Faculty Bibliography

Most animals orient their bodies with respect to gravity to facilitate locomotion and perception. The neural circuits responsible for these orienting movements have long served as a model to address fundamental questions in systems neuroscience. Though postural control is vital, we know little about development of either balance reflexes or the neural circuitry that produces them. Recent work in a genetically and optically accessible vertebrate, the larval zebrafish, has begun to reveal the mechanisms by which such vestibular behaviors and circuits come to function. Here we highlight recent work that leverages the particular advantages of the larval zebrafish to illuminate mechanisms of postural development, the role of sensation for balance circuit development, and the organization of developing vestibular circuits. Further, we frame open questions regarding the developmental mechanisms for functional circuit assembly and maturation where studying the zebrafish vestibular system is likely to open new frontiers.

Context modulates the brain's response to sensory input. Recent work has identified the neurons that implement contextual gating of a startle behavior in zebrafish and suggests a synaptic mechanism for this modulation.

Walking is the predominant locomotor behavior expressed by land-dwelling vertebrates, but it is unknown when the neural circuits that are essential for limb control first appeared. Certain fish species display walking-like behaviors, raising the possibility that the underlying circuitry originated in primitive marine vertebrates. We show that the neural substrates of bipedalism are present in the little skate Leucoraja erinacea, whose common ancestor with tetrapods existed ∼420 million years ago. Leucoraja exhibits core features of tetrapod locomotor gaits, including left-right alternation and reciprocal extension-flexion of the pelvic fins. Leucoraja also deploys a remarkably conserved Hox transcription factor-dependent program that is essential for selective innervation of fin/limb muscle. This network encodes peripheral connectivity modules that are distinct from those used in axial muscle-based swimming and has apparently been diminished in most modern fish. These findings indicate that the circuits that are essential for walking evolved through adaptation of a genetic regulatory network shared by all vertebrates with paired appendages. VIDEO ABSTRACT.

Expansion microscopy (ExM) allows scalable imaging of preserved 3D biological specimens with nanoscale resolution on fast diffraction-limited microscopes. Here, we explore the utility of ExM in the larval and embryonic zebrafish, an important model organism for the study of neuroscience and development. Regarding neuroscience, we found that ExM enabled the tracing of fine processes of radial glia, which are not resolvable with diffraction-limited microscopy. ExM further resolved putative synaptic connections, as well as molecular differences between densely packed synapses. Finally, ExM could resolve subsynaptic protein organization, such as ring-like structures composed of glycine receptors. Regarding development, we used ExM to characterize the shapes of nuclear invaginations and channels, and to visualize cytoskeletal proteins nearby. We detected nuclear invagination channels at late prophase and telophase, potentially suggesting roles for such channels in cell division. Thus, ExM of the larval and embryonic zebrafish may enable systematic studies of how molecular components are configured in multiple contexts of interest to neuroscience and developmental biology.

Within reflex circuits, specific anatomical projections allow central neurons to relay sensations to effectors that generate movements. A major challenge is to relate anatomical features of central neural populations -- such as asymmetric connectivity -- to the computations the populations perform. To address this problem, we mapped the anatomy, modeled the function, and discovered a new behavioral role for a genetically-defined population of central vestibular neurons in rhombomeres 5-7 of larval zebrafish. First, we found that neurons within this central population project preferentially to motoneurons that move the eyes downward. Concordantly, when the entire population of asymmetrically-projecting neurons was stimulated collectively, only downward eye rotations were observed, demonstrating a functional correlate of the anatomical bias. When these neurons are ablated, fish failed to rotate their eyes following either nose-up or nose-down body tilts. This asymmetrically-projecting central population thus participates in both up and downward gaze stabilization. In addition to projecting to motoneurons, central vestibular neurons also receive direct sensory input from peripheral afferents. To infer whether asymmetric projections can facilitate sensory encoding or motor output, we modeled differentially-projecting sets of central vestibular neurons. Whereas motor command strength was independent of projection allocation, asymmetric projections enabled more accurate representation of nose-up stimuli. The model shows how asymmetric connectivity could enhance the representation of imbalance during nose-up postures while preserving gaze-stabilization performance. Finally, we found that central vestibular neurons were necessary for a vital behavior requiring maintenance of a nose-up posture: swim bladder inflation. These observations suggest that asymmetric connectivity in the vestibular system facilitates representation of ethologically-relevant stimuli without compromising reflexive behavior.SIGNIFICANCE STATEMENTInterneuron populations use specific anatomical projections to transform sensations into reflexive actions. Here we examined how the anatomical composition of a genetically-defined population of balance interneurons in the larval zebrafish relates to the computations it performs. First, we found that the population of interneurons that stabilize gaze preferentially project to motoneurons that move the eyes downward. Next, we discovered through modeling that such projection patterns can enhance the encoding of nose-up sensations without compromising gaze stabilization. Finally we found that loss of these interneurons impairs a vital behavior, swim bladder inflation, that relies on maintaining a nose-up posture. These observations suggest that anatomical specialization permits neural circuits to represent relevant features of the environment without compromising behavior.

