The neuropeptide Pth2 dynamically senses others via mechanosensation.

Species that depend on membership in social groups for survival exhibit changes in neuronal gene expression and behaviour when they face restricted social interactions or isolation1-3. Here we show that, across the lifespan of zebrafish (Danio rerio), social isolation specifically decreased the level of transcription of pth2, the gene that encodes the vertebrate-specific neuropeptide Pth2. However, 30 minutes of exposure to conspecifics was sufficient to initiate a significant rescue of pth2 transcript levels in previously isolated zebrafish. Transcription of pth2 exhibited bidirectional dynamics; following the acute isolation of socially reared fish, a rapid reduction in the levels of pth2 was observed. The expression of pth2 tracked not only the presence of other fish but also the density of the group. The sensory modality that controls the expression of pth2 was neither visual nor chemosensory in origin but instead was mechanical, induced by the movements of neighbouring fish. Chemical ablation of the mechanosensitive neuromast cells within the lateral line of fish prevented the rescue of pth2 levels that was induced by the social environment. In addition, mechanical perturbation of the water at frequencies similar to the movements of the zebrafish tail was sufficient to rescue the levels of pth2 in previously isolated fish. These data indicate a previously underappreciated role for the relatively unexplored neuropeptide Pth2 in both tracking and responding to the population density of the social environment of an animal.

Fear circuit-based neurobehavioral signatures mirror resilience to chronic social stress in mouse.

Consistent evidence from human data points to successful threat-safety discrimination and responsiveness to extinction of fear memories as key characteristics of resilient individuals. To promote valid cross-species approaches for the identification of resilience mechanisms, we establish a translationally informed mouse model enabling the stratification of mice into three phenotypic subgroups following chronic social defeat stress, based on their individual ability for threat-safety discrimination and conditioned learning: the Discriminating-avoiders, characterized by successful social threat-safety discrimination and extinction of social aversive memories; the Indiscriminate-avoiders, showing aversive response generalization and resistance to extinction, in line with findings on susceptible individuals; and the Non-avoiders displaying impaired aversive conditioned learning. To explore the neurobiological mechanisms underlying the stratification, we perform transcriptome analysis within three key target regions of the fear circuitry. We identify subgroup-specific differentially expressed genes and gene networks underlying the behavioral phenotypes, i.e., the individual ability to show threat-safety discrimination and respond to extinction training. Our approach provides a translationally informed template with which to characterize the behavioral, molecular, and circuit bases of resilience in mice.

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.

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.

Larval Zebrafish Proteome Regulation in Response to an Environmental Challenge.

Adaptation to the environment during development influences the life-long survival of an animal. While brain-wide proteomic changes are expected to underlie such experience-driven physiological and behavioral flexibility, a comprehensive overview of the nature and extent of the proteomic regulation following an environmental challenge during development is currently lacking. In this study, the brain proteome of larval zebrafish is identified and it is determined how it is altered by an exposure to a natural and physical environmental challenge, namely prolonged exposure to strong water currents. A comprehensive larval zebrafish brain proteome is presented here. Furthermore, 57 proteins that are regulated by the exposure to an environmental challenge are identified, which cover multiple functions including neuronal plasticity, the stress response, axonal growth and guidance, spatial learning, and energy metabolism. These represent candidate proteins that may play crucial roles for the adaption to an environmental challenge during development.

iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins.

DNA adenine methyltransferase identification (DamID) has emerged as an alternative method to profile protein-DNA interactions; however, critical issues limit its widespread applicability. Here, we present iDamIDseq, a protocol that improves specificity and sensitivity by inverting the steps DpnI-DpnII and adding steps that involve a phosphatase and exonuclease. To determine genome-wide protein-DNA interactions efficiently, we present the analysis tool iDEAR (iDamIDseq Enrichment Analysis with R). The combination of DamID and iDEAR permits the establishment of consistent profiles for transcription factors, even in transient assays, as we exemplify using the small teleost medaka (Oryzias latipes). We report that the bacterial Dam-coding sequence induces aberrant splicing when it is used with different promoters to drive tissue-specific expression. Here, we present an optimization of the sequence to avoid this problem. This and our other improvements will allow researchers to use DamID effectively in any organism, in a general or targeted manner.

Anatomy, development, and plasticity of the neurosecretory hypothalamus in zebrafish.

