A single-cell resolution gene expression atlas of the larval zebrafish brain.

The advent of multimodal brain atlases promises to accelerate progress in neuroscience by allowing in silico queries of neuron morphology, connectivity, and gene expression. We used multiplexed fluorescent in situ RNA hybridization chain reaction (HCR) technology to generate expression maps across the larval zebrafish brain for a growing set of marker genes. The data were registered to the Max Planck Zebrafish Brain (mapzebrain) atlas, thus allowing covisualization of gene expression, single-neuron tracings, and expertly curated anatomical segmentations. Using post hoc HCR labeling of the immediate early gene cfos, we mapped responses to prey stimuli and food ingestion across the brain of freely swimming larvae. This unbiased approach revealed, in addition to previously described visual and motor areas, a cluster of neurons in the secondary gustatory nucleus, which express the marker calb2a, as well as a specific neuropeptide Y receptor, and project to the hypothalamus. This discovery exemplifies the power of this new atlas resource for zebrafish neurobiology.

The microbiota promotes social behavior by modulating microglial remodeling of forebrain neurons.

Host-associated microbiotas guide the trajectory of developmental programs, and altered microbiota composition is linked to neurodevelopmental conditions such as autism spectrum disorder. Recent work suggests that microbiotas modulate behavioral phenotypes associated with these disorders. We discovered that the zebrafish microbiota is required for normal social behavior and reveal a molecular pathway linking the microbiota, microglial remodeling of neural circuits, and social behavior in this experimentally tractable model vertebrate. Examining neuronal correlates of behavior, we found that the microbiota restrains neurite complexity and targeting of forebrain neurons required for normal social behavior and is necessary for localization of forebrain microglia, brain-resident phagocytes that remodel neuronal arbors. The microbiota also influences microglial molecular functions, including promoting expression of the complement signaling pathway and the synaptic remodeling factor c1q. Several distinct bacterial taxa are individually sufficient for normal microglial and neuronal phenotypes, suggesting that host neuroimmune development is sensitive to a feature common among many bacteria. Our results demonstrate that the microbiota influences zebrafish social behavior by stimulating microglial remodeling of forebrain circuits during early neurodevelopment and suggest pathways for new interventions in multiple neurodevelopmental disorders.

Visual recognition of social signals by a tectothalamic neural circuit.

Social affiliation emerges from individual-level behavioural rules that are driven by conspecific signals1-5. Long-distance attraction and short-distance repulsion, for example, are rules that jointly set a preferred interanimal distance in swarms6-8. However, little is known about their perceptual mechanisms and executive neural circuits3. Here we trace the neuronal response to self-like biological motion9,10, a visual trigger for affiliation in developing zebrafish2,11. Unbiased activity mapping and targeted volumetric two-photon calcium imaging revealed 21 activity hotspots distributed throughout the brain as well as clustered biological-motion-tuned neurons in a multimodal, socially activated nucleus of the dorsal thalamus. Individual dorsal thalamus neurons encode local acceleration of visual stimuli mimicking typical fish kinetics but are insensitive to global or continuous motion. Electron microscopic reconstruction of dorsal thalamus neurons revealed synaptic input from the optic tectum and projections into hypothalamic areas with conserved social function12-14. Ablation of the optic tectum or dorsal thalamus selectively disrupted social attraction without affecting short-distance repulsion. This tectothalamic pathway thus serves visual recognition of conspecifics, and dissociates neuronal control of attraction from repulsion during social affiliation, revealing a circuit underpinning collective behaviour.

Egr1 Is Necessary for Forebrain Dopaminergic Signaling during Social Behavior.

Finding the link between behaviors and their regulatory molecular pathways is a major obstacle in treating neuropsychiatric disorders. The immediate early gene (IEG) EGR1 is implicated in the etiology of neuropsychiatric disorders, and is linked to gene pathways associated with social behavior. Despite extensive knowledge of EGR1 gene regulation at the molecular level, it remains unclear how EGR1 deficits might affect the social component of these disorders. Here, we examined the social behavior of zebrafish with a mutation in the homologous gene egr1 Mutant fish exhibited reduced social approach and orienting, whereas other sensorimotor behaviors were unaffected. On a molecular level, expression of the dopaminergic biosynthetic enzyme, tyrosine hydroxylase (TH), was strongly decreased in TH-positive neurons of the anterior parvocellular preoptic nucleus. These neurons are connected with basal forebrain (BF) neurons associated with social behavior. Chemogenetic ablation of around 30% of TH-positive neurons in this preoptic region reduced social attraction to a similar extent as the egr1 mutation. These results demonstrate the requirement of Egr1 and dopamine signaling during social interactions, and identify novel circuitry underlying this behavior.

