Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.     

Long-Term Live Cell Imaging and Automated 4D Analysis of Drosophila Neuroblast Lineages

The developing Drosophila brain is a well-studied model system for neurogenesis and stem cell biology. In the Drosophila central brain, around 200 neural stem cells called neuroblasts undergo repeated rounds of asymmetric cell division. These divisions typically generate a larger self-renewing neuroblast and a smaller ganglion mother cell that undergoes one terminal division to create two differentiating neurons. Although single mitotic divisions of neuroblasts can easily be imaged in real time, the lack of long term imaging procedures has limited the use of neuroblast live imaging for lineage analysis. Here we describe a method that allows live imaging of cultured Drosophila neuroblasts over multiple cell cycles for up to 24 hours. We describe a 4D image analysis protocol that can be used to extract cell cycle times and growth rates from the resulting movies in an automated manner. We use it to perform lineage analysis in type II neuroblasts where clonal analysis has indicated the presence of a transit-amplifying population that potentiates the number of neurons. Indeed, our experiments verify type II lineages and provide quantitative parameters for all cell types in those lineages. As defects in type II neuroblast lineages can result in brain tumor formation, our lineage analysis method will allow more detailed and quantitative analysis of tumorigenesis and asymmetric cell division in the Drosophila brain.     

Localizing Protein in 3D Neural Stem Cell Culture: a Hybrid Visualization Methodology

The importance of 3-dimensional (3D) topography in influencing neural stem and progenitor cell (NPC) phenotype is widely acknowledged yet challenging to study. When dissociated from embryonic or post-natal brain, single NPCs will proliferate in suspension to form neurospheres. Daughter cells within these cultures spontaneously adopt distinct developmental lineages (neurons, oligodendrocytes, and astrocytes) over the course of expansion despite being exposed to the same extracellular milieu. This progression recapitulates many of the stages observed over the course of neurogenesis and gliogenesis in post-natal brain and is often used to study basic NPC biology within a controlled environment. Assessing the full impact of 3D topography and cellular positioning within these cultures on NPC fate is, however, difficult. To localize target proteins and identify NPC lineages by immunocytochemistry, free-floating neurospheres must be plated on a substrate or serially sectioned. This processing is required to ensure equivalent cell permeabilization and antibody access throughout the sphere. As a result, 2D epifluorescent images of cryosections or confocal reconstructions of 3D Z-stacks can only provide spatial information about cell position within discrete physical or digital 3D slices and do not visualize cellular position in the intact sphere. Here, to reiterate the topography of the neurosphere culture and permit spatial analysis of protein expression throughout the entire culture, we present a protocol for isolation, expansion, and serial sectioning of post-natal hippocampal neurospheres suitable for epifluorescent or confocal immunodetection of target proteins. Connexin29 (Cx29) is analyzed as an example. Next, using a hybrid of graphic editing and 3D modelling softwares rigorously applied to maintain biological detail, we describe how to re-assemble the 3D structural positioning of these images and digitally map labelled cells within the complete neurosphere. This methodology enables visualization and analysis of the cellular position of target proteins and cells throughout the entire 3D culture topography and will facilitate a more detailed analysis of the spatial relationships between cells over the course of neurogenesis and gliogenesis in vitro.Both Imbeault and Valenzuela contributed equally and should be considered joint first authors.     

Light Scattering Properties Vary across Different Regions of the Adult Mouse Brain

Recently developed optogenetic tools provide powerful approaches to optically excite or inhibit neural activity. In a typical in-vivo experiment, light is delivered to deep nuclei via an implanted optical fiber. Light intensity attenuates with increasing distance from the fiber tip, determining the volume of tissue in which optogenetic proteins can successfully be activated. However, whether and how this volume of effective light intensity varies as a function of brain region or wavelength has not been systematically studied. The goal of this study was to measure and compare how light scatters in different areas of the mouse brain. We delivered different wavelengths of light via optical fibers to acute slices of mouse brainstem, midbrain and forebrain tissue. We measured light intensity as a function of distance from the fiber tip, and used the data to model the spread of light in specific regions of the mouse brain. We found substantial differences in effective attenuation coefficients among different brain areas, which lead to substantial differences in light intensity demands for optogenetic experiments. The use of light of different wavelengths additionally changes how light illuminates a given brain area. We created a brain atlas of effective attenuation coefficients of the adult mouse brain, and integrated our data into an application that can be used to estimate light scattering as well as required light intensity for optogenetic manipulation within a given volume of tissue.     