The paraventricular nucleus (PVN) of the hypothalamus harbors diverse neurosecretory cells with critical physiological roles for the homeostasis. Decades of research in rodents have provided a large amount of information on the anatomy, development, and function of this important hypothalamic nucleus. However, since the hypothalamus lies deep within the brain in mammals and is difficult to access, many questions regarding development and plasticity of this nucleus still remain. In particular, how different environmental conditions, including stress exposure, shape the development of this important nucleus has been difficult to address in animals that develop in utero. To address these open questions, the transparent larval zebrafish with its rapid external development and excellent genetic toolbox offers exciting opportunities. In this review, we summarize recent information on the anatomy and development of the neurosecretory preoptic area (NPO), which represents a similar structure to the mammalian PVN in zebrafish. We will then review recent studies on the development of different cell types in the neurosecretory hypothalamus both in mouse and in fish. Lastly, we discuss stress-induced plasticity of the PVN mainly discussing the data obtained in rodents, but pointing out tools and approaches available in zebrafish for future studies. This review serves as a primer for the currently available information relevant for studying the development and plasticity of this important brain region using zebrafish.

The Severity of Acute Stress Is Represented by Increased Synchronous Activity and Recruitment of Hypothalamic CRH Neurons.

The hypothalamo-pituitary-adrenocortical (HPA) axis regulates stress physiology and behavior. To achieve an optimally tuned adaptive response, it is critical that the magnitude of the stress response matches the severity of the threat. Corticotropin-releasing hormone (CRH) released from the paraventricular nucleus of the hypothalamus is a major regulator of the HPA axis. However, how CRH-producing neurons in an intact animal respond to different stressor intensities is currently not known. Using two-photon calcium imaging on intact larval zebrafish, we recorded the activity of CRH cells, while the larvae were exposed to stressors of varying intensity. By combining behavioral and physiological measures, we first determined how sudden alterations in environmental conditions lead to different levels of stress axis activation. Then, we measured changes in the frequency and amplitude of Ca(2+) transients in individual CRH neurons in response to such stressors. The response magnitude of individual CRH cells covaried with stressor intensity. Furthermore, stressors caused the recruitment of previously inactive CRH neurons in an intensity-dependent manner, thus increasing the pool of responsive CRH cells. Strikingly, stressor-induced activity appeared highly synchronized among CRH neurons, and also across hemispheres. Thus, the stressor strength-dependent output of CRH neurons emerges by a dual mechanism that involves both the increased activity of individual cells and the recruitment of a larger pool of responsive cells. The synchronicity of CRH neurons within and across hemispheres ensures that the overall output of the HPA axis matches the severity of the threat. SIGNIFICANCE STATEMENT: Stressors trigger adaptive responses in the body that are essential for survival. How the brain responds to acute stressors of varying intensity in an intact animal, however, is not well understood. We address this question using two-photon Ca(2+) imaging in larval zebrafish with transgenically labeled corticotropin-releasing hormone (CRH) cells, which represent a major regulator of the stress axis. We show that stressor strength-dependent responses of CRH neurons emerge via an intensity-dependent increase in the activity of individual CRH cells, and by an increase in the pool of responsive CRH cells at the population level. Furthermore, we report striking synchronicity among CRH neurons even across hemispheres, which suggests tight intrahypothalamic and interhypothalamic coordination. Thus, our work reveals how CRH neurons respond to different levels of acute stress in vivo.

Engineering of a red-light-activated human cAMP/cGMP-specific phosphodiesterase.

Sensory photoreceptors elicit vital physiological adaptations in response to incident light. As light-regulated actuators, photoreceptors underpin optogenetics, which denotes the noninvasive, reversible, and spatiotemporally precise perturbation by light of living cells and organisms. Of particular versatility, naturally occurring photoactivated adenylate cyclases promote the synthesis of the second messenger cAMP under blue light. Here, we have engineered a light-activated phosphodiesterase (LAPD) with complementary light sensitivity and catalytic activity by recombining the photosensor module of Deinococcus radiodurans bacterial phytochrome with the effector module of Homo sapiens phosphodiesterase 2A. Upon red-light absorption, LAPD up-regulates hydrolysis of cAMP and cGMP by up to sixfold, whereas far-red light can be used to down-regulate activity. LAPD also mediates light-activated cAMP and cGMP hydrolysis in eukaryotic cell cultures and in zebrafish embryos; crucially, the biliverdin chromophore of LAPD is available endogenously and does not need to be provided exogenously. LAPD thus establishes a new optogenetic modality that permits light control over diverse cAMP/cGMP-mediated physiological processes. Because red light penetrates tissue more deeply than light of shorter wavelengths, LAPD appears particularly attractive for studies in living organisms.

Specification of posterior hypothalamic neurons requires coordinated activities of Fezf2, Otp, Sim1a and Foxb1.2.

