Florian Alois Engert - US grants

[unreadable] DESCRIPTION (provided by applicant): Over the next 5 years, the main objective of this project will be to analyze visually evoked synaptic activity in tectal neurons of Xenopus tadpoles. In particular, the temporal and spatial patterns of active regions on the dendritic trees will be examined during development in general and also during periods of induced changes in synaptic strength. To this end patterned visual stimulation and multiple patch-clamp recordings of tectal cells will be combined with simultaneous multi-photon laser scanning calcium imaging. Patch clamp recordings will provide the amplitude and the fast temporal dynamics of synaptic responses and two-photon calcium imaging will reveal the spatial pattern in three dimensions. Synapses will be activated by direct electrical stimulation of retinal ganglion cells as well as by visual images presented to the retina. To induce changes in synaptic connectivity several electrical and visual stimulation paradigms will be used. These experiments will provide new information about dendritic integration of complex physiological inputs and about the development of receptive fields in the vertebrate visual system. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Project Abstract/Summary Elucidating the structure and function of brain circuits is arguably this century's greatest scientific challenge. Pioneering studies in invertebrates have established that this challenge can be met by combining anatomical circuit diagrams ("connectomes"), neuronal activity maps and defined behavioral assays. Recent technological progress now makes it possible to apply this approach to the vertebrate nervous system. The main goal of this proposal is to generate image data sets that are based on serial electron microscopy and have sufficient scale and resolution to create maps of the full set of neurons and synaptic connections in the larval zebrafish brain. The ability to reconstruct local circuits within that data set will be tested for small and well- described sub-networks, the trigeminal sensory ganglion and the optic tectum. To relate structure and function in these dedicated sub-circuits, functional calcium imaging will be combined with EM reconstruction. The combination of the translucence, relative simplicity and small size of the larval zebrafish's nervous system brings this otherwise daunting task into the realm of feasibility. We have assembled a team of four groups: Drs. Reid and Lichtman provide expertise and facilities for serial EM reconstruction. Drs. Engert and Schier provide the functional imaging technology based on in vivo 2-photon laser scanning. The experiments proposed here will provide the foundation to reconstruct the circuit diagram of an entire vertebrate brain and combine sub-circuit anatomy and function in the context of a behaving animal. The EM datasets will be available through open access via a web-based data storage and retrieval system. PUBLIC HEALTH RELEVANCE: Project Narrative We propose to examine the structure and function of the larval zebrafish brain at sub- cellular resolution. We will combine functional imaging using calcium indicators with serial electron microscopy-assisted reconstruction of the zebrafish brain.

DESCRIPTION (provided by applicant): Existing techniques for monitoring neural activity in awake, behaving vertebrates are invasive and often require restraining the animal. Here we propose the use of bioluminescence to non- invasively monitor the activity of genetically specified neurons in freely behaving zebrafish. The photoprotein GFP-apoAequorin (Ga) will be expressed in neurons of larval zebrafish and constituted in vivo with its substrate coelenterazine (CLZN) to form the Ca2+-sensitive bioluminescent sensor GFP-Aequorin (GA). Flashes of luminescence will then report spontaneous and evoked Ca2+ signals in the targeted neurons. These 'neuroluminescence'responses can be recorded with a large-area photon-counting detector while simultaneously monitoring behavior with an infrared-sensitive camera. Pilot studies have shown that transgenic, pan-neuronal GA fish produced large and fast neuroluminescent signals that could be recorded continuously for many days. The relationship of these light signals with the neurons underlying electrical activity will be explored by targeting patch-clamp recordings to individual neurons expressing the protein under pan-neuronal promoters. To explore the limits and the sensitivity of this technique, GA will specifically be targeted to the hypocretin-positive neurons of the hypothalamus, the serotonergic neurons of the raphe nuclei and the dopaminergic neurons of the ventral hypothalamus. To overcome a major limitation of existing bioluminescence monitoring strategies, which require a completely dark environment, we propose an extension of this method for fast temporal gating that is able to count single photons during normal lighting conditions. Thus, this assay will allow us to monitor, with high temporal resolution and stability, the activity of small subsets of neurons during unrestrained, visual behavior over a time period of many days. We believe that the fast, stable properties of GA's report of neural activity along with non- imaging detection strategies can provide a useful, easily implemented tool for monitoring the activity of genetically specified cell types during natural behavior;an attractive alternative to other more technically challenging imaging approaches currently being pursued. PUBLIC HEALTH RELEVANCE: The ability to record from genetically identified neurons in freely behaving animals is highly desirable in modern neuroscience. We propose here a technology based on bioluminescence that exploits the translucence of the larval zebrafish combined with its availability for genetic manipulation. Bioluminescent and calcium sensitive proteins can be targeted to specific neurons of interest and will report neural activity with high temporal resolution and stability, during unrestrained, visual behavior over a time period of many days.