Mammalian neurogenesis requires Treacle-Plk1 for precise control of spindle orientation, mitotic progression, and maintenance of neural progenitor cells

The cerebral cortex is a specialized region of the brain that processes cognitive, motor, somatosensory, auditory, and visual functions. Its characteristic architecture and size is dependent upon the number of neurons generated during embryogenesis and has been postulated to be governed by symmetric versus asymmetric cell divisions, which mediate the balance between progenitor cell maintenance and neuron differentiation, respectively. The mechanistic importance of spindle orientation remains controversial, hence there is considerable interest in understanding how neural progenitor cell mitosis is controlled during neurogenesis. We discovered that Treacle, which is encoded by the Tcof1 gene, is a novel centrosome- and kinetochore-associated protein that is critical for spindle fidelity and mitotic progression. Tcof1/Treacle loss-of-function disrupts spindle orientation and cell cycle progression, which perturbs the maintenance, proliferation, and localization of neural progenitors during cortical neurogenesis. Consistent with this, Tcof1(+/-) mice exhibit reduced brain size as a consequence of defects in neural progenitor maintenance. We determined that Treacle elicits its effect via a direct interaction with Polo-like kinase1 (Plk1), and furthermore we discovered novel in vivo roles for Plk1 in governing mitotic progression and spindle orientation in the developing mammalian cortex. Increased asymmetric cell division, however, did not promote increased neuronal differentiation. Collectively our research has therefore identified Treacle and Plk1 as novel in vivo regulators of spindle fidelity, mitotic progression, and proliferation in the maintenance and localization of neural progenitor cells. Together, Treacle and Plk1 are critically required for proper cortical neurogenesis, which has important implications in the regulation of mammalian brain size and the pathogenesis of congenital neurodevelopmental disorders such as microcephaly.     

Neural progenitor diversity and their therapeutic potential for spinal cord repair

Development of the central nervous system (CNS) requires progressive differentiation of neural stem cells, which generate a variety of neural progenitors with distinct properties and differentiation potentials in a spatiotemporally restricted manner. The underlying mechanisms of neural progenitor diversification during development started to be unraveled over the past years. We have addressed these questions by v-myc immortalization method and generation of neural progenitor clones. These clones are served as in vitro models of neural differentiation and cellular tools for transplantation in animal models of neurological disorders including spinal cord injury. In this review, we will discuss features of two neural progenitor types (radial glia and GABAergic interneuron progenitor) and diversification even within each progenitor type. We will also discuss pathophysiology of spinal cord injury and our ongoing research to address both motor and sensory malfunctions by transplantation of these neural progenitors.     

Ex vivo time-lapse confocal imaging of the mouse embryo aorta

Time-lapse confocal microscopy of mouse embryo slices was developed to access and image the living aorta. In this paper, we explain how to label all hematopoietic and endothelial cells inside the intact mouse aorta with fluorescent directly labeled antibodies. Then we describe the technique to cut nonfixed labeled embryos into thick slices that are further imaged by time-lapse confocal imaging. This approach allows direct observation of the dynamic cell behavior in the living aorta, which was previously inaccessible because of its location deep inside the opaque mouse embryo. In particular, this approach is sensitive enough to allow the experimenter to witness the transition from endothelial cells into hematopoietic stem/progenitor cells in the aorta, the first site of hematopoietic stem cell generation during development. The protocol can be applied to observe other embryonic sites throughout mouse development. A complete experiment requires ～2 d of practical work.     

