Zebrafish (Danio rerio) as a translational model for neuro-immune interactions in the enteric nervous system in autism spectrum disorders.

Autism spectrum disorders (ASD) affect about 1% of the population and are strongly associated with gastrointestinal diseases creating shortcomings in quality of life. Multiple factors contribute to the development of ASD and although neurodevelopmental deficits are central, the pathogenesis of the condition is complex and the high prevalence of intestinal disorders is poorly understood. In agreement with the prominent research establishing clear bidirectional interactions between the gut and the brain, several studies have made it evident that such a relation also exists in ASD. Thus, dysregulation of the gut microbiota and gut barrier integrity may play an important role in ASD. However, only limited research has investigated how the enteric nervous system (ENS) and intestinal mucosal immune factors may impact on the development of ASD-related intestinal disorders. This review focuses on the mechanistic studies that elucidate the regulation and interactions between enteric immune cells, residing gut microbiota and the ENS in models of ASD. Especially the multifaceted properties and applicability of zebrafish (Danio rerio) for the study of ASD pathogenesis are assessed in comparison to studies conducted in rodent models and humans. Advances in molecular techniques and in vivo imaging, combined with genetic manipulation and generation of germ-free animals in a controlled environment, appear to make zebrafish an underestimated model of choice for the study of ASD. Finally, we establish the research gaps that remain to be explored to further our understanding of the complexity of ASD pathogenesis and associated mechanisms that may lead to intestinal disorders.

Optopharmacological tools for precise spatiotemporal control of oxytocin signaling in the central nervous system and periphery.

Oxytocin is a neuropeptide critical for maternal physiology and social behavior, and is thought to be dysregulated in several neuropsychiatric disorders. Despite the biological and neurocognitive importance of oxytocin signaling, methods are lacking to activate oxytocin receptors with high spatiotemporal precision in the brain and peripheral mammalian tissues. Here we developed and validated caged analogs of oxytocin which are functionally inert until cage release is triggered by ultraviolet light. We examined how focal versus global oxytocin application affected oxytocin-driven Ca2+ wave propagation in mouse mammary tissue. We also validated the application of caged oxytocin in the hippocampus and auditory cortex with electrophysiological recordings in vitro, and demonstrated that oxytocin uncaging can accelerate the onset of mouse maternal behavior in vivo. Together, these results demonstrate that optopharmacological control of caged peptides is a robust tool with spatiotemporal precision for modulating neuropeptide signaling throughout the brain and body.

Brain-wide visual habituation networks in wild type and fmr1 zebrafish.

Habituation is a form of learning during which animals stop responding to repetitive stimuli, and deficits in habituation are characteristic of several psychiatric disorders. Due to technical challenges, the brain-wide networks mediating habituation are poorly understood. Here we report brain-wide calcium imaging during larval zebrafish habituation to repeated visual looming stimuli. We show that different functional categories of loom-sensitive neurons are located in characteristic locations throughout the brain, and that both the functional properties of their networks and the resulting behavior can be modulated by stimulus saliency and timing. Using graph theory, we identify a visual circuit that habituates minimally, a moderately habituating midbrain population proposed to mediate the sensorimotor transformation, and downstream circuit elements responsible for higher order representations and the delivery of behavior. Zebrafish larvae carrying a mutation in the fmr1 gene have a systematic shift toward sustained premotor activity in this network, and show slower behavioral habituation.

From calcium imaging to graph topology.

Systems neuroscience is facing an ever-growing mountain of data. Recent advances in protein engineering and microscopy have together led to a paradigm shift in neuroscience; using fluorescence, we can now image the activity of every neuron through the whole brain of behaving animals. Even in larger organisms, the number of neurons that we can record simultaneously is increasing exponentially with time. This increase in the dimensionality of the data is being met with an explosion of computational and mathematical methods, each using disparate terminology, distinct approaches, and diverse mathematical concepts. Here we collect, organize, and explain multiple data analysis techniques that have been, or could be, applied to whole-brain imaging, using larval zebrafish as an example model. We begin with methods such as linear regression that are designed to detect relations between two variables. Next, we progress through network science and applied topological methods, which focus on the patterns of relations among many variables. Finally, we highlight the potential of generative models that could provide testable hypotheses on wiring rules and network progression through time, or disease progression. While we use examples of imaging from larval zebrafish, these approaches are suitable for any population-scale neural network modeling, and indeed, to applications beyond systems neuroscience. Computational approaches from network science and applied topology are not limited to larval zebrafish, or even to systems neuroscience, and we therefore conclude with a discussion of how such methods can be applied to diverse problems across the biological sciences.

