Norepinephrine changes behavioral state via astroglial purinergic signaling.

Both neurons and glia communicate via diffusible neuromodulatory substances, but the substrates of computation in such neuromodulatory networks are unclear. During behavioral transitions in the larval zebrafish, the neuromodulator norepinephrine drives fast excitation and delayed inhibition of behavior and circuit activity. We find that the inhibitory arm of this feedforward motif is implemented by astroglial purinergic signaling. Neuromodulator imaging, behavioral pharmacology, and perturbations of neurons and astroglia reveal that norepinephrine triggers astroglial release of adenosine triphosphate, extracellular conversion into adenosine, and behavioral suppression through activation of hindbrain neuronal adenosine receptors. This work, along with a companion piece by Lefton and colleagues demonstrating an analogous pathway mediating the effect of norepinephrine on synaptic connectivity in mice, identifies a computational and behavioral role for an evolutionarily conserved astroglial purinergic signaling axis in norepinephrine-mediated behavioral and brain state transitions.

Functional neuronal circuits emerge in the absence of developmental activity.

The complex neuronal circuitry of the brain develops from limited information contained in the genome. After the genetic code instructs the birth of neurons, the emergence of brain regions, and the formation of axon tracts, it is believed that temporally structured spiking activity shapes circuits for behavior. Here, we challenge the learning-dominated assumption that spiking activity is required for circuit formation by quantifying its contribution to the development of visually-guided swimming in the larval zebrafish. We found that visual experience had no effect on the emergence of the optomotor response (OMR) in dark-reared zebrafish. We then raised animals while pharmacologically silencing action potentials with the sodium channel blocker tricaine. After washout of the anesthetic, fish could swim and performed with 75-90% accuracy in the OMR paradigm. Brain-wide imaging confirmed that neuronal circuits came 'online' fully tuned, without requiring activity-dependent plasticity. Thus, complex sensory-guided behaviors can emerge through activity-independent developmental mechanisms.

Seeing stars: Astroglia modulate visual circuits during behavioral-state transitions.

In this issue of Neuron, Uribe-Arias et al.1 show that, in larval zebrafish, astrocyte-like cells exhibit calcium responses to norepinephrine during behavioral-state transitions and alter neuronal response properties. Thus, astroglia can sculpt neuronal dynamics in behaviorally meaningful ways.

Functional and pharmacological analyses of visual habituation learning in larval zebrafish.

Habituation allows animals to learn to ignore persistent but inconsequential stimuli. Despite being the most basic form of learning, a consensus model on the underlying mechanisms has yet to emerge. To probe relevant mechanisms, we took advantage of a visual habituation paradigm in larval zebrafish, where larvae reduce their reactions to abrupt global dimming (a dark flash). We used Ca2+ imaging during repeated dark flashes and identified 12 functional classes of neurons that differ based on their rate of adaptation, stimulus response shape, and anatomical location. While most classes of neurons depressed their responses to repeated stimuli, we identified populations that did not adapt or that potentiated their response. These neurons were distributed across brain areas, consistent with a distributed learning process. Using a small-molecule screening approach, we confirmed that habituation manifests from multiple distinct molecular mechanisms, and we have implicated molecular pathways in habituation, including melatonin, oestrogen, and GABA signalling. However, by combining anatomical analyses and pharmacological manipulations with Ca2+ imaging, we failed to identify a simple relationship between pharmacology, altered activity patterns, and habituation behaviour. Collectively, our work indicates that habituation occurs via a complex and distributed plasticity processes that cannot be captured by a simple model. Therefore, untangling the mechanisms of habituation will likely require dedicated approaches aimed at sub-component mechanisms underlying this multidimensional learning process.

Olfactory detection of viruses shapes brain immunity and behavior in zebrafish.

