Larval zebrafish as a model for studying individual variability in translational neuroscience research

The larval zebrafish is a popular model for translational research into neurological and psychiatric disorders due to its conserved vertebrate brain structures, ease of genetic and experimental manipulation and small size and scalability to large numbers. The possibility of obtaining in vivo whole-brain cellular resolution neural data is contributing important advances into our understanding of neural circuit function and their relation to behavior. Here we argue that the larval zebrafish is ideally poised to push our understanding of how neural circuit function relates to behavior to the next level by including considerations of individual differences. Understanding variability across individuals is particularly relevant for tackling the variable presentations that neuropsychiatric conditions frequently show, and it is equally elemental if we are to achieve personalized medicine in the future. We provide a blueprint for investigating variability by covering examples from humans and other model organisms as well as existing examples from larval zebrafish. We highlight recent studies where variability may be hiding in plain sight and suggest how future studies can take advantage of existing paradigms for further exploring individual variability. We conclude with an outlook on how the field can harness the unique strengths of the zebrafish model to advance this important impending translational question

Glucocorticoid effects on the brain:from adaptive developmental plasticity to allostatic overload

Exposure to stress during early life may alter the developmental trajectory of an animal by a mechanism known as adaptive plasticity. For example, to enhance reproductive success in an adverse environment, it is known that animals accelerate their growth during development. However, these short-term fitness benefits are often associated with reduced longevity, a phenomenon known as the growth rate–lifespan trade-off. In humans, early life stress exposure compromises health later in life and increases disease susceptibility. Glucocorticoids (GCs) are major stress hormones implicated in these processes. This Review discusses the evidence for GC-mediated adaptive plasticity in development, leading to allostatic overload in later life. We focus on GC-induced effects on brain structure and function, including neurogenesis; highlight the need for longitudinal studies; and discuss approaches to identify molecular mechanisms mediating GC-induced alteration of the brain developmental trajectory leading to adult dysfunctions. Further understanding of how stress and GC exposure can alter developmental trajectories at the molecular and cellular level is of critical importance to reduce the burden of mental and physical ill health across the life course

Understanding and predicting antidepressant response : using animal models to move toward precision psychiatry

There are two important gaps of knowledge in depression treatment, namely the lack of biomarkers predicting response to antidepressants and the limited knowledge of the molecular mechanisms underlying clinical improvement. However, individually tailored treatment strategies and individualized prescription are greatly needed given the huge socio-economic burden of depression, the latency until clinical improvement can be observed and the response variability to a particular compound. Still, individual patient-level antidepressant treatment outcomes are highly unpredictable. In contrast to other therapeutic areas and despite tremendous efforts during the past years, the genomics era so far has failed to provide biological or genetic predictors of clinical utility for routine use in depression treatment. Specifically, we suggest to 1) shift the focus from the group patterns to individual outcomes, 2) use dimensional classifications such as Research Domain Criteria, 3) envision better planning and improved connections between pre-clinical and clinical studies within translational research units. In contrast to studies in patients, animal models enable both searches for peripheral biosignatures predicting treatment response and in depth analyses of the neurobiological pathways shaping individual antidepressant response in the brain. While there is a considerable number of animal models available aiming at mimicking disease-like conditions such as those seen in depressive disorder, only a limited number of preclinical or truly translational investigations is dedicated to the issue of heterogeneity seen in response to antidepressant treatment. In this mini-review, we provide an overview on the current state of knowledge and propose a framework for successful translational studies into antidepressant treatment response

Single-Cell Reconstruction of Oxytocinergic Neurons Reveals Separate Hypophysiotropic and Encephalotropic Subtypes in Larval Zebrafish

Oxytocin regulates a diverse set of processes including stress, analgesia, metabolism, and social behavior. How such diverse functions are mediated by a single hormonal system is not well understood. Different functions of oxytocin could be mediated by distinct cell groups, yet it is currently unknown whether different oxytocinergic cell types exist that specifically mediate peripheral neuroendocrine or various central neuromodulatory processes via dedicated pathways. Using the Brainbow technique to map the morphology and projections of individual oxytocinergic cells in the larval zebrafish brain, we report here the existence of two main types of oxytocinergic cells: those that innervate the pituitary and those that innervate diverse brain regions. Similar to the situation in the adult rat and the adult midshipman, but in contrast to the situation in the adult trout, these two cell types are mutually exclusive and can be distinguished based on morphological and anatomical criteria. Further, our results reveal that complex oxytocinergic innervation patterns are already established in the larval zebrafish brain