Modeling Neural Immune Signaling of Episodic and Chronic Migraine Using Spreading Depression In Vitro

Migraine and its transformation to chronic migraine are healthcare burdens in need of improved treatment options. We seek to define how neural immune signaling modulates the susceptibility to migraine, modeled in vitro using spreading depression (SD), as a means to develop novel therapeutic targets for episodic and chronic migraine. SD is the likely cause of migraine aura and migraine pain. It is a paroxysmal loss of neuronal function triggered by initially increased neuronal activity, which slowly propagates within susceptible brain regions. Normal brain function is exquisitely sensitive to, and relies on, coincident low-level immune signaling. Thus, neural immune signaling likely affects electrical activity of SD, and therefore migraine. Pain perception studies of SD in whole animals are fraught with difficulties, but whole animals are well suited to examine systems biology aspects of migraine since SD activates trigeminal nociceptive pathways. However, whole animal studies alone cannot be used to decipher the cellular and neural circuit mechanisms of SD. Instead, in vitro preparations where environmental conditions can be controlled are necessary. Here, it is important to recognize limitations of acute slices and distinct advantages of hippocampal slice cultures. Acute brain slices cannot reveal subtle changes in immune signaling since preparing the slices alone triggers: pro-inflammatory changes that last days, epileptiform behavior due to high levels of oxygen tension needed to vitalize the slices, and irreversible cell injury at anoxic slice centers. In contrast, we examine immune signaling in mature hippocampal slice cultures since the cultures closely parallel their in vivo counterpart with mature trisynaptic function; show quiescent astrocytes, microglia, and cytokine levels; and SD is easily induced in an unanesthetized preparation. Furthermore, the slices are long-lived and SD can be induced on consecutive days without injury, making this preparation the sole means to-date capable of modeling the neuroimmune consequences of chronic SD, and thus perhaps chronic migraine. We use electrophysiological techniques and non-invasive imaging to measure neuronal cell and circuit functions coincident with SD. Neural immune gene expression variables are measured with qPCR screening, qPCR arrays, and, importantly, use of cDNA preamplification for detection of ultra-low level targets such as interferon-gamma using whole, regional, or specific cell enhanced (via laser dissection microscopy) sampling. Cytokine cascade signaling is further assessed with multiplexed phosphoprotein related targets with gene expression and phosphoprotein changes confirmed via cell-specific immunostaining. Pharmacological and siRNA strategies are used to mimic and modulate SD immune signaling.     

Refining the Ciona intestinalis Model of Central Nervous System Regeneration

Background

New, practical models of central nervous system regeneration are required and should provide molecular tools and resources. We focus here on the tunicate Ciona intestinalis, which has the capacity to regenerate nerves and a complete adult central nervous system, a capacity unusual in the chordate phylum. We investigated the timing and sequence of events during nervous system regeneration in this organism.

Methodology/Principal Findings

We developed techniques for reproducible ablations and for imaging live cellular events in tissue explants. Based on live observations of more than 100 regenerating animals, we subdivided the regeneration process into four stages. Regeneration was functional, as shown by the sequential recovery of reflexes that established new criteria for defining regeneration rates. We used transgenic animals and labeled nucleotide analogs to describe in detail the early cellular events at the tip of the regenerating nerves and the first appearance of the new adult ganglion anlage.

Conclusions/Significance

The rate of regeneration was found to be negatively correlated with adult size. New neural structures were derived from the anterior and posterior nerve endings. A blastemal structure was implicated in the formation of new neural cells. This work demonstrates that Ciona intestinalis is as a useful system for studies on regeneration of the brain, brain-associated organs and nerves.     

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation^{1, 2}, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.This assay may be modified to image a range of embryonic tissues^{3, 4} In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling⁵) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.     

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.     

CNS Targets Support and Sustain Differentiation of Cultured Neuronal and Retinal Progenitor Cells

Superior colliculus (SC) is the target of retinal neurons, allowing them to form connections. Cultured stem cells/progenitors can potentially be used as donor tissue to reconstruct degenerated retina including perhaps replacing lost ganglion cells in glaucoma. In which case, it will be essential for these cells to integrate with the central nervous system targets. Here, we have investigated if the mid-brain region containing superior colliculus (SC) provides a permissive environment for the survival and differentiation of neural progenitors, including retinal progenitor cells propagated in cultures. Neural (NPCs) and retinal progenitor cells (RPCs) from green fluorescent protein (GFP) transgenic mice were cultured. Passage two through four neural and retinal progenitor cells were subsequently cocultured with the SC organotypic slices and maintained in culture for 17 and eight days respectively. Differentiation of the neurons was studied by immunocytochemistry for retinotypic neuronal markers. Retinal progenitor cells cocultured with SC slices were able to differentiate into various neuronal morphologies. Some cocultured progenitor cells differentiated into neurons as suggested by class III β tubulin immunoreactivity. In addition, specific retinotypic neuronal differentiation of RPC was detected by immunoreactivity for calbindin and PKC. SC provides a permissive environment that supports survival and differentiation of the progenitor cells.     