Contributions of Luminance and Motion to Visual Escape and Habituation in Larval Zebrafish.

Animals from insects to humans perform visual escape behavior in response to looming stimuli, and these responses habituate if looms are presented repeatedly without consequence. While the basic visual processing and motor pathways involved in this behavior have been described, many of the nuances of predator perception and sensorimotor gating have not. Here, we have performed both behavioral analyses and brain-wide cellular-resolution calcium imaging in larval zebrafish while presenting them with visual loom stimuli or stimuli that selectively deliver either the movement or the dimming properties of full loom stimuli. Behaviorally, we find that, while responses to repeated loom stimuli habituate, no such habituation occurs when repeated movement stimuli (in the absence of luminance changes) are presented. Dim stimuli seldom elicit escape responses, and therefore cannot habituate. Neither repeated movement stimuli nor repeated dimming stimuli habituate the responses to subsequent full loom stimuli, suggesting that full looms are required for habituation. Our calcium imaging reveals that motion-sensitive neurons are abundant in the brain, that dim-sensitive neurons are present but more rare, and that neurons responsive to both stimuli (and to full loom stimuli) are concentrated in the tectum. Neurons selective to full loom stimuli (but not to movement or dimming) were not evident. Finally, we explored whether movement- or dim-sensitive neurons have characteristic response profiles during habituation to full looms. Such functional links between baseline responsiveness and habituation rate could suggest a specific role in the brain-wide habituation network, but no such relationships were found in our data. Overall, our results suggest that, while both movement- and dim-sensitive neurons contribute to predator escape behavior, neither plays a specific role in brain-wide visual habituation networks or in behavioral habituation.

RNA-induced inflammation and migration of precursor neurons initiates neuronal circuit regeneration in zebrafish.

Tissue regeneration and functional restoration after injury are considered as stem- and progenitor-cell-driven processes. In the central nervous system, stem cell-driven repair is slow and problematic because function needs to be restored rapidly for vital tasks. In highly regenerative vertebrates, such as zebrafish, functional recovery is rapid, suggesting a capability for fast cell production and functional integration. Surprisingly, we found that migration of dormant "precursor neurons" to the injury site pioneers functional circuit regeneration after spinal cord injury and controls the subsequent stem-cell-driven repair response. Thus, the precursor neurons make do before the stem cells make new. Furthermore, RNA released from the dying or damaged cells at the site of injury acts as a signal to attract precursor neurons for repair. Taken together, our data demonstrate an unanticipated role of neuronal migration and RNA as drivers of neural repair.

Broad frequency sensitivity and complex neural coding in the larval zebrafish auditory system.

Most animals have complex auditory systems that identify salient features of the acoustic landscape to direct appropriate responses. In fish, these features include the volume, frequency, complexity, and temporal structure of acoustic stimuli transmitted through water. Larval fish have simple brains compared to adults but swim freely and depend on sophisticated sensory processing for survival.1-5 Zebrafish larvae, an important model for studying brain-wide neural networks, have thus far been found to possess a rudimentary auditory system, sensitive to a narrow range of frequencies and without evident sensitivity to acoustic features that are salient and ethologically important to adult fish.6,7 Here, we have combined a novel method for delivering water-borne sounds, a diverse assembly of acoustic stimuli, and whole-brain calcium imaging to describe the responses of individual auditory-responsive neurons across the brains of zebrafish larvae. Our results reveal responses to frequencies ranging from 100 Hz to 4 kHz, with evidence of frequency discrimination from 100 Hz to 2.5 kHz. Frequency-selective neurons are located in numerous regions of the brain, and neurons responsive to the same frequency are spatially grouped in some regions. Using functional clustering, we identified categories of neurons that are selective for a single pure-tone frequency, white noise, the sharp onset of acoustic stimuli, and stimuli involving a gradual crescendo. These results suggest a more nuanced auditory system than has previously been described in larval fish and provide insights into how a young animal's auditory system can both function acutely and serve as the scaffold for a more complex adult system.