Neural circuitry for stimulus selection in the zebrafish visual system.

When navigating the environment, animals need to prioritize responses to the most relevant stimuli. Although a theoretical framework for selective visual attention exists, its circuit implementation has remained obscure. Here we investigated how larval zebrafish select between simultaneously presented visual stimuli. We found that a mix of winner-take-all (WTA) and averaging strategies best simulates behavioral responses. We identified two circuits whose activity patterns predict the relative saliencies of competing visual objects. Stimuli presented to only one eye are selected by WTA computation in the inner retina. Binocularly presented stimuli, on the other hand, are processed by reciprocal, bilateral connections between the nucleus isthmi (NI) and the tectum. This interhemispheric computation leads to WTA or averaging responses. Optogenetic stimulation and laser ablation of NI neurons disrupt stimulus selection and behavioral action selection. Thus, depending on the relative locations of competing stimuli, a combination of retinotectal and isthmotectal circuits enables selective visual attention.

Rapid Effects of Selection on Brain-wide Activity and Behavior.

Interindividual variation in behavior and brain activity is universal and provides substrates for natural selection [1-9]. Selective pressures shift the expression of behavioral traits at the population level [10, 11], but the accompanying changes of the underlying neural circuitry have rarely been identified [12, 13]. Selection likely acts through the genetic and/or epigenetic underpinnings of neural activity controlling the selected behavior [14]. Endocrine and neuromodulatory systems participate in behavioral diversity and could provide the substrate for evolutionary modifications [15-21]. Here, we examined brain-wide patterns of activity in larval zebrafish selectively bred over two generations for extreme differences in habituation of the acoustic startle response (ASR) [22]. The ASR is an evolutionarily conserved defensive behavior induced by strong acoustic/vibrational stimuli. ASR habituation shows great individual variability that is stable over days and heritable [4, 22]. Selection for high ASR habituation leads to stronger sound-evoked activation of ASR-processing brain areas. In contrast, animals selected for low habituation displayed stronger spontaneous activity in ASR-processing centers. Ablation of dopaminergic tyrosine hydroxylase (TH) neurons decreased ASR sensitivity. Independently selected ASR habituation lineages link the effect of behavioral selection to dopaminergic caudal hypothalamus (HC) neurons [23]. High ASR habituation co-segregated with decreased spontaneous swimming phenotypes, but visual startle responses were unaffected. Furthermore, high- and low-habituation larvae differed in stress responses as adults. Thus, selective pressure over a couple of generations on ASR habituation behavior is able to induce substantial differences in brain activity, carrying along additional behaviors as piggyback traits that might further affect fitness in the wild. VIDEO ABSTRACT.

Learning: Complexities of Habituation in Escaping Zebrafish Larvae.

Animals decrease responses to repeating stimuli through habituation. New research has revealed independent tuning of multiple parameters of zebrafish escape behavior during habituation.

Biological Motion as an Innate Perceptual Mechanism Driving Social Affiliation.

Collective behavior, such as shoaling in teleost fish, is driven by the perceptual recognition of conspecific animals. Because social interactions are mutual, it has been difficult to disentangle the exact sensory cues that trigger affiliation in the first place from those that are emitted by receptive and responsive shoal mates. Here, we overcome this challenge in a virtual reality assay in zebrafish. We discovered that simple visual features of conspecific biological motion provide a potent shoaling cue. Individual juvenile fish shoal for hours with circular black dots projected onto a screen, provided these virtual objects mimic the characteristic kinetics of zebrafish swim bouts. Other naturalistic cues previously implicated in shoaling, such as fish-like shape, pigmentation pattern, or non-visual sensory modalities are not required. During growth, the animals' stimulus preferences shift gradually, matching self-like kinetics, and this tuning exists even in fish raised in isolation. Virtual group interactions and our multi-agent model implementation of this perceptual mechanism demonstrate that kinetic cues can drive assortative shoaling, a phenomenon commonly observed in field studies. Coordinated behavior can emerge from autonomous interactions, such as collective odor avoidance in Drosophila, or from reciprocal interactions, such as the codified turn-taking in wren duet singing. We found that individual zebrafish shoal autonomously without evidence for a reciprocal choreography. Our results reveal individual-level, innate perceptual rules of engagement in mutual affiliation and provide experimental access to the neural mechanisms of social recognition. VIDEO ABSTRACT.

Oscillatory stimuli differentiate adapting circuit topologies.

