscopy (LSM) is a powerful imaging technique that uses a planar illumination oriented orthogonally to the detection axis. Two-photon (2P) LSM is a variant of LSM that exploits the 2P absorption effect for sample excitation. The light polarization state plays a significant, and often overlooked, role in 2P absorption processes. The scope of this work is to test whether using different polarization states for excitation light can affect the detected signal levels in 2P LSM imaging of typical biological samples with a spatially unordered dye population. Supported by a theoretical model, we compared the fluorescence signals obtained using different polarization states with various fluorophores (fluorescein, EGFP and GCaMP6s) and different samples (liquid solution and fixed or living zebrafish larvae). In all conditions, in agreement with our theoretical expectations, linear polarization oriented parallel to the detection plane provided the largest signal levels, while perpendicularly-oriented polarization gave low fluorescence signal with the biological samples, but a large signal for the fluorescein solution. Finally, circular polarization generally provided lower signal levels. These results highlight the importance of controlling the light polarization state in 2P LSM of biological samples. Furthermore, this characterization represents a useful guide to choose the best light polarization state when maximization of signal levels is needed, e.g. in high-speed 2P LSM.

Dual-beam confocal light-sheet microscopy via flexible acousto-optic deflector.

Confocal detection in digital scanned laser light-sheet fluorescence microscopy (DSLM) has been established as a gold standard method to improve image quality. The selective line detection of a complementary metal­oxide­semiconductor camera (CMOS) working in rolling shutter mode allows the rejection of out-of-focus and scattered light, thus reducing background signal during image formation. Most modern CMOS have two rolling shutters, but usually only a single illuminating beam is used, halving the maximum obtainable frame rate. We report on the capability to recover the full image acquisition rate via dual confocal DSLM by using an acousto-optic deflector. Such a simple solution enables us to independently generate, control and synchronize two beams with the two rolling slits on the camera. We show that the doubling of the imaging speed does not affect the confocal detection high contrast.

In-vivo and ex-vivo optical clearing methods for biological tissues: review.

Every optical imaging technique is limited in its penetration depth by scattering occurring in biological tissues. Possible solutions to overcome this problem consist of limiting the detrimental effects of scattering by reducing optical inhomogeneities within the sample. This can be achieved either by using physical methods (such as refractive index matching solutions) or by chemical methods (such as the removal of scatterers), based on tissue transformation protocols. This review provides an overview of the current state-of-the-art methods used for both ex-vivo and in-vivo optical clearing of biological tissues. We start with a brief history of the development of the most widespread clearing methods across the new millennium, then we describe the working principles of both physical and chemical methods. Clearing methods are then reviewed, pointing the attention of the reader on both physical and chemical methods, classified based on the tissue size and type for each specific application. A small section is reserved for methods that have already found in-vivo applications at the research level. Finally, a detailed discussion highlighting both the most relevant results achieved and the new ongoing developments in this field is reported in the last part, together with future perspectives for the clearing methodology.

Corrigendum: Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy.

[This corrects the article DOI: 10.3389/fncel.2018.00315.].

Flexible Multi-Beam Light-Sheet Fluorescence Microscope for Live Imaging Without Striping Artifacts.

The development of light-sheet fluorescence microscopy (LSFM) has greatly expanded the experimental capabilities in many biological and biomedical research fields, enabling for example live studies of murine and zebrafish neural activity or of cell growth and division. The key feature of the method is the selective illumination of a sample single plane, providing an intrinsic optical sectioning and allowing direct 2D image recording. On the other hand, this excitation scheme is more affected by absorption or scattering artifacts in comparison to point scanning methods, leading to un-even illumination. We present here an easily implementable method, based on acousto-optical deflectors (AOD), to overcome this obstacle. We report the advantages provided by flexible and fast AODs in generating simultaneous angled multiple beams from a single laser beam and in fast light sheet pivoting and we demonstrate the suppression of illumination artifacts.