Sound generation in zebrafish with Bio-Opto-Acoustics.

Hearing is a crucial sense in underwater environments for communication, hunting, attracting mates, and detecting predators. However, the tools currently used to study hearing are limited, as they cannot controllably stimulate specific parts of the auditory system. To date, the contributions of hearing organs have been identified through lesion experiments that inactivate an organ, making it difficult to gauge the specific stimuli to which each organ is sensitive, or the ways in which inputs from multiple organs are combined during perception. Here, we introduce Bio-Opto-Acoustic (BOA) stimulation, using optical forces to generate localized vibrations in vivo, and demonstrate stimulation of the auditory system of zebrafish larvae with precise control. We use a rapidly oscillated optical trap to generate vibrations in individual otolith organs that are perceived as sound, while adjacent otoliths are either left unstimulated or similarly stimulated with a second optical laser trap. The resulting brain-wide neural activity is characterized using fluorescent calcium indicators, thus linking each otolith organ to its individual neuronal network in a way that would be impossible using traditional sound delivery methods. The results reveal integration and cooperation of the utricular and saccular otoliths, which were previously described as having separate biological functions, during hearing.

Deep conservation of the enhancer regulatory code in animals.

Interactions of transcription factors (TFs) with DNA regulatory sequences, known as enhancers, specify cell identity during animal development. Unlike TFs, the origin and evolution of enhancers has been difficult to trace. We drove zebrafish and mouse developmental transcription using enhancers from an evolutionarily distant marine sponge. Some of these sponge enhancers are located in highly conserved microsyntenic regions, including an Islet enhancer in the Islet-Scaper region. We found that Islet enhancers in humans and mice share a suite of TF binding motifs with sponges, and that they drive gene expression patterns similar to those of sponge and endogenous Islet enhancers in zebrafish. Our results suggest the existence of an ancient and conserved, yet flexible, genomic regulatory syntax that has been repeatedly co-opted into cell type-specific gene regulatory networks across the animal kingdom.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Altered brain-wide auditory networks in a zebrafish model of fragile X syndrome.

BACKGROUND: Loss or disrupted expression of the FMR1 gene causes fragile X syndrome (FXS), the most common monogenetic form of autism in humans. Although disruptions in sensory processing are core traits of FXS and autism, the neural underpinnings of these phenotypes are poorly understood. Using calcium imaging to record from the entire brain at cellular resolution, we investigated neuronal responses to visual and auditory stimuli in larval zebrafish, using fmr1 mutants to model FXS. The purpose of this study was to model the alterations of sensory networks, brain-wide and at cellular resolution, that underlie the sensory aspects of FXS and autism. RESULTS: Combining functional analyses with the neurons' anatomical positions, we found that fmr1-/- animals have normal responses to visual motion. However, there were several alterations in the auditory processing of fmr1-/- animals. Auditory responses were more plentiful in hindbrain structures and in the thalamus. The thalamus, torus semicircularis, and tegmentum had clusters of neurons that responded more strongly to auditory stimuli in fmr1-/- animals. Functional connectivity networks showed more inter-regional connectivity at lower sound intensities (a - 3 to - 6 dB shift) in fmr1-/- larvae compared to wild type. Finally, the decoding capacities of specific components of the ascending auditory pathway were altered: the octavolateralis nucleus within the hindbrain had significantly stronger decoding of auditory amplitude while the telencephalon had weaker decoding in fmr1-/- mutants. CONCLUSIONS: We demonstrated that fmr1-/- larvae are hypersensitive to sound, with a 3-6 dB shift in sensitivity, and identified four sub-cortical brain regions with more plentiful responses and/or greater response strengths to auditory stimuli. We also constructed an experimentally supported model of how auditory information may be processed brain-wide in fmr1-/- larvae. Our model suggests that the early ascending auditory pathway transmits more auditory information, with less filtering and modulation, in this model of FXS.