Balance arises from the interplay of external forces acting on the body and internally generated movements. Many animal bodies are inherently unstable, necessitating corrective locomotion to maintain stability. Understanding how developing animals come to balance remains a challenge. Here we study the interplay among environment, sensation, and action as balance develops in larval zebrafish. We first model the physical forces that challenge underwater balance and experimentally confirm that larvae are subject to constant destabilization. Larvae propel in swim bouts that, we find, tend to stabilize the body. We confirm the relationship between locomotion and balance by changing larval body composition, exacerbating instability and eliciting more frequent swimming. Intriguingly, developing zebrafish come to control the initiation of locomotion, swimming preferentially when unstable, thus restoring preferred postures. To test the sufficiency of locomotor-driven stabilization and the developing control of movement timing, we incorporate both into a generative model of swimming. Simulated larvae recapitulate observed postures and movement timing across early development, but only when locomotor-driven stabilization and control of movement initiation are both utilized. We conclude the ability to move when unstable is the key developmental improvement to balance in larval zebrafish. Our work informs how emerging sensorimotor ability comes to impact how and why animals move when they do.

Both spatial and temporal cues determine the fate of immature neurons. A major challenge at the interface of developmental and systems neuroscience is to relate this spatiotemporal trajectory of maturation to circuit-level functional organization. This study examined the development of two extraocular motor nuclei (nIII and nIV), structures in which a motoneuron's identity, or choice of muscle partner, defines its behavioral role. We used retro-orbital dye fills, in combination with fluorescent markers for motoneuron location and birthdate, to probe spatial and temporal organization of the oculomotor (nIII) and trochlear (nIV) nuclei in the larval zebrafish. We describe a dorsoventral organization of the four nIII motoneuron pools, in which inferior and medial rectus motoneurons occupy dorsal nIII, while inferior oblique and superior rectus motoneurons occupy distinct divisions of ventral nIII. Dorsal nIII motoneurons are, moreover, born before motoneurons of ventral nIII and nIV. The order of neurogenesis can therefore account for the dorsoventral organization of nIII and may play a primary role in determining motoneuron identity. We propose that the temporal development of extraocular motoneurons plays a key role in assembling a functional oculomotor circuit. J. Comp. Neurol. 525:65-78, 2017. (c) 2016 The Authors The Journal of Comparative Neurology Published by Wiley Periodicals, Inc.

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.

The Mauthner cell (M-cell) is a command-like neuron in teleost fish whose firing in response to aversive stimuli is correlated with short-latency escapes [1-3]. M-cells have been proposed as evolutionary ancestors of startle response neurons of the mammalian reticular formation [4], and studies of this circuit have uncovered important principles in neurobiology that generalize to more complex vertebrate models [3]. The main excitatory input was thought to originate from multisensory afferents synapsing directly onto the M-cell dendrites [3]. Here, we describe an additional, convergent pathway that is essential for the M-cell-mediated startle behavior in larval zebrafish. It is composed of excitatory interneurons called spiral fiber neurons, which project to the M-cell axon hillock. By in vivo calcium imaging, we found that spiral fiber neurons are active in response to aversive stimuli capable of eliciting escapes. Like M-cell ablations, bilateral ablations of spiral fiber neurons largely eliminate short-latency escapes. Unilateral spiral fiber neuron ablations shift the directionality of escapes and indicate that spiral fiber neurons excite the M-cell in a lateralized manner. Their optogenetic activation increases the probability of short-latency escapes, supporting the notion that spiral fiber neurons help activate M-cell-mediated startle behavior. These results reveal that spiral fiber neurons are essential for the function of the M-cell in response to sensory cues and suggest that convergent excitatory inputs that differ in their input location and timing ensure reliable activation of the M-cell, a feedforward excitatory motif that may extend to other neural circuits.

Animals modulate their arousal state to ensure that their sensory responsiveness and locomotor activity match environmental demands. Neuropeptides can regulate arousal, but studies of their roles in vertebrates have been constrained by the vast array of neuropeptides and their pleiotropic effects. To overcome these limitations, we systematically dissected the neuropeptidergic modulation of arousal in larval zebrafish. We quantified spontaneous locomotor activity and responsiveness to sensory stimuli after genetically induced expression of seven evolutionarily conserved neuropeptides, including adenylate cyclase activating polypeptide 1b (adcyap1b), cocaine-related and amphetamine-related transcript (cart), cholecystokinin (cck), calcitonin gene-related peptide (cgrp), galanin, hypocretin, and nociceptin. Our study reveals that arousal behaviors are dissociable: neuropeptide expression uncoupled spontaneous activity from sensory responsiveness, and uncovered modality-specific effects upon sensory responsiveness. Principal components analysis and phenotypic clustering revealed both shared and divergent features of neuropeptidergic functions: hypocretin and cgrp stimulated spontaneous locomotor activity, whereas galanin and nociceptin attenuated these behaviors. In contrast, cart and adcyap1b enhanced sensory responsiveness yet had minimal impacts on spontaneous activity, and cck expression induced the opposite effects. Furthermore, hypocretin and nociceptin induced modality-specific differences in responsiveness to changes in illumination. Our study provides the first systematic and high-throughput analysis of neuropeptidergic modulation of arousal, demonstrates that arousal can be partitioned into independent behavioral components, and reveals novel and conserved functions of neuropeptides in regulating arousal.