The hypothalamus is a key integrative center in the brain that consists of diverse cell types required for a variety of functions including homeostasis, reproduction, stress response, social and cognitive behavior. Despite our knowledge of several transcription factors crucial for hypothalamic development, it is not known how the wide diversity of neuron types in the hypothalamus is produced. In particular, almost nothing is known about the mechanisms that specify neurons in the posteriormost part of the hypothalamus, the mammillary area. Here, we investigated the specification of two distinct neuron types in the mammillary area that produce the hypothalamic hormones Vasoactive intestinal peptide (Vip) and Urotensin 1 (Uts1). We show that Vip- and Uts1-positive neurons develop in distinct domains in the mammillary area defined by the differential expression of the transcription factors Fezf2, Otp, Sim1a and Foxb1.2. Coordinated activities of these factors are crucial for the establishment of the mammillary area subdomains and the specification of Vip- and Uts1-positive neurons. In addition, Fezf2 is important for early development of the posterior hypothalamus. Thus, our study provides the first molecular anatomical map of the posterior hypothalamus in zebrafish and identifies, for the first time, molecular requirements underlying the specification of distinct posterior hypothalamic neuron types.

A vertebrate-conserved cis-regulatory module for targeted expression in the main hypothalamic regulatory region for the stress response.

BACKGROUND: The homeodomain transcription factor orthopedia (Otp) is an evolutionarily conserved regulator of neuronal fates. In vertebrates, Otp is necessary for the proper development of different regions of the brain and is required in the diencephalon to specify several hypothalamic cell types, including the cells that control the stress response. To understand how this widely expressed transcription factor accomplishes hypothalamus-specific functions, we performed a comprehensive screening of otp cis-regulatory regions in zebrafish. RESULTS: Here, we report the identification of an evolutionarily conserved vertebrate enhancer module with activity in a restricted area of the forebrain, which includes the region of the hypothalamus that controls the stress response. This region includes neurosecretory cells producing Corticotropin-releasing hormone (Crh), Oxytocin (Oxt) and Arginine vasopressin (Avp), which are key components of the stress axis. Lastly, expression of the bacterial nitroreductase gene under this specific enhancer allowed pharmacological attenuation of the stress response in zebrafish larvae. CONCLUSION: Vertebrates share many cellular and molecular components of the stress response and our work identified a striking conservation at the cis-regulatory level of a key hypothalamic developmental gene. In addition, this enhancer provides a useful tool to manipulate and visualize stress-regulatory hypothalamic cells in vivo with the long-term goal of understanding the ontogeny of the stress axis in vertebrates.

Exposure to elevated glucocorticoid during development primes altered transcriptional responses to acute stress in adulthood.

Early life stress (ELS) is a major risk factor for developing psychiatric disorders, with glucocorticoids (GCs) implicated in mediating its effects in shaping adult phenotypes. In this process, exposure to high levels of developmental GC (hdGC) is thought to induce molecular changes that prime differential adult responses. However, identities of molecules targeted by hdGC exposure are not completely known. Here, we describe lifelong molecular consequences of hdGC exposure using a newly developed zebrafish double-hit stress model, which shows altered behaviors and stress hypersensitivity in adulthood. We identify a set of primed genes displaying altered expression only upon acute stress in hdGC-exposed adult fish brains. Interestingly, this gene set is enriched in risk factors for psychiatric disorders in humans. Lastly, we identify altered epigenetic regulatory elements following hdGC exposure. Thus, our study provides comprehensive datasets delineating potential molecular targets mediating the impact of hdGC exposure on adult responses.

Elevated glucocorticoid alters the developmental dynamics of hypothalamic neurogenesis in zebrafish.

Exposure to excess glucocorticoid (GC) during early development is implicated in adult dysfunctions. Reduced adult hippocampal neurogenesis is a well-known consequence of exposure to early life stress or elevated GC, however the effects on neurogenesis during development and effects on other brain regions are not well understood. Using an optogenetic zebrafish model, here we analyse the effects of GC exposure on neurogenesis during development in the whole brain. We identify that the hypothalamus is a highly GC-sensitive region where elevated GC causes precocious development. This is followed by failed maturation and early decline accompanied by impaired feeding, growth, and survival. In GC-exposed animals, the developmental trajectory of hypothalamic progenitor cells is strikingly altered, potentially mediated by direct regulation of transcription factors such as rx3 by GC. Our data provide cellular and molecular level insight into GC-induced alteration of the hypothalamic developmental trajectory, a process crucial for health across the life-course.

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.

Acute Stress Modulates Social Approach and Social Maintenance in Adult Zebrafish.