Olfactory sensory neurons (OSNs) are constantly exposed to pathogens, including viruses. However, serious brain infection via the olfactory route rarely occurs. When OSNs detect a virus, they coordinate local antiviral immune responses to stop virus progression to the brain. Despite effective immune control in the olfactory periphery, pathogen-triggered neuronal signals reach the CNS via the olfactory bulb (OB). We hypothesized that neuronal detection of a virus by OSNs initiates neuroimmune responses in the OB that prevent pathogen invasion. Using zebrafish ( Danio rerio ) as a model, we demonstrate viral-specific neuronal activation of OSNs projecting into the OB, indicating that OSNs are electrically activated by viruses. Further, behavioral changes are seen in both adult and larval zebrafish after viral exposure. By profiling the transcription of single cells in the OB after OSNs are exposed to virus, we found that both microglia and neurons enter a protective state. Microglia and macrophage populations in the OB respond within minutes of nasal viral delivery followed decreased expression of neuronal differentiation factors and enrichment of genes in the neuropeptide signaling pathway in neuronal clusters. Pituitary adenylate-cyclase-activating polypeptide ( pacap ), a known antimicrobial, was especially enriched in a neuronal cluster. We confirm that PACAP is antiviral in vitro and that PACAP expression increases in the OB 1 day post-viral treatment. Our work reveals how encounters with viruses in the olfactory periphery shape the vertebrate brain by inducing antimicrobial programs in neurons and by altering host behavior.

A Visual Interface for Exploring Hypotheses about Neural Circuits.

One of the fundamental problems in neurobiological research is to understand how neural circuits generate behaviors in response to sensory stimuli. Elucidating such neural circuits requires anatomical and functional information about the neurons that are active during the processing of the sensory information and generation of the respective response, as well as an identification of the connections between these neurons. With modern imaging techniques, both morphological properties of individual neurons as well as functional information related to sensory processing, information integration and behavior can be obtained. Given the resulting information, neurobiologists are faced with the task of identifying the anatomical structures down to individual neurons that are linked to the studied behavior and the processing of the respective sensory stimuli. Here, we present a novel interactive tool that assists neurobiologists in the aforementioned task by allowing them to extract hypothetical neural circuits constrained by anatomical and functional data. Our approach is based on two types of structural data: brain regions that are anatomically or functionally defined, and morphologies of individual neurons. Both types of structural data are interlinked and augmented with additional information. The presented tool allows the expert user to identify neurons using Boolean queries. The interactive formulation of these queries is supported by linked views, using, among other things, two novel 2D abstractions of neural circuits. The approach was validated in two case studies investigating the neural basis of vision-based behavioral responses in zebrafish larvae. Despite this particular application, we believe that the presented tool will be of general interest for exploring hypotheses about neural circuits in other species, genera and taxa.

Neural circuits underlying habituation of visually evoked escape behaviors in larval zebrafish.

Larval zebrafish that are exposed repeatedly to dark looming stimuli will quickly habituate to these aversive signals and cease to respond with their stereotypical escape swims. A dark looming stimulus can be separated into two independent components: one that is characterized by an overall spatial expansion, where overall luminance is maintained at the same level, and a second, that represents an overall dimming within the whole visual field in the absence of any motion energy. Using specific stimulation patterns that isolate these independent components, we first extracted the behavioral algorithms that dictate how these separate information channels interact with each other and across the two eyes during the habituation process. Concurrent brain wide imaging experiments then permitted the construction of circuit models that suggest the existence of two separate neural pathways. The first is a looming channel which responds specifically to expanding edges presented to the contralateral eye and relays that information to the brain stem escape network to generate directed escapes. The second is a dimming-specific channel that could be either monocular or binocularly responsive, and that appears to specifically inhibit escape response when activated. We propose that this second channel is under strong contextual modulation and that it is primarily responsible for the incremental silencing of successive dark looming-evoked escapes.

Visual object detection biases escape trajectories following acoustic startle in larval zebrafish.

In this study, we investigated whether the larval zebrafish is sensitive to the presence of obstacles in its environment. Zebrafish execute fast escape swims when in danger of predation. We posited that collisions with solid objects during escape would be maladaptive to the fish, and therefore, the direction of escape swims should be informed by the locations of barriers. To test this idea, we developed a closed-loop imaging rig outfitted with barriers of various qualities. We show that when larval zebrafish escape in response to a non-directional vibrational stimulus, they use visual scene information to avoid collisions with obstacles. Our study demonstrates that barrier avoidance rate corresponds to the absolute distance of obstacles, as distant barriers outside of collision range elicit less bias than nearby collidable barriers that occupy the same amount of visual field. The computation of barrier avoidance is covert: the fact that fish will avoid barriers during escape cannot be predicted by its routine swimming behavior in the barrier arena. Finally, two-photon laser ablation experiments suggest that excitatory bias is provided to the Mauthner cell ipsilateral to approached barriers, either via direct excitation or a multi-step modulation process. We ultimately propose that zebrafish detect collidable objects via an integrative visual computation that is more complex than retinal occupancy alone, laying a groundwork for understanding how cognitive physical models observed in humans are implemented in an archetypal vertebrate brain. VIDEO ABSTRACT.