Centrosome defects cause microcephaly by activating the 53BP1‐USP28‐TP53 mitotic surveillance pathway

Mutations in centrosome genes deplete neural progenitor cells (NPCs) during brain development, causing microcephaly. While NPC attrition is linked to TP53‐mediated cell death in several microcephaly models, how TP53 is activated remains unclear. In cultured cells, mitotic delays resulting from centrosome loss prevent the growth of unfit daughter cells by activating a pathway involving 53BP1, USP28, and TP53, termed the mitotic surveillance pathway. Whether this pathway is active in the developing brain is unknown. Here, we show that the depletion of centrosome proteins in NPCs prolongs mitosis and increases TP53‐mediated apoptosis. Cell death after a delayed mitosis was rescued by inactivation of the mitotic surveillance pathway. Moreover, 53BP1 or USP28 deletion restored NPC proliferation and brain size without correcting the upstream centrosome defects or extended mitosis. By contrast, microcephaly caused by the loss of the non‐centrosomal protein SMC5 is also TP53‐dependent but is not rescued by loss of 53BP1 or USP28. Thus, we propose that mutations in centrosome genes cause microcephaly by delaying mitosis and pathologically activating the mitotic surveillance pathway in the developing brain.     

The zebrafish brain in research and teaching: a simple in vivo and in vitro model for the study of spontaneous neural activity

Recently, the zebrafish (Danio rerio) has been established as a key animal model in neuroscience. Behavioral, genetic, and immunohistochemical techniques have been used to describe the connectivity of diverse neural circuits. However, few studies have used zebrafish to understand the function of cerebral structures or to study neural circuits. Information about the techniques used to obtain a workable preparation is not readily available. Here, we describe a complete protocol for obtaining in vitro and in vivo zebrafish brain preparations. In addition, we performed extracellular recordings in the whole brain, brain slices, and immobilized nonanesthetized larval zebrafish to evaluate the viability of the tissue. Each type of preparation can be used to detect spontaneous activity, to determine patterns of activity in specific brain areas with unknown functions, or to assess the functional roles of different neuronal groups during brain development in zebrafish. The technique described offers a guide that will provide innovative and broad opportunities to beginner students and researchers who are interested in the functional analysis of neuronal activity, plasticity, and neural development in the zebrafish brain.     

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function^1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.Download video file.^{(49M, mov)}     

Improved biocytin labeling and neuronal 3D reconstruction

In this report, we describe a reliable protocol for biocytin labeling of neuronal tissue and diaminobenzidine (DAB)-based processing of brain slices. We describe how to embed tissues in different media and how to subsequently histochemically label the tissues for light or electron microscopic examination. We provide a detailed dehydration and embedding protocol using Eukitt that avoids the common problem of tissue distortion and therefore prevents fading of cytoarchitectural features (in particular, lamination) of brain tissue; as a result, additional labeling methods (such as cytochrome oxidase staining) become unnecessary. In addition, we provide correction factors for tissue shrinkage in all spatial dimensions so that a realistic neuronal morphology can be obtained from slice preparations. Such corrections were hitherto difficult to calculate because embedding in viscous media resulted in highly nonlinear tissue deformation. Fixation, immunocytochemistry and embedding procedures for light microscopy (LM) can be completed within 42-48 h. Subsequent reconstructions and morphological analyses take an additional 24 h or more.     

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution^1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described^4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm¹, (2) a high survival rate of > 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days² and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter¹ in stable transgenic lines and the exon trap line FasII-GFP¹. (4) In contrast to other methods^4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days¹. The accompanying video details the function of individual parts of the in vivo imaging chamber^2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.