DESCRIPTION Abstract: In order to study the neural basis of navigation, learning, and memory, it is important to simultaneously record the activity of large populations of neurons involved in execution of behavior. This has been difficult or impossible in awake, freely moving preparations and the anesthetics and paralytics used to facilitate recordings often alter or abolish the relevant behaviors. I propose to establish a fictive swimming assay that allows paralyzed larval zebrafish to navigate through- and interact with a virtual environment. The sensory feedback provided to the animals will consist of visual cues through video projection screens and water flow provided by a computer controlled valve system. The fish are allowed to control these stimuli via activity in their motor-neurons recorded by a set of external suction electrodes. This arrangement makes it possible for immobilized fish to navigate a virtual environment entirely through power of activity in their motorneurons. As a first step we will combine this assay with 2-photon laser-scanning microscopy and allow transgenic fish lines that express genetically encoded calcium indicators in all neurons to navigate through virtual environments while neuronal activity is monitored. The small size and perfect translucence of the larval zebrafish provides in principle no barrier or restriction to these recordings and thus we have access to the whole brain at single cell resolution during these behavioral tasks. We can thus independently monitor the activities of hundreds or thousands of neurons as animals navigate their virtual worlds. As a subsequent step we will implement different learning assays that the animals have to execute in this virtual environment. In principle this will allow us to follow the flow of neural information in a single animal, before, during and after specific training sessions. The large volumes of resulting data will be analyzed and distilled with automated algorithms and the results used to sketc

? DESCRIPTION (provided by applicant): We propose to combine whole brain 2-photon imaging of neural activity in behaving larval zebrafish with detailed anatomical and connectivity information extracted from the same animals. The final goal is to generate quantitative models of brain wide neural circuits that explain the dynamic processing of sensory information as well as the generation of motor output by these circuits. Anatomical data will be generated by two complementary technologies: 1) whole brain EM data sets will be prepared from the same fish that were used for calcium imaging. Respective data sets will be registered to each other, functionally relevant neuronal ensembles will then be identified in the EM stacks and connectivity will be analyzed in these sub-networks via sparse reconstruction. 2) EM based connectivity information will be supplemented by trans-synaptic viral tracing technology. These two technologies for identifying synaptic connections have complementary strengths and weaknesses and are thus ideally suited for combination with in-vivo 2-photon calcium imaging studies. The specific power of this approach is that all three techniques, whole brain calcium imaging, viral tracing and EM reconstruction, can be done in the same animal. Functional, anatomical and behavioral data can then be analyzed in the context of the specific stimuli and quantified behavioral output and subsequently synthesized into a theoretical framework. To that end we will start with quantitative models of simple reflex behaviors, like the optomotor and optokinetic reflex, where the transformation of sensory input to motor output is relatively straightforward and well defined. These elementary models will serve as a scaffold that can be refined and complemented by additional data from structure function studies from fish performing in more sophisticated behavioral assays that involve more complex stimuli, different modalities and plastic changes. As such the process of building such a virtual fish will be an iterative, open ended process that requires continuous and bidirectional exchange of information between the theoretical and experimental groups of the research team.