Social isolation modulates appetite and avoidance behavior via a common oxytocinergic circuit in larval zebrafish.

Animal brains have evolved to encode social stimuli and transform these representations into advantageous behavioral responses. The commonalities and differences of these representations across species are not well-understood. Here, we show that social isolation activates an oxytocinergic (OXT), nociceptive circuit in the larval zebrafish hypothalamus and that chemical cues released from conspecific animals are potent modulators of this circuit's activity. We delineate an olfactory to subpallial pathway that transmits chemical social cues to OXT circuitry, where they are transformed into diverse outputs simultaneously regulating avoidance and feeding behaviors. Our data allow us to propose a model through which social stimuli are integrated within a fundamental neural circuit to mediate diverse adaptive behaviours.

Voltage imaging identifies spinal circuits that modulate locomotor adaptation in zebrafish.

Motor systems must continuously adapt their output to maintain a desired trajectory. While the spinal circuits underlying rhythmic locomotion are well described, little is known about how the network modulates its output strength. A major challenge has been the difficulty of recording from spinal neurons during behavior. Here, we use voltage imaging to map the membrane potential of large populations of glutamatergic neurons throughout the spinal cord of the larval zebrafish during fictive swimming in a virtual environment. We characterized a previously undescribed subpopulation of tonic-spiking ventral V3 neurons whose spike rate correlated with swimming strength and bout length. Optogenetic activation of V3 neurons led to stronger swimming and longer bouts but did not affect tail beat frequency. Genetic ablation of V3 neurons led to reduced locomotor adaptation. The power of voltage imaging allowed us to identify V3 neurons as a critical driver of locomotor adaptation in zebrafish.

Functional and ultrastructural analysis of reafferent mechanosensation in larval zebrafish.

All animals need to differentiate between exafferent stimuli, which are caused by the environment, and reafferent stimuli, which are caused by their own movement. In the case of mechanosensation in aquatic animals, the exafferent inputs are water vibrations in the animal's proximity, which need to be distinguishable from the reafferent inputs arising from fluid drag due to locomotion. Both of these inputs are detected by the lateral line, a collection of mechanosensory organs distributed along the surface of the body. In this study, we characterize in detail how hair cells-the receptor cells of the lateral line-in zebrafish larvae discriminate between such reafferent and exafferent signals. Using dye labeling of the lateral line nerve, we visualize two parallel descending inputs that can influence lateral line sensitivity. We combine functional imaging with ultra-structural EM circuit reconstruction to show that cholinergic signals originating from the hindbrain transmit efference copies (copies of the motor command that cancel out self-generated reafferent stimulation during locomotion) and that dopaminergic signals from the hypothalamus may have a role in threshold modulation, both in response to locomotion and salient stimuli. We further gain direct mechanistic insight into the core components of this circuit by loss-of-function perturbations using targeted ablations and gene knockouts. We propose that this simple circuit is the core implementation of mechanosensory reafferent suppression in these young animals and that it might form the first instantiation of state-dependent modulation found at later stages in development.

Precise visuomotor transformations underlying collective behavior in larval zebrafish.

Complex schooling behaviors result from local interactions among individuals. Yet, how sensory signals from neighbors are analyzed in the visuomotor stream of animals is poorly understood. Here, we studied aggregation behavior in larval zebrafish and found that over development larvae transition from overdispersed groups to tight shoals. Using a virtual reality assay, we characterized the algorithms fish use to transform visual inputs from neighbors into movement decisions. We found that young larvae turn away from virtual neighbors by integrating and averaging retina-wide visual occupancy within each eye, and by using a winner-take-all strategy for binocular integration. As fish mature, their responses expand to include attraction to virtual neighbors, which is based on similar algorithms of visual integration. Using model simulations, we show that the observed algorithms accurately predict group structure over development. These findings allow us to make testable predictions regarding the neuronal circuits underlying collective behavior in zebrafish.