Bessel Beam Illumination Reduces Random and Systematic Errors in Quantitative Functional Studies Using Light-Sheet Microscopy.

Light-sheet microscopy (LSM), in combination with intrinsically transparent zebrafish larvae, is a method of choice to observe brain function with high frame rates at cellular resolution. Inherently to LSM, however, residual opaque objects cause stripe artifacts, which obscure features of interest and, during functional imaging, modulate fluorescence variations related to neuronal activity. Here, we report how Bessel beams reduce streaking artifacts and produce high-fidelity quantitative data demonstrating a fivefold increase in sensitivity to calcium transients and a 20-fold increase in accuracy in the detection of activity correlations in functional imaging. Furthermore, using principal component analysis, we show that measurements obtained with Bessel beams are clean enough to reveal in one-shot experiments correlations that can not be averaged over trials after stimuli as is the case when studying spontaneous activity. Our results not only demonstrate the contamination of data by systematic and random errors through conventional Gaussian illumination and but,furthermore, quantify the increase in fidelity of such data when using Bessel beams.

Sliding of a single lac repressor protein along DNA is tuned by DNA sequence and molecular switching.

In any living cell, genome maintenance is carried out by DNA-binding proteins that recognize specific sequences among a vast amount of DNA. This includes fundamental processes such as DNA replication, DNA repair, and gene expression and regulation. Here, we study the mechanism of DNA target search by a single lac repressor protein (LacI) with ultrafast force-clamp spectroscopy, a sub-millisecond and few base-pair resolution technique based on laser tweezers. We measure 1D-diffusion of proteins on DNA at physiological salt concentrations with 20 bp resolution and find that sliding of LacI along DNA is sequence dependent. We show that only allosterically activated LacI slides along non-specific DNA sequences during target search, whereas the inhibited conformation does not support sliding and weakly interacts with DNA. Moreover, we find that LacI undergoes a load-dependent conformational change when it switches between sliding and strong binding to the target sequence. Our data reveal how DNA sequence and molecular switching regulate LacI target search process and provide a comprehensive model of facilitated diffusion for LacI.

Loss of cardiac Wnt/ß-catenin signalling in desmoplakin-deficient AC8 zebrafish models is rescuable by genetic and pharmacological intervention.

Aims: Arrhythmogenic cardiomyopathy (AC) is an inherited heart disease characterized by life-threatening ventricular arrhythmias and fibro-fatty replacement of the myocardium. More than 60% of AC patients show pathogenic mutations in genes encoding for desmosomal proteins. By focusing our attention on the AC8 form, linked to the junctional protein desmoplakin (DSP), we present here a zebrafish model of DSP deficiency, exploited to identify early changes of cell signalling in the cardiac region. Methods and results: To obtain an embryonic model of Dsp deficiency, we first confirmed the orthologous correspondence of zebrafish Dsp genes (dspa and dspb) to the human DSP counterpart. Then, we verified their cardiac expression, at embryonic and adult stages, and subsequently we targeted them by antisense morpholino strategy, confirming specific and disruptive effects on desmosomes, like those identified in AC patients. Finally, we exploited our Dsp-deficient models for an in vivo cell signalling screen, using pathway-specific reporter transgenes. Out of nine considered, three pathways (Wnt/ß-catenin, TGFß/Smad3, and Hippo/YAP-TAZ) were significantly altered, with Wnt as the most dramatically affected. Interestingly, under persistent Dsp deficiency, Wnt signalling is rescuable both by a genetic and a pharmacological approach. Conclusion: Our data point to Wnt/ß-catenin as the final common pathway underlying different desmosomal AC forms and support the zebrafish as a suitable model for detecting early signalling pathways involved in the pathogenesis of DSP-associated diseases, possibly responsive to pharmacological or genetic rescue.

A versatile clearing agent for multi-modal brain imaging.

