Escape behaviors deliver organisms away from imminent catastrophe. Here, we characterize behavioral responses of freely swimming larval zebrafish to looming visual stimuli simulating predators. We ...report that the visual system alone can recruit lateralized, rapid escape motor programs, similar to those elicited by mechanosensory modalities. Two-photon calcium imaging of retino-recipient midbrain regions isolated the optic tectum as an important center processing looming stimuli, with ensemble activity encoding the critical image size determining escape latency. Furthermore, we describe activity in retinal ganglion cell terminals and superficial inhibitory interneurons in the tectum during looming and propose a model for how temporal dynamics in tectal periventricular neurons might arise from computations between these two fundamental constituents. Finally, laser ablations of hindbrain circuitry confirmed that visual and mechanosensory modalities share the same premotor output network. We establish a circuit for the processing of aversive stimuli in the context of an innate visual behavior.
•Larval zebrafish escape from looming stimuli after a critical image size is reached•Population activity of neurons in the optic tectum encodes critical image size•Modeling predicts the critical role of characterized cell types in the retina and tectum•Motor output is conveyed via multimodal circuitry in the hindbrain
Dunn et al. characterize the parameters influencing visually evoked escape behavior in larval zebrafish. Via large-scale functional imaging, the authors identify the neural circuits underlying the behavior and provide a mechanistic model that incorporates newly classified neural response types.
Detailed descriptions of brain-scale sensorimotor circuits underlying vertebrate behavior remain elusive. Recent advances in zebrafish neuroscience offer new opportunities to dissect such circuits ...via whole-brain imaging, behavioral analysis, functional perturbations, and network modeling. Here, we harness these tools to generate a brain-scale circuit model of the optomotor response, an orienting behavior evoked by visual motion. We show that such motion is processed by diverse neural response types distributed across multiple brain regions. To transform sensory input into action, these regions sequentially integrate eye- and direction-specific sensory streams, refine representations via interhemispheric inhibition, and demix locomotor instructions to independently drive turning and forward swimming. While experiments revealed many neural response types throughout the brain, modeling identified the dimensions of functional connectivity most critical for the behavior. We thus reveal how distributed neurons collaborate to generate behavior and illustrate a paradigm for distilling functional circuit models from whole-brain data.
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•Optomotor response is driven asymmetrically by visual motion to each eye•Dedicated circuits differentially process eye- and direction-specific motion•Neural representations are distributed over select overrepresented response types•Behavior and neural activity are captured by realistic whole-brain circuit model
Whole-brain imaging and behavioral analysis combined with network modeling reveal key circuit elements contributing to a complex sensorimotor behavior in zebrafish larvae and provide a framework for building brain-level circuit models.
In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of ...larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.
In the absence of salient sensory cues to guide behavior, animals must still execute sequences of motor actions in order to forage and explore. How such successive motor actions are coordinated to ...form global locomotion trajectories is unknown. We mapped the structure of larval zebrafish swim trajectories in homogeneous environments and found that trajectories were characterized by alternating sequences of repeated turns to the left and to the right. Using whole-brain light-sheet imaging, we identified activity relating to the behavior in specific neural populations that we termed the anterior rhombencephalic turning region (ARTR). ARTR perturbations biased swim direction and reduced the dependence of turn direction on turn history, indicating that the ARTR is part of a network generating the temporal correlations in turn direction. We also find suggestive evidence for ARTR mutual inhibition and ARTR projections to premotor neurons. Finally, simulations suggest the observed turn sequences may underlie efficient exploration of local environments.
•Zebrafish brain-scale imaging is routinely used to identify relevant regions.•Many neurons in multiple brain areas cooperate to generate behaviors.•Specific circuit mechanisms underlying various ...behaviors are being elucidated.•Causal interrogation, dynamic monitoring, and modelling will be necessary.•Computational tools should integrate acquisition, analysis, and data sharing.
Detailed quantification of neural dynamics across the entire brain will be the key to genuinely understanding perception and behavior. With the recent developments in microscopy and biosensor engineering, the zebrafish has made a grand entrance in neuroscience as its small size and optical transparency enable imaging access to its entire brain at cellular and even subcellular resolution. However, until recently many neurobiological insights were largely correlational or provided little mechanistic insight into the brain-wide population dynamics generated by diverse types of neurons. Now with increasingly sophisticated behavioral, imaging, and causal intervention paradigms, zebrafish are revealing how entire vertebrate brains function. Here we review recent research that fulfills promises made by the early wave of technical advances. These studies reveal new features of brain-wide neural processing and the importance of integrative investigation and computational modelling. Moreover, we outline the future tools necessary for solving broader brain-scale circuit problems.
Existing techniques for monitoring neural activity in awake, freely behaving vertebrates are invasive and difficult to target to genetically identified neurons. We used bioluminescence to ...non-invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. Transgenic fish with the Ca(2+)-sensitive photoprotein green fluorescent protein (GFP)-Aequorin in most neurons generated large and fast bioluminescent signals that were related to neural activity, neuroluminescence, which could be recorded continuously for many days. To test the limits of this technique, we specifically targeted GFP-Aequorin to the hypocretin-positive neurons of the hypothalamus. We found that neuroluminescence generated by this group of approximately 20 neurons was associated with periods of increased locomotor activity and identified two classes of neural activity corresponding to distinct swim latencies. Our neuroluminescence assay can report, with high temporal resolution and sensitivity, the activity of small subsets of neurons during unrestrained behavior.