Stress alters social functioning in a complex manner. An important variable determining the final effects of stress is stressor intensity. However, the precise relationship between stressor intensity and social behavior is not well understood. Here, we investigate the effects of varying acute stressor intensity exposure on social behavior using adult zebrafish. We first establish a novel test using adult zebrafish that allows distinguishing fish's drive to approach a social cue and its ability to engage and maintain social interaction within the same behavioral paradigm. Next, we combined this test with a new method to deliver an acute stress stimulus of varying intensities. Our results show that both social approach and social maintenance are reduced in adult zebrafish on acute stress exposure in an intensity-dependent manner. Interestingly, lower stress intensity reduces social maintenance without affecting the social approach, while a higher stress level is required to alter social approach. These results provide evidence for a direct correlation between acute stressor intensity and social functioning and suggest that distinct steps in social behavior are modulated differentially by the acute stress level.

Altered glucocorticoid reactivity and behavioral phenotype in rx3-/- larval zebrafish.

Introduction: The transcription factor rx3 is important for the formation of the pituitary and parts of the hypothalamus. Mutant animals lacking rx3 function have been well characterized in developmental studies, but relatively little is known about their behavioral phenotypes. Methods: We used cell type staining to reveal differences in stress axis architecture, and performed cortisol measurements and behavior analysis to study both hormonal and behavioral stress responses in rx3 mutants. Results and Discussion: Consistent with the role of rx3 in hypothalamus and pituitary development, we show a distinct loss of corticotrope cells involved in stress regulation, severe reduction of pituitary innervation by hypothalamic cells, and lack of stress-induced cortisol release in rx3 mutants. Interestingly, despite these deficits, we report that rx3-/- larval zebrafish can still display nominal behavioral responses to both stressful and non-stressful stimuli. However, unlike wildtypes, mutants lacking proper pituitary-interrenal function do not show enhanced behavioral performance under moderate stress level, supporting the view that corticotroph cells are not required for behavioral responses to some types of stressful stimuli but modulate subtle behavioral adjustments under moderate stress.

Zebrafish as a model to investigate the CRH axis and interactions with DISC1.

Release of corticotropin-releasing hormone (CRH) from CRH neurons activates the hypothalamo-pituitary-adrenal (HPA) axis, one of the main physiological stress response systems. Complex feedback loops operate in the HPA axis and understanding the neurobiological mechanisms regulating CRH neurons is of great importance in the context of stress disorders. In this article, we review how in vivo studies in zebrafish have advanced knowledge of the neurobiology of CRH neurons. Disrupted-in-schizophrenia 1 (DISC1) mutant zebrafish have blunted stress responses and can be used to model human stress disorders. We propose that DISC1 influences the development and functioning of CRH neurons as a mechanism linking DISC1 to psychiatric disorders.

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).

Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio).

Zebrafish are highly social teleost fish and an excellent model to study social behavior. The neuropeptide Oxytocin is associated different social behaviors as well as disorders resulting in social impairment like autism spectrum disorder. However, how Oxytocin receptor signaling affects the development and expression kinetics of social behavior is not known. In this study we investigated the role of the two oxytocin receptors, Oxtr and Oxtrl, in the development and maintenance of social preference and shoaling behavior in 2- to 8-week-old zebrafish. Using CRISPR/Cas9 mediated oxtr and oxtrl knock-out fish, we found that the development of social preference is accelerated if one of the Oxytocin receptors is knocked-out and that the knock-out fish reach significantly higher levels of social preference. Moreover, oxtr-/- fish showed impairments in the maintenance of social preference. Social isolation prior to testing led to impaired maintenance of social preference in both wild-type and oxtr and oxtrl knock-out fish. Knocking-out either of the Oxytocin receptors also led to increased group spacing and reduced polarization in a 20-fish shoal at 8 weeks post fertilization, but not at 4. These results show that the development and maintenance of social behavior is influenced by the Oxytocin receptors and that the effects are not just pro- or antisocial, but dependent on both the age and social context of the fish.

The neuropeptide Pth2 modulates social behavior and anxiety in zebrafish.

Behavior is context-dependent and often modulated by an animal's internal state. In particular, different social contexts can alter anxiety levels and modulate social behavior. The vertebrate-specific neuropeptide parathyroid hormone 2 (pth2) is regulated by the presence of conspecifics in zebrafish. As its cognate receptor, the parathyroid hormone 2 receptor (pth2r), is widely expressed across the brain, we tested fish lacking the functional Pth2 peptide in several anxiety-related and social behavior paradigms. Here, we show that the propensity to react to sudden stimuli with an escape response was increased in pth2 -/- zebrafish, consistent with an elevated anxiety level. While overall social preference for conspecifics was maintained in pth2 -/- fish until the early juvenile stage, we found that both social preference and shoaling were altered later in development. The data presented suggest that the neuropeptide Pth2 modulates several conserved behaviors and may thus enable the animal to react appropriately in different social contexts.