Extensive mapping of neuronal connections in the central nervous system requires high-throughput µm-scale imaging of large volumes. In recent years, different approaches have been developed to overcome the limitations due to tissue light scattering. These methods are generally developed to improve the performance of a specific imaging modality, thus limiting comprehensive neuroanatomical exploration by multi-modal optical techniques. Here, we introduce a versatile brain clearing agent (2,2'-thiodiethanol; TDE) suitable for various applications and imaging techniques. TDE is cost-efficient, water-soluble and low-viscous and, more importantly, it preserves fluorescence, is compatible with immunostaining and does not cause deformations at sub-cellular level. We demonstrate the effectiveness of this method in different applications: in fixed samples by imaging a whole mouse hippocampus with serial two-photon tomography; in combination with CLARITY by reconstructing an entire mouse brain with light sheet microscopy and in translational research by imaging immunostained human dysplastic brain tissue.

Combining single-molecule manipulation and imaging for the study of protein-DNA interactions.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Decoupling polarization of the Golgi apparatus and GM1 in the plasma membrane.

Cell polarization is a process of coordinated cellular rearrangements that prepare the cell for migration. GM1 is synthesized in the Golgi apparatus and localized in membrane microdomains that appear at the leading edge of polarized cells, but the mechanism by which GM1 accumulates asymmetrically is unknown. The Golgi apparatus itself becomes oriented toward the leading edge during cell polarization, which is thought to contribute to plasma membrane asymmetry. Using quantitative image analysis techniques, we measure the extent of polarization of the Golgi apparatus and GM1 in the plasma membrane simultaneously in individual cells subject to a wound assay. We find that GM1 polarization starts just 10 min after stimulation with growth factors, while Golgi apparatus polarization takes 30 min. Drugs that block Golgi polarization or function have no effect on GM1 polarization, and, conversely, inhibiting GM1 polarization does not affect Golgi apparatus polarization. Evaluation of Golgi apparatus and GM1 polarization in single cells reveals no correlation between the two events. Our results indicate that Golgi apparatus and GM1 polarization are controlled by distinct intracellular cascades involving the Ras/Raf/MEK/ERK and the PI3K/Akt/mTOR pathways, respectively. Analysis of cell migration and invasion suggest that MEK/ERK activation is crucial for two dimensional migration, while PI3K activation drives three dimensional invasion, and no cumulative effect is observed from blocking both simultaneously. The independent biochemical control of GM1 polarity by PI3K and Golgi apparatus polarity by MEK/ERK may act synergistically to regulate and reinforce directional selection in cell migration.

Optical Methods to Study Protein-DNA Interactions in Vitro and in Living Cells at the Single-Molecule Level.

The maintenance of intact genetic information, as well as the deployment of transcription for specific sets of genes, critically rely on a family of proteins interacting with DNA and recognizing specific sequences or features. The mechanisms by which these proteins search for target DNA are the subject of intense investigations employing a variety of methods in biology. A large interest in these processes stems from the faster-than-diffusion association rates, explained in current models by a combination of 3D and 1D diffusion. Here, we present a review of the single-molecule approaches at the forefront of the study of protein-DNA interaction dynamics and target search in vitro and in vivo. Flow stretch, optical and magnetic manipulation, single fluorophore detection and localization as well as combinations of different methods are described and the results obtained with these techniques are discussed in the framework of the current facilitated diffusion model.

Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke.

We describe a dual-trap force-clamp configuration that applies constant loads between a binding protein and an intermittently interacting biological polymer. The method has a measurement delay of only ∼10 µs, allows detection of interactions as brief as ∼100 µs and probes sub-nanometer conformational changes with a time resolution of tens of microseconds. We tested our method on molecular motors and DNA-binding proteins. We could apply constant loads to a single motor domain of myosin before its working stroke was initiated (0.2-1 ms), thus directly measuring its load dependence. We found that, depending on the applied load, myosin weakly interacted (<1 ms) with actin without production of movement, fully developed its working stroke or prematurely detached (<5 ms), thus reducing the working stroke size with load. Our technique extends single-molecule force-clamp spectroscopy and opens new avenues for investigating the effects of forces on biological processes.