Habenula Circuit Development: Past, Present, and Future

The habenular neural circuit is attracting increasing attention from researchers in fields as diverse as neuroscience, medicine, behavior, development, and evolution. Recent studies have revealed that this part of the limbic system in the dorsal diencephalon is involved in reward, addiction, and other behaviors and its impairment is associated with various neurological conditions and diseases. Since the initial description of the dorsal diencephalic conduction system (DDC) with the habenulae in its center at the end of the nineteenth century, increasingly sophisticated techniques have resolved much of its anatomy and have shown that these pathways relay information from different parts of the forebrain to the tegmentum, midbrain, and hindbrain. The first part of this review gives a brief historical overview on how the improving experimental approaches have allowed the stepwise uncovering much of the architecture of the habenula circuit as we know it today. Our brain distributes tasks differentially between left and right and it has become a paradigm that this functional lateralization is a universal feature of vertebrates. Moreover, task dependent differential brain activities have been linked to anatomical differences across the left–right axis in humans. A good way to further explore this fundamental issue will be to study the functional consequences of subtle changes in neural network formation, which requires that we fully understand DDC system development. As the habenular circuit is evolutionarily highly conserved, researchers have the option to perform such difficult experiments in more experimentally amenable vertebrate systems. Indeed, research in the last decade has shown that the zebrafish is well suited for the study of DDC system development and the phenomenon of functional lateralization. We will critically discuss the advantages of the zebrafish model, available techniques, and others that are needed to fully understand habenular circuit development

Optogenetic induction of chronic glucocorticoid exposure in early-life leads to blunted stress-response in larval zebrafish

Early life stress (ELS) exposure alters stress susceptibility in later life and affects vulnerability to stress-related disorders, but how ELS changes the long-lasting responsiveness of the stress system is not well understood. Zebrafish provides an opportunity to study conserved mechanisms underlying the development and function of the stress response that is regulated largely by the neuroendocrine hypothalamus-pituitary-adrenal/interrenal (HPA/I) axis, with glucocorticoids (GC) as the final effector. In this study, we established a method to chronically elevate endogenous GC levels during early life in larval zebrafish. To this end, we employed an optogenetic actuator, beggiatoa photoactivated adenylyl cyclase, specifically expressed in the interrenal cells of zebrafish and demonstrate that its chronic activation leads to hypercortisolaemia and dampens the acute-stress evoked cortisol levels, across a variety of stressor modalities during early life. This blunting of stress-response was conserved in ontogeny at a later developmental stage. Furthermore, we observe a strong reduction of proopiomelanocortin (pomc)-expression in the pituitary as well as upregulation of fkbp5 gene expression. Going forward, we propose that this model can be leveraged to tease apart the mechanisms underlying developmental programming of the HPA/I axis by early-life GC exposure and its implications for vulnerability and resilience to stress in adulthood

Targeting retinal dopaminergic neurons in tyrosine hydroxylase-driven green fluorescent protein transgenic zebrafish

Purpose: Dopamine plays key roles in a variety of basic functions in the central nervous system. To study developmental and functional roles of dopaminergic cells in zebrafish, we have generated a transgenic line of zebrafish expressing green fluorescent protein (GFP) under the control of the tyrosine hydroxylase (th1) promoter. Methods: A 12 kb gene fragment that contains the th1 promoter was isolated and ligated to the MmGFP coding sequence, linearized, microinjected into 1−2 cell stage embryos and the founders crossed with wild−type fish to screen for transgenic lines. Tg(−12th:MmGFP) embryos were visualized under fluorescence microscopy for GFP expression during development. Confocal microscopy was used to visualize GFP−labeled cells in the living whole mount retina and immunostained vertical sections of adult zebrafish retina. Single−cell reverse transcription polymerase chain reaction (RT−PCR) was performed on individual GFP+ cells collected from dispersed retinal cell cultures for th1 and dopamine transporter (dat). Loose−patch recordings of spike activity of GFP+ neurons were made in isolated whole mount retinas. Results: th1 promoter−driven GFP exhibited robust expression in the brain and retina during zebrafish development. In juvenile and adult fish retinas, GFP was expressed in cells located in the inner nuclear layer. Immunocytochemistry with antibodies for GFP and TH showed that 29±2% of GFP−labeled cells also expressed TH. Two subpopulations of GFP−labeled cells were identified by fluorescent microscopy: bright GFP−expressing cells and dim GFP−expressing cells. Seminested single−cell RT−PCR showed that 71% of dim GFP−expressing cells expressed both th and dat mRNA. Loose−patch voltage−clamp recording from dim GFP−labeled cells in retinal whole mounts revealed that many of these dopaminergic neurons are spontaneously active in darkness. Conclusions: Although this Tg(−12th:MmGFP) line is not a completely specific reporter for dopaminergic neurons, using relative GFP intensity we are able to enrich for the selection of retinal dopaminergic cells in vitro and in situ in molecular and electrophysiological experiments. This transgenic line provides a useful tool for studying retinal dopaminergic cells in the zebrafish