Collective behavior emerges from genetically controlled simple behavioral motifs in zebrafish.

It is not understood how changes in the genetic makeup of individuals alter the behavior of groups of animals. Here, we find that, even at early larval stages, zebrafish regulate their proximity and alignment with each other. Two simple visual responses, one that measures relative visual field occupancy and one that accounts for global visual motion, suffice to account for the group behavior that emerges. Mutations in genes known to affect social behavior in humans perturb these simple reflexes in individual larval zebrafish and change their emergent collective behaviors in the predicted fashion. Model simulations show that changes in these two responses in individual mutant animals predict well the distinctive collective patterns that emerge in a group. Hence, group behaviors reflect in part genetically defined primitive sensorimotor "motifs," which are evident even in young larvae.

Coordination of two enhancers drives expression of olfactory trace amine-associated receptors.

Olfactory sensory neurons (OSNs) are functionally defined by their expression of a unique odorant receptor (OR). Mechanisms underlying singular OR expression are well studied, and involve a massive cross-chromosomal enhancer interaction network. Trace amine-associated receptors (TAARs) form a distinct family of olfactory receptors, and here we find that mechanisms regulating Taar gene choice display many unique features. The epigenetic signature of Taar genes in TAAR OSNs is different from that in OR OSNs. We further identify that two TAAR enhancers conserved across placental mammals are absolutely required for expression of the entire Taar gene repertoire. Deletion of either enhancer dramatically decreases the expression probabilities of different Taar genes, while deletion of both enhancers completely eliminates the TAAR OSN populations. In addition, both of the enhancers are sufficient to drive transgene expression in the partially overlapped TAAR OSNs. We also show that the TAAR enhancers operate in cis to regulate Taar gene expression. Our findings reveal a coordinated control of Taar gene choice in OSNs by two remote enhancers, and provide an excellent model to study molecular mechanisms underlying formation of an olfactory subsystem.

Algorithms underlying flexible phototaxis in larval zebrafish.

To thrive, organisms must maintain physiological and environmental variables in suitable ranges. Given that these variables undergo constant fluctuations over varying time scales, how do biological control systems maintain control over these values? We explored this question in the context of phototactic behavior in larval zebrafish. We demonstrate that larval zebrafish use phototaxis to maintain environmental luminance at a set point, that the value of this set point fluctuates on a time scale of seconds when environmental luminance changes, and that it is determined by calculating the mean input across both sides of the visual field. These results expand on previous studies of flexible phototaxis in larval zebrafish; they suggest that larval zebrafish exert homeostatic control over the luminance of their surroundings, and that feedback from the surroundings drives allostatic changes to the luminance set point. As such, we describe a novel behavioral algorithm with which larval zebrafish exert control over a sensory variable.

Study of locomotion response and development in zebrafish (Danio rerio) embryos and larvae exposed to enniatin A, enniatin B, and beauvericin.

Mycotoxins are secondary metabolites produced by a variety of fungi that contaminate food and feed resources, and are capable of inducing a wide range of toxicity. Here, we studied the developmental and behavioral toxicity in zebrafish (Danio rerio) embryos and larvae exposed to three mycotoxins: beauvericin (BEA), Enniatin A (ENN A), and Ennitain B (ENN B). Zebrafish embryos were collected after fertilization, treated individually from 1 to 6 dpf with BEA at 8, 16, 32 and, 64 µM and for both enniatins at 3.12, 6.25, 12.5 and, 25 µM. Mixture of mycotoxins were assayed as follows: i) for BEA + ENN A and BEA + ENN B at [32 + 12.5] µM and [16 + 6.25] µM; ii) for ENN A + ENN B at [12.5 + 12.5] µM and [6.25 + 6.25] µM and, iii) for BEA + ENN A + ENN B at [32 + 12.5 + 12.5] µM and [16 + 6.25 + 6.25] µM. Response was collected after a white light-flash intermittent coming on for 5 s during 2 h with a imaging platform. Outcomes measured were: time to death, response to light, and circadian rhythm. This last outcome was measured in a plate where embryos had evolved in natural intervals of light and dark until day 7 or in a plate maintained in darkness. Images of all stages and evolution were collected. Results indicated that mycotoxins induced toxicity at the concentrations tested. All exposed zebrafish induced developmental defects, specifically hatching time and motion activity. After exposure, fish showed enhanced baseline activity but they lost their responsiveness to light.