The dynamics of living organisms are organized across many spatial scales. However, current cost-effective imaging systems can measure only a subset of these scales at once. We have created a ...scalable multi-camera array microscope (MCAM) that enables comprehensive high-resolution recording from multiple spatial scales simultaneously, ranging from structures that approach the cellular scale to large-group behavioral dynamics. By collecting data from up to 96 cameras, we computationally generate gigapixel-scale images and movies with a field of view over hundreds of square centimeters at an optical resolution of 18 µm. This allows us to observe the behavior and fine anatomical features of numerous freely moving model organisms on multiple spatial scales, including larval zebrafish, fruit flies, nematodes, carpenter ants, and slime mold. Further, the MCAM architecture allows stereoscopic tracking of the z-position of organisms using the overlapping field of view from adjacent cameras. Overall, by removing the bottlenecks imposed by single-camera image acquisition systems, the MCAM provides a powerful platform for investigating detailed biological features and behavioral processes of small model organisms across a wide range of spatial scales.
Schizophrenia is a highly heritable disorder with diverse mental and somatic symptoms. The molecular mechanisms leading from genes to disease pathology in schizophrenia remain largely unknown. ...Genome-wide association studies (GWASs) have shown that common single-nucleotide polymorphisms associated with specific diseases are enriched in the recognition sequences of transcription factors that regulate physiological processes relevant to the disease. We have used a "bottom-up" approach and tracked a developmental trajectory from embryology to physiological processes and behavior and recognized that the transcription factor NK2 homeobox 1 (NKX2-1) possesses properties of particular interest for schizophrenia. NKX2-1 is selectively expressed from prenatal development to adulthood in the brain, thyroid gland, parathyroid gland, lungs, skin, and enteric ganglia, and has key functions at the interface of the brain, the endocrine-, and the immune system. In the developing brain, NKX2-1-expressing progenitor cells differentiate into distinct subclasses of forebrain GABAergic and cholinergic neurons, astrocytes, and oligodendrocytes. The transcription factor is highly expressed in mature limbic circuits related to context-dependent goal-directed patterns of behavior, social interaction and reproduction, fear responses, responses to light, and other homeostatic processes. It is essential for development and mature function of the thyroid gland and the respiratory system, and is involved in calcium metabolism and immune responses. NKX2-1 interacts with a number of genes identified as susceptibility genes for schizophrenia. We suggest that NKX2-1 may lie at the core of several dose dependent pathways that are dysregulated in schizophrenia. We correlate the symptoms seen in schizophrenia with the temporal and spatial activities of NKX2-1 in order to highlight promising future research areas.
In optogenetics, light-activated proteins are used to monitor and modulate cellular behaviour with light. Combining genetic targeting of distinct cellular populations with defined patterns of optical ...stimulation enables one to study specific cell classes in complex biological tissues. In the current study we attempted to investigate the functional relevance of heterocellular electrotonic coupling in cardiac tissue in situ. In order to do that, we used a Cre-Lox approach to express the light-gated cation channel Channelrhodopsin-2 (ChR2) specifically in either cardiac myocytes or non-myocytes. Despite high specificity when using the same Cre driver lines in a previous study in combination with a different optogenetic probe, we found patchy off-target ChR2 expression in cryo-sections and extended z-stack imaging through the ventricular wall of hearts cleared using CLARITY. Based on immunohistochemical analysis, single-cell electrophysiological recordings and whole-genome sequencing, we reason that non-specificity is caused on the Cre recombination level. Our study highlights the importance of careful design and validation of the Cre recombination targets for reliable cell class specific expression of optogenetic tools.
One of the primary goals of systems neuroscience is to relate the structure of neural circuits to their function, yet patterns of connectivity are difficult to establish when recording from large ...populations in behaving organisms. Many previous approaches have attempted to estimate functional connectivity between neurons using statistical modeling of observational data, but these approaches rely heavily on parametric assumptions and are purely correlational. Recently, however, holographic photostimulation techniques have made it possible to precisely target selected ensembles of neurons, offering the possibility of establishing direct causal links. Here, we propose a method based on noisy group testing that drastically increases the efficiency of this process in sparse networks. By stimulating small ensembles of neurons, we show that it is possible to recover binarized network connectivity with a number of tests that grows only logarithmically with population size under minimal statistical assumptions. Moreover, we prove that our approach, which reduces to an efficiently solvable convex optimization problem, can be related to Variational Bayesian inference on the binary connection weights, and we derive rigorous bounds on the posterior marginals. This allows us to extend our method to the streaming setting, where continuously updated posteriors allow for optional stopping, and we demonstrate the feasibility of inferring connectivity for networks of up to tens of thousands of neurons online. Finally, we show how our work can be theoretically linked to compressed sensing approaches, and compare results for connectivity inference in different settings.
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