A versatile transcription factor: Multiple roles of orthopedia a (otpa) beyond its restricted localization in dopaminergic systems of developing and adult zebrafish (Danio rerio) brains

Many transcription factors boost neural development and differentiation in specific directions and serve for identifying similar or homologous structures across species. The expression of Orthopedia (Otp) is critical for the development of certain cell groups along the vertebrate neuraxis, for example, the medial amygdala or hypothalamic neurosecretory neurons. Therefore, the primary focus of the present study is the distribution of Orthopedia a (Otpa) in the larval and adult zebrafish (Danio rerio) brain. Since Otpa is also critical for the development of zebrafish basal diencephalic dopaminergic cells, colocalization of Otpa with the catecholamine synthesizing enzyme tyrosine hydroxylase (TH) is studied. Cellular colocalization of Otpa and dopamine is only seen in magnocellular neurons of the periventricular posterior tubercular nucleus and in the posterior tuberal nucleus. Otpa-positive cells occur in many additional structures along the zebrafish neuraxis, from the secondary prosencephalon down to the hindbrain. Furthermore, Otpa expression is studied in shh-GFP and islet1-GFP transgenic zebrafish. Otpa-positive cells only express shh in dopaminergic magnocellular periventricular posterior tubercular cells, and only colocalize with islet1-GFP in the ventral zone and prerecess caudal periventricular hypothalamic zone and the perilemniscal nucleus. The scarcity of cellular colocalization of Otpa in islet1-GFP cells indicates that the Shh-islet1 neurogenetic pathway is not active in most Otpa-expressing domains. Our analysis reveals detailed correspondences between mouse and zebrafish forebrain territories including the zebrafish intermediate nucleus of the ventral telencephalon and the mouse medial amygdala. The zebrafish preoptic Otpa-positive domain represents the neuropeptidergic supraopto-paraventricular region of all tetrapods. Otpa domains in the zebrafish basal plate hypothalamus suggest that the ventral periventricular hypothalamic zone corresponds to the otp-expressing basal hypothalamic tuberal field in the mouse. Furthermore, the mouse otp domain in the mammillary hypothalamus compares partly to our Otpa-positive domain in the prerecess caudal periventricular hypothalamic zone (Hc-a)

Understanding and Predicting Antidepressant Response: Using Animal Models to Move Toward Precision Psychiatry

There are two important gaps of knowledge in depression treatment, namely the lack of biomarkers predicting response to antidepressants and the limited knowledge of the molecular mechanisms underlying clinical improvement. However, individually tailored treatment strategies and individualized prescription are greatly needed given the huge socio-economic burden of depression, the latency until clinical improvement can be observed and the response variability to a particular compound. Still, individual patient-level antidepressant treatment outcomes are highly unpredictable. In contrast to other therapeutic areas and despite tremendous efforts during the past years, the genomics era so far has failed to provide biological or genetic predictors of clinical utility for routine use in depression treatment. Specifically, we suggest to (1) shift the focus from the group patterns to individual outcomes, (2) use dimensional classifications such as Research Domain Criteria, and (3) envision better planning and improved connections between pre-clinical and clinical studies within translational research units. In contrast to studies in patients, animal models enable both searches for peripheral biosignatures predicting treatment response and in depth-analyses of the neurobiological pathways shaping individual antidepressant response in the brain. While there is a considerable number of animal models available aiming at mimicking disease-like conditions such as those seen in depressive disorder, only a limited number of preclinical or truly translational investigations is dedicated to the issue of heterogeneity seen in response to antidepressant treatment. In this mini-review, we provide an overview on the current state of knowledge and propose a framework for successful translational studies into antidepressant treatment response

Revealing salt-expedited reduction mechanism for hollow silicon microsphere formation in bi-functional halide melts

The thermochemical reduction of silica to silicon using chemical reductants requires high temperature and has a high activation energy, which depends on the melting temperature of the reductant. The addition of bi-functional molten salts with a low melting temperature may reduce the required energy, and several examples using molten salts have been demonstrated. Here we study the mechanism of reduction of silica in the presence of aluminum metal reductant and aluminum chloride as bi-functional molten salts. An aluminum-aluminum chloride complex plays a key role in the reduction mechanism, reacting with the oxygen of the silica surfaces to lower the heat of reaction and subsequently survives a recycling step in the reaction. This experimentally and theoretically validated reaction mechanism may open a new pathway using bi-functional molten salts. Furthermore, the as-synthesized hollow porous silicon microsphere anodes show structural durability on cycling in both half/full cell tests, attributed to the high volume-accommodating ability