The Trypanosoma Brucei KIFC1 Kinesin Ensures the Fast Antibody Clearance Required for Parasite Infectivity.

Human innate immunity to Trypanosoma brucei involves the trypanosome C-terminal kinesin TbKIFC1, which transports internalized trypanolytic factor apolipoprotein L1 (APOL1) within the parasite. We show that TbKIFC1 preferentially associates with cholesterol-containing membranes and is indispensable for mammalian infectivity. Knockdown of TbKIFC1 did not affect trypanosome growth in vitro but rendered the parasites unable to infect mice unless antibody synthesis was compromised. Surface clearance of Variant Surface Glycoprotein (VSG)-antibody complexes was far slower in these cells, which were more susceptible to capture by macrophages. This phenotype was not due to defects in VSG expression or trafficking but to decreased VSG mobility in a less fluid, stiffer surface membrane. This change can be attributed to increased cholesterol level in the surface membrane in TbKIFC1 knockdown cells. Clearance of surface-bound antibodies by T. brucei is therefore essential for infectivity and depends on high membrane fluidity maintained by the cholesterol-trafficking activity of TbKIFC1.

Brain-Wide Mapping of Water Flow Perception in Zebrafish.

Information about water flow, detected by lateral line organs, is critical to the behavior and survival of fish and amphibians. While certain aspects of water flow processing have been revealed through electrophysiology, we lack a comprehensive description of the neurons that respond to water flow and the network that they form. Here, we use brain-wide calcium imaging in combination with microfluidic stimulation to map out, at cellular resolution, neuronal responses involved in perceiving and processing water flow information in larval zebrafish. We find a diverse array of neurons responding to head-to-tail (h-t) flow, tail-to-head (t-h) flow, or both. Early in this pathway, in the lateral line ganglia, neurons respond almost exclusively to the simple presence of h-t or t-h flow, but later processing includes neurons responding specifically to flow onset, representing the accumulated displacement of flow during a stimulus, or encoding the speed of the flow. The neurons reporting on these more nuanced details are located across numerous brain regions, including some not previously implicated in water flow processing. A graph theory-based analysis of the brain-wide water flow network shows that a majority of this processing is dedicated to h-t flow detection, and this is reinforced by our finding that details like flow velocity and the total accumulated flow are only encoded for the h-t direction. The results represent the first brain-wide description of processing for this important modality, and provide a departure point for more detailed studies of the flow of information through this network.SIGNIFICANCE STATEMENT In aquatic animals, the lateral line is important for detecting water flow stimuli, but the brain networks that interpret this information remain mysterious. Here, we have imaged the activity of individual neurons across the entire brains of larval zebrafish, revealing all response types and their brain locations as water flow processing occurs. We find neurons that respond to the simple presence of water flow, and others attuned to the direction, speed, and duration of flow, or the accumulated displacement of water that has passed during the stimulus. With this information, we modeled the underlying network, describing a system that is nuanced in its processing of water flow simulating head-to-tail motion but rudimentary in processing flow in the tail-to-head direction.

Calcium Imaging and the Curse of Negativity.