DESCRIPTION (provided by applicant): The zebra fish has emerged as a powerful model system to study neural development, physiology, and disease. It is ideal for this particular purpose since it is a small and translucent vertebrate with a wide behavioral repertoire. Despite this progress, there are still comparatively few transgenic lines that allow the full exploitation f optogenetic approaches. For example, the field still lacks specific drivers and reporters for most neurotransmitters or peptides, and existing lines that mark specific brain regions or nuclei often lack the necessary specificity. The zebra fish as a model system is thus still lacking the essential tool set that the Drosophila community has built in the last decade. To overcome these limitations, we propose to generate transgenic lines that allow the specific expression of proteins of interest in restricted subpopulations of neurons that are specified by brain region and/or by neurotransmitter subtype. We will use enhancer trapping to generate 240 lines that drive region- and cell- type specific neural expression via a modified Gal4 vector (Aim 1), and we will use genome engineering to generate 12 lines that drive expression in neurons producing the major classes of neurotransmitters (Aim 2). An essential component of the enterprise will be the design and establishment of an online interface that will provide details of each transgenic line. Researchers will be provided with extensive details of each line's characteristics and well-annotated image datasets. The Engert and Schier lab have extensive expertise in anatomical and functional imaging of the zebra fish nervous system as well as the molecular and genetic toolbox that is necessary for the proposed project. The project will be further supported by an advisory committee consisting of leaders in the field of zebra fish neurobiology.

Administrative Core-Abstract The overall goal of the Administrative Core is to integrate the multiple components of this multi-institutional, interdisciplinary team science proposal. The approach focuses on three specific aims, to manage the research endeavor, to manage the finances and reporting, and to form a coherent community of scientists. The Administrative Core will be integrated within the existing professional administration of the Center for Brain Science, augmented by a full-time administrator dedicate to this project. This Administrative Core will be organized within a clear governance structure and explicit procedural guidelines will be put in place for decision-making and conflict resolution.

Project 2 - Quantitative behavioral analysis and imaging - Abstract Behavior is the foundation for systems neuroscience. Its quantitative description and algorithmic analyses need to be the first and essential step on the journey to generating a realistic circuit model of any brain. Whether fly, fish, rat or human, for an animal to exhibit purposeful behavior, it must be endowed with the computational ability to map patterns of sensory input to patterns of motor output. The quantifiable rules of sensory-motor mapping define the algorithms of behavioral strategy. These computational rules determine what the underlying neural circuits are actually doing. The critical test of a nervous system is whether it can provide computational solutions for behavioral challenges that the animal faces in natural environments. As such, it is the behavioral algorithms that dictate the questions and framework for any model of neural implementation and it is therefore necessary to turn behavior into a conceptual framework that can fully integrate genetic, anatomical, and physiological data. In the first part of Project 2, we describe a fleet of behavioral set-ups and assays that allow the quantitative description of fish behavior at various levels of detail and throughput. To perform a neural implementation of these algorithms and generate a realistic whole-brain model for larval zebrafish we need, in addition to knowledge about the anatomical structure of the circuits, detailed information about neural activity patterns during behavior. To that end, we have designed a variety of assays that are compatible with brainwide imaging of neural activity in freely swimming and tethered larval zebrafish. These assays in conjunction with Ca2? + imaging are described in the second part of Project 2. Furthermore, tests for causality and the rigorous validation of circuit models require targeted perturbation experiments, where identified neuronal cell types can be silenced or activated in a targeted way. The tethered behavioral assays described in this project provide the ideal setting to perform such perturbations in the context of a controlled behavioral paradigm. We conclude, in the third part of Project 2, by describing a specific application of these assays that shed light on the role that various modulatory neurotransmitters, such as oxytocin and serotonin, play in regulating internal states involving hunger, stress or loneliness.