Elevated preoptic brain activity in zebrafish glial glycine transporter mutants is linked to lethargy-like behaviors and delayed emergence from anesthesia.

Delayed emergence from anesthesia was previously reported in a case study of a child with Glycine Encephalopathy. To investigate the neural basis of this delayed emergence, we developed a zebrafish glial glycine transporter (glyt1 - / -) mutant model. We compared locomotor behaviors; dose-response curves for tricaine, ketamine, and 2,6-diisopropylphenol (propofol); time to emergence from these anesthetics; and time to emergence from propofol after craniotomy in glyt1-/- mutants and their siblings. To identify differentially active brain regions in glyt1-/- mutants, we used pERK immunohistochemistry as a proxy for brain-wide neuronal activity. We show that glyt1-/- mutants initiated normal bouts of movement less frequently indicating lethargy-like behaviors. Despite similar anesthesia dose-response curves, glyt1-/- mutants took over twice as long as their siblings to emerge from ketamine or propofol, mimicking findings from the human case study. Reducing glycine levels rescued timely emergence in glyt1-/- mutants, pointing to a causal role for elevated glycine. Brain-wide pERK staining showed elevated activity in hypnotic brain regions in glyt1-/- mutants under baseline conditions and a delay in sensorimotor integration during emergence from anesthesia. Our study links elevated activity in preoptic brain regions and reduced sensorimotor integration to lethargy-like behaviors and delayed emergence from propofol in glyt1-/- mutants.

Distributed chromatic processing at the interface between retina and brain in the larval zebrafish.

Larval zebrafish (Danio rerio) are an ideal organism for studying color vision, as their retina possesses four types of cone photoreceptors, covering most of the visible range and into the UV.1,2 Additionally, their eye and nervous systems are accessible to imaging, given that they are naturally transparent.3-5 Recent studies have found that, through a set of wavelength-range-specific horizontal, bipolar, and retinal ganglion cells (RGCs),6-9 the eye relays tetrachromatic information to several retinorecipient areas (RAs).10-13 The main RA is the optic tectum, receiving 97% of the RGC axons via the neuropil mass termed arborization field 10 (AF10).14,15 Here, we aim to understand the processing of chromatic signals at the interface between RGCs and their major brain targets. We used 2-photon calcium imaging to separately measure the responses of RGCs and neurons in the brain to four different chromatic stimuli in awake animals. We find that chromatic information is widespread throughout the brain, with a large variety of responses among RGCs, and an even greater diversity in their targets. Specific combinations of response types are enriched in specific nuclei, but there is no single color processing structure. In the main interface in this pathway, the connection between AF10 and tectum, we observe key elements of neural processing, such as enhanced signal decorrelation and improved chromatic decoding.16,17 A richer stimulus set revealed that these enhancements occur in the context of a more distributed code in tectum, facilitating chromatic signal association in this small vertebrate brain.

Larval Zebrafish Use Olfactory Detection of Sodium and Chloride to Avoid Salt Water.

Salinity levels constrain the habitable environment of all aquatic organisms. Zebrafish are freshwater fish that cannot tolerate high-salt environments and would therefore benefit from neural mechanisms that enable the navigation of salt gradients to avoid high salinity. Yet zebrafish lack epithelial sodium channels, the primary conduit land animals use to taste sodium. This suggests fish may possess novel, undescribed mechanisms for salt detection. In the present study, we show that zebrafish indeed respond to small temporal increases in salt by reorienting more frequently. Further, we use calcium imaging techniques to identify the olfactory system as the primary sense used for salt detection, and we find that a specific subset of olfactory receptor neurons encodes absolute salinity concentrations by detecting monovalent anions and cations. In summary, our study establishes that zebrafish larvae have the ability to navigate and thus detect salinity gradients and that this is achieved through previously undescribed sensory mechanisms for salt detection.

Neuromodulation: How Dopaminergic Neurons Shape and Modulate Behavior.

The roles of dopaminergic neurons in the modulation, structuring and execution of behavioral elements are still unclear. A new study of larval zebrafish has identified the various classes of these neurons based on their projection patterns and functional properties, and uncovered the respective roles of these neurons in the initiation of specific motor sequences.