The imaging of neuronal activity using calcium indicators has become a staple of modern neuroscience. However, without ground truths, there is a real risk of missing a significant portion of the real responses. Here, we show that a common assumption, the non-negativity of the neuronal responses as detected by calcium indicators, biases all levels of the frequently used analytical methods for these data. From the extraction of meaningful fluorescence changes to spike inference and the analysis of inferred spikes, each step risks missing real responses because of the assumption of non-negativity. We first show that negative deviations from baseline can exist in calcium imaging of neuronal activity. Then, we use simulated data to test three popular algorithms for image analysis, CaImAn, suite2p, and CellSort, finding that suite2p may be the best suited to large datasets. We also tested the spike inference algorithms included in CaImAn, suite2p, and Cellsort, as well as the dedicated inference algorithms MLspike and CASCADE, and found each to have limitations in dealing with inhibited neurons. Among these spike inference algorithms, FOOPSI, from CaImAn, performed the best on inhibited neurons, but even this algorithm inferred spurious spikes upon the return of the fluorescence signal to baseline. As such, new approaches will be needed before spikes can be sensitively and accurately inferred from calcium data in inhibited neurons. We further suggest avoiding data analysis approaches that, by assuming non-negativity, ignore inhibited responses. Instead, we suggest a first exploratory step, using k-means or PCA for example, to detect whether meaningful negative deviations are present. Taking these steps will ensure that inhibition, as well as excitation, is detected in calcium imaging datasets.

Cellular-Resolution Imaging of Vestibular Processing across the Larval Zebrafish Brain.

The vestibular system, which reports on motion and gravity, is essential to postural control, balance, and egocentric representations of movement and space. The motion needed to stimulate the vestibular system complicates studying its circuitry, so we previously developed a method for fictive vestibular stimulation in zebrafish, using optical trapping to apply physical forces to the otoliths. Here, we combine this approach with whole-brain calcium imaging at cellular resolution, delivering a comprehensive map of the brain regions and cellular responses involved in basic vestibular processing. We find responses broadly distributed across the brain, with unique profiles of cellular responses and topography in each region. The most widespread and abundant responses involve excitation that is graded to the stimulus strength. Other responses, localized to the telencephalon and habenulae, show excitation that is only weakly correlated to stimulus strength and that is sensitive to weak stimuli. Finally, numerous brain regions contain neurons that are inhibited by vestibular stimuli, and these neurons are often tightly localized spatially within their regions. By exerting separate control over the left and right otoliths, we explore the laterality of brain-wide vestibular processing, distinguishing between neurons with unilateral and bilateral vestibular sensitivity and revealing patterns whereby conflicting signals from the ears mutually cancel. Our results confirm previously identified vestibular responses in specific regions of the larval zebrafish brain while revealing a broader and more extensive network of vestibular responsive neurons than has previously been described. This provides a departure point for more targeted studies of the underlying functional circuits.

Luminance Changes Drive Directional Startle through a Thalamic Pathway.

Looming visual stimuli result in escape responses that are conserved from insects to humans. Despite their importance for survival, the circuits mediating visual startle have only recently been explored in vertebrates. Here we show that the zebrafish thalamus is a luminance detector critical to visual escape. Thalamic projection neurons deliver dim-specific information to the optic tectum, and ablations of these projections disrupt normal tectal responses to looms. Without this information, larvae are less likely to escape from dark looming stimuli and lose the ability to escape away from the source of the loom. Remarkably, when paired with an isoluminant loom stimulus to the opposite eye, dimming is sufficient to increase startle probability and to reverse the direction of the escape so that it is toward the loom. We suggest that bilateral comparisons of luminance, relayed from the thalamus to the tectum, facilitate escape responses and are essential for their directionality.

Diffuse light-sheet microscopy for stripe-free calcium imaging of neural populations.

Light-sheet microscopy is used extensively in developmental biology and neuroscience. One limitation of this approach is that absorption and scattering produces shadows in the illuminating light sheet, resulting in stripe artifacts. Here, we introduce diffuse light-sheet microscopes that use a line diffuser to randomize the light propagation within the image plane, allowing the light sheets to reform after obstacles. We incorporate diffuse light sheets in two existing configurations: selective plane illumination microscopy in which the sample is illuminated with a static sheet of light, and digitally scanned light sheet (DSLS) in which a thin Gaussian beam is scanned across the image plane during each acquisition. We compare diffuse light-sheet microscopes to their conventional counterparts for calcium imaging of neural activity in larval zebrafish. We show that stripe artifacts can cast deep shadows that conceal some neurons, and that the stripes can flicker, producing spurious signals that could be interpreted as biological activity. Diffuse light-sheets mitigate these problems, illuminating the blind spots produced by stripes and removing artifacts produced by the stripes' movements. The upgrade to diffuse light sheets is simple and inexpensive, especially in the case of DSLS, where it requires the addition of one optical element.