Project Summary - A realistic multiscale circuit model of the larval zebrafish brain The working group of the BRAIN initiative (BRAIN 2025, a Scientific Vision) identified ?the analysis of circuits of interacting neurons as being particularly rich in opportunity, with potential for revolutionary advances?. They further pointed out that ?truly understanding a circuit requires identifying and characterizing the component cells, defining their synaptic connections with one another, observing their dynamic patterns of activity as their circuit functions in vivo during behavior, and perturbing these patterns to test their significance. It also requires an understanding of the algorithms that govern information processing within a circuit and between interacting circuits in the brain as a whole?. We propose to generate a realistic multiscale circuit model of the larval zebrafish brain ? the multiscale virtual fish (MVF), which is well aligned with the BRAIN initiative's guidelines. The model will be based on algorithms inferred from behavioral assays and it will span spatial ranges across three levels: from the nanoscale at the synaptic level, to the microscale describing local circuits, to the macroscale brain-wide activity patterns distributed across many regions. The model will be constrained and validated by optogenetic interrogation and sparse connectomics of identified circuit elements 1? ,2?. The ultimate purpose is to explain and simulate the quantitative and qualitative nature of behavioral outputs in response to sensory inputs across various timescales, and to explore how these findings might integrate with parallel work in two other important behavioral model systems, ? the ?Drosophila larva and the rat. Our prior U01 project achieved the first instantiation of this model, whereby we successfully dissected the optomotor response (OMR)1? ?, where a larval zebrafish will turn and swim to match the direction of a whole-field visual stimulus ?3?5.? We will build on this model by achieving three further aims: First, we will expand the OMR project with four additional ethologically relevant behaviors: phototaxis, rheotaxis, escape, and hunting. We will extract the precise algorithms underlying each behavior and develop a version of the circuit model to understand their neural implementation. Second, we will further refine the model to account for multimodal integration and decision making, events that naturally happen when conflicting stimuli driving different behaviors are presented simultaneously. For example, a fish might be driven to execute a left turn by whole field motion moving to the left (OMR), while simultaneously being induced to turn right by increased brightness on its right side (phototaxis). Third, we will examine how internal brain states, such as hunger or stress, influence and modulate the specific behaviors (Aim 1) or behavioral interactions (Aim 2). Implementation of neurochemical modulation into the framework of the MVF will be achieved through simulation of highly conserved neuromodulatory neurotransmitter systems such as serotonin, acetylcholine, epinephrine and dopamine. To uncover generalizable principles of circuit design and function, we will compare our findings with those from two other model systems, the fruit fly larva and the rat. This will serve to elucidate the rules, motifs and algorithms of neural circuit function that transcend the potential idiosyncrasies of any given model.

In recent years, the zebrafish has emerged as an important model organism as researchers have become able to obtain detailed information about the connectivity and activity of its brain. The laboratory of the Principal Investigator recently presented work that makes use of this wealth of available information to better understand zebrafish behavior at the level of specific neural circuitry. To that end, a realistic network model, composed of experimental verified functional cell types, was developed that accurately models the fish's tendency to swim in a particular direction in response to a moving visual stimulus. The purpose of this collaboration project is to develop a similar theoretical circuit model for mechanosensory-based rheotaxis, a more sophisticated behavior that also is displayed readily by larval zebrafish. The project combines experimental and computational approaches, and constitutes a significant step towards establishing the larval zebrafish as a vertebrate model where whole-brain imaging can be combined with whole-brain circuit modelling in order to generate a unified theoretical basis for the holistic study of neural circuits. The project also provides undergraduate and graduate students from diverse backgrounds opportunities to receive hands-on laboratory experiences.

As a first step towards the scientific goal of the project, the investigators demonstrated that larval zebrafish can perform efficient rheotaxis in complete darkness and in the absence of any other direct cues from the external reference frame. Furthermore, the investigators showed that this behavior requires the presence of a flow velocity gradient, and presented behavioral data that support a novel algorithm that fish use to efficiently navigate laminar flow: detailed behavioral analysis shows that fish use the hair-cells of their lateral line to measure (1) the curl of the local velocity vector field to detect the presence of flow, and (2) the temporal change in curl magnitude following swim bouts to deduce flow direction. As such, a precise and predictive algorithm is presented, whose quantitative description allows to examine its neural implementation, and eventually to generate a realistic and testable circuit model of all brain areas involved in executing this computation. The complementary expertise of the researchers of the two participating laboratories allows testing, verification, and refinement of this model-circuit based on an iterative combination of whole-brain imaging, genetic perturbations and quantitative modeling. A companion project is being funded by the Federal Ministry of Education and Research, Germany (BMBF).

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.