Biology emerges from interactions between molecules, which are challenging to elucidate with current techniques. An orthogonal approach is to probe for 'response signatures' that identify specific circuit motifs. For example, bistability, hysteresis, or irreversibility are used to detect positive feedback loops. For adapting systems, such signatures are not known. Only two circuit motifs generate adaptation: negative feedback loops (NFLs) and incoherent feed-forward loops (IFFLs). On the basis of computational testing and mathematical proofs, we propose differential signatures: in response to oscillatory stimulation, NFLs but not IFFLs show refractory-period stabilization (robustness to changes in stimulus duration) or period skipping. Applying this approach to yeast, we identified the circuit dominating cell cycle timing. In Caenorhabditis elegans AWA neurons, which are crucial for chemotaxis, we uncovered a Ca2+ NFL leading to adaptation that would be difficult to find by other means. These response signatures allow direct access to the outlines of the wiring diagrams of adapting systems.

A Circuit for Gradient Climbing in C. elegans Chemotaxis.

Animals have a remarkable ability to track dynamic sensory information. For example, the nematode Caenorhabditis elegans can locate a diacetyl odor source across a 100,000-fold concentration range. Here, we relate neuronal properties, circuit implementation, and behavioral strategies underlying this robust navigation. Diacetyl responses in AWA olfactory neurons are concentration and history dependent; AWA integrates over time at low odor concentrations, but as concentrations rise, it desensitizes rapidly through a process requiring cilia transport. After desensitization, AWA retains sensitivity to small odor increases. The downstream AIA interneuron amplifies weak odor inputs and desensitizes further, resulting in a stereotyped response to odor increases over three orders of magnitude. The AWA-AIA circuit drives asymmetric behavioral responses to odor increases that facilitate gradient climbing. The adaptation-based circuit motif embodied by AWA and AIA shares computational properties with bacterial chemotaxis and the vertebrate retina, each providing a solution for maintaining sensitivity across a dynamic range.

MultiFocus Polarization Microscope (MF-PolScope) for 3D polarization imaging of up to 25 focal planes simultaneously.

We have developed an imaging system for 3D time-lapse polarization microscopy of living biological samples. Polarization imaging reveals the position, alignment and orientation of submicroscopic features in label-free as well as fluorescently labeled specimens. Optical anisotropies are calculated from a series of images where the sample is illuminated by light of different polarization states. Due to the number of images necessary to collect both multiple polarization states and multiple focal planes, 3D polarization imaging is most often prohibitively slow. Our MF-PolScope system employs multifocus optics to form an instantaneous 3D image of up to 25 simultaneous focal-planes. We describe this optical system and show examples of 3D multi-focus polarization imaging of biological samples, including a protein assembly study in budding yeast cells.

High-throughput imaging of neuronal activity in Caenorhabditis elegans.

Neuronal responses to sensory inputs can vary based on genotype, development, experience, or stochastic factors. Existing neuronal recording techniques examine a single animal at a time, limiting understanding of the variability and range of potential responses. To scale up neuronal recordings, we here describe a system for simultaneous wide-field imaging of neuronal calcium activity from at least 20 Caenorhabditis elegans animals under precise microfluidic chemical stimulation. This increased experimental throughput was used to perform a systematic characterization of chemosensory neuron responses to multiple odors, odor concentrations, and temporal patterns, as well as responses to pharmacological manipulation. The system allowed recordings from sensory neurons and interneurons in freely moving animals, whose neuronal responses could be correlated with behavior. Wide-field imaging provides a tool for comprehensive circuit analysis with elevated throughput in C. elegans.

Serotonin and the neuropeptide PDF initiate and extend opposing behavioral states in C. elegans.

Foraging animals have distinct exploration and exploitation behaviors that are organized into discrete behavioral states. Here, we characterize a neuromodulatory circuit that generates long-lasting roaming and dwelling states in Caenorhabditis elegans. We find that two opposing neuromodulators, serotonin and the neuropeptide pigment dispersing factor (PDF), each initiate and extend one behavioral state. Serotonin promotes dwelling states through the MOD-1 serotonin-gated chloride channel. The spontaneous activity of serotonergic neurons correlates with dwelling behavior, and optogenetic modulation of the critical MOD-1-expressing targets induces prolonged dwelling states. PDF promotes roaming states through a Gαs-coupled PDF receptor; optogenetic activation of cAMP production in PDF receptor-expressing cells induces prolonged roaming states. The neurons that produce and respond to each neuromodulator form a distributed circuit orthogonal to the classical wiring diagram, with several essential neurons that express each molecule. The slow temporal dynamics of this neuromodulatory circuit supplement fast motor circuits to organize long-lasting behavioral states.