Integrative whole-brain neuroscience in larval zebrafish.

Due to their small size and transparency, zebrafish larvae are amenable to a range of fluorescence microscopy techniques. With the development of sensitive genetically encoded calcium indicators, this has extended to the whole-brain imaging of neural activity with cellular resolution. This technique has been used to study brain-wide population dynamics accompanying sensory processing and sensorimotor transformations, and has spurred the development of innovative closed-loop behavioral paradigms in which stimulus-response relationships can be studied. More recently, microscopes have been developed that allow whole-brain calcium imaging in freely swimming and behaving larvae. In this review, we highlight the technologies underlying whole-brain functional imaging in zebrafish, provide examples of the sensory and motor processes that have been studied with this technique, and discuss the need to merge data from whole-brain functional imaging studies with neurochemical and anatomical information to develop holistic models of functional neural circuits.

APOLs with low pH dependence can kill all African trypanosomes.

The primate-specific serum protein apolipoprotein L1 (APOL1) is the only secreted member of a family of cell death promoting proteins 1-4 . APOL1 kills the bloodstream parasite Trypanosoma brucei brucei, but not the human sleeping sickness agents T.b. rhodesiense and T.b. gambiense 3 . We considered the possibility that intracellular members of the APOL1 family, against which extracellular trypanosomes could not have evolved resistance, could kill pathogenic T. brucei subspecies. Here we show that recombinant APOL3 (rAPOL3) kills all African trypanosomes, including T.b. rhodesiense, T.b. gambiense and the animal pathogens Trypanosoma evansi, Trypanosoma congolense and Trypanosoma vivax. However, rAPOL3 did not kill more distant trypanosomes such as Trypanosoma theileri or Trypanosoma cruzi. This trypanolytic potential was partially shared by rAPOL1 from Papio papio (rPpAPOL1). The differential killing ability of rAPOL3 and rAPOL1 was associated with a distinct dependence on acidic pH for activity. Due both to its instability and toxicity when injected into mice, rAPOL3 cannot be used for the treatment of infection, but an experimental rPpAPOL1 mutant inspired by APOL3 exhibited enhanced trypanolytic activity in vitro and the ability to completely inhibit T.b. gambiense infection in mice. We conclude that pH dependence influences the trypanolytic potential of rAPOLs.

Trypanosoma Infection Favors Brucella Elimination via IL-12/IFNγ-Dependent Pathways.

This study develops an original co-infection model in mice using Brucella melitensis, the most frequent cause of human brucellosis, and Trypanosoma brucei, the agent of African trypanosomiasis. Although the immunosuppressive effects of T. brucei in natural hosts and mice models are well established, we observed that the injection of T. brucei in mice chronically infected with B. melitensis induces a drastic reduction in the number of B. melitensis in the spleen, the main reservoir of the infection. Similar results are obtained with Brucella abortus- and Brucella suis-infected mice and B. melitensis-infected mice co-infected with Trypanosoma cruzi, demonstrating that this phenomenon is not due to antigenic cross-reactivity. Comparison of co-infected wild-type and genetically deficient mice showed that Brucella elimination required functional IL-12p35/IFNγ signaling pathways and the presence of CD4+ T cells. However, the impact of wild type and an attenuated mutant of T. brucei on B. melitensis were similar, suggesting that a chronic intense inflammatory reaction is not required to eliminate B. melitensis. Finally, we also tested the impact of T. brucei infection on the course of Mycobacterium tuberculosis infection. Although T. brucei strongly increases the frequency of IFNγ+CD4+ T cells, it does not ameliorate the control of M. tuberculosis infection, suggesting that it is not controlled by the same effector mechanisms as Brucella. Thus, whereas T. brucei infections are commonly viewed as immunosuppressive and pathogenic, our data suggest that these parasites can specifically affect the immune control of Brucella infection, with benefits for the host.