The Visual Systems of Zebrafish.

The zebrafish visual system has become a paradigmatic preparation for behavioral and systems neuroscience. Around 40 types of retinal ganglion cells (RGCs) serve as matched filters for stimulus features, including light, optic flow, prey, and objects on a collision course. RGCs distribute their signals via axon collaterals to 13 retinorecipient areas in forebrain and midbrain. The major visuomotor hub, the optic tectum, harbors nine RGC input layers that combine information on multiple features. The retinotopic map in the tectum is locally adapted to visual scene statistics and visual subfield-specific behavioral demands. Tectal projections to premotor centers are topographically organized according to behavioral commands. The known connectivity in more than 20 processing streams allows us to dissect the cellular basis of elementary perceptual and cognitive functions. Visually evoked responses, such as prey capture or loom avoidance, are controlled by dedicated multistation pathways that-at least in the larva-resemble labeled lines. This architecture serves the neuronal code's purpose of driving adaptive behavior.

Multiscale imaging of basal cell dynamics in the functionally mature mammary gland.

The mammary epithelium is indispensable for the continued survival of more than 5,000 mammalian species. For some, the volume of milk ejected in a single day exceeds their entire blood volume. Here, we unveil the spatiotemporal properties of physiological signals that orchestrate the ejection of milk from alveolar units and its passage along the mammary ductal network. Using quantitative, multidimensional imaging of mammary cell ensembles from GCaMP6 transgenic mice, we reveal how stimulus evoked Ca2+ oscillations couple to contractions in basal epithelial cells. Moreover, we show that Ca2+-dependent contractions generate the requisite force to physically deform the innermost layer of luminal cells, compelling them to discharge the fluid that they produced and housed. Through the collective action of thousands of these biological positive-displacement pumps, each linked to a contractile ductal network, milk begins its passage toward the dependent neonate, seconds after the command.

Brain-Wide Mapping of Water Flow Perception in Zebrafish.

Information about water flow, detected by lateral line organs, is critical to the behavior and survival of fish and amphibians. While certain aspects of water flow processing have been revealed through electrophysiology, we lack a comprehensive description of the neurons that respond to water flow and the network that they form. Here, we use brain-wide calcium imaging in combination with microfluidic stimulation to map out, at cellular resolution, neuronal responses involved in perceiving and processing water flow information in larval zebrafish. We find a diverse array of neurons responding to head-to-tail (h-t) flow, tail-to-head (t-h) flow, or both. Early in this pathway, in the lateral line ganglia, neurons respond almost exclusively to the simple presence of h-t or t-h flow, but later processing includes neurons responding specifically to flow onset, representing the accumulated displacement of flow during a stimulus, or encoding the speed of the flow. The neurons reporting on these more nuanced details are located across numerous brain regions, including some not previously implicated in water flow processing. A graph theory-based analysis of the brain-wide water flow network shows that a majority of this processing is dedicated to h-t flow detection, and this is reinforced by our finding that details like flow velocity and the total accumulated flow are only encoded for the h-t direction. The results represent the first brain-wide description of processing for this important modality, and provide a departure point for more detailed studies of the flow of information through this network.SIGNIFICANCE STATEMENT In aquatic animals, the lateral line is important for detecting water flow stimuli, but the brain networks that interpret this information remain mysterious. Here, we have imaged the activity of individual neurons across the entire brains of larval zebrafish, revealing all response types and their brain locations as water flow processing occurs. We find neurons that respond to the simple presence of water flow, and others attuned to the direction, speed, and duration of flow, or the accumulated displacement of water that has passed during the stimulus. With this information, we modeled the underlying network, describing a system that is nuanced in its processing of water flow simulating head-to-tail motion but rudimentary in processing flow in the tail-to-head direction.

Altered brain-wide auditory networks in a zebrafish model of fragile X syndrome.

BACKGROUND: Loss or disrupted expression of the FMR1 gene causes fragile X syndrome (FXS), the most common monogenetic form of autism in humans. Although disruptions in sensory processing are core traits of FXS and autism, the neural underpinnings of these phenotypes are poorly understood. Using calcium imaging to record from the entire brain at cellular resolution, we investigated neuronal responses to visual and auditory stimuli in larval zebrafish, using fmr1 mutants to model FXS. The purpose of this study was to model the alterations of sensory networks, brain-wide and at cellular resolution, that underlie the sensory aspects of FXS and autism. RESULTS: Combining functional analyses with the neurons' anatomical positions, we found that fmr1-/- animals have normal responses to visual motion. However, there were several alterations in the auditory processing of fmr1-/- animals. Auditory responses were more plentiful in hindbrain structures and in the thalamus. The thalamus, torus semicircularis, and tegmentum had clusters of neurons that responded more strongly to auditory stimuli in fmr1-/- animals. Functional connectivity networks showed more inter-regional connectivity at lower sound intensities (a - 3 to - 6 dB shift) in fmr1-/- larvae compared to wild type. Finally, the decoding capacities of specific components of the ascending auditory pathway were altered: the octavolateralis nucleus within the hindbrain had significantly stronger decoding of auditory amplitude while the telencephalon had weaker decoding in fmr1-/- mutants. CONCLUSIONS: We demonstrated that fmr1-/- larvae are hypersensitive to sound, with a 3-6 dB shift in sensitivity, and identified four sub-cortical brain regions with more plentiful responses and/or greater response strengths to auditory stimuli. We also constructed an experimentally supported model of how auditory information may be processed brain-wide in fmr1-/- larvae. Our model suggests that the early ascending auditory pathway transmits more auditory information, with less filtering and modulation, in this model of FXS.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

Limitations of Neural Map Topography for Decoding Spatial Information.

UNLABELLED: Topographic maps are common throughout the nervous system, yet their functional role is still unclear. In particular, whether they are necessary for decoding sensory stimuli is unknown. Here we examined this question by recording population activity at the cellular level from the larval zebrafish tectum in response to visual stimuli at three closely spaced locations in the visual field. Due to map imprecision, nearby stimulus locations produced intermingled tectal responses, and decoding based on map topography yielded an accuracy of only 64%. In contrast, maximum likelihood decoding of stimulus location based on the statistics of the evoked activity, while ignoring any information about the locations of neurons in the map, yielded an accuracy close to 100%. A simple computational model of the zebrafish visual system reproduced these results. Although topography is a useful initial decoding strategy, we suggest it may be replaced by better methods following visual experience. SIGNIFICANCE STATEMENT: A very common feature of brain wiring is that neighboring points on a sensory surface (eg, the retina) are connected to neighboring points in the brain. It is often assumed that this "topography" of wiring is essential for decoding sensory stimuli. However, here we show in the developing zebrafish that topographic decoding performs very poorly compared with methods that do not rely on topography. This suggests that, although wiring topography could provide a starting point for decoding at a very early stage in development, it may be replaced by more accurate methods as the animal gains experience of the world.

Quantitative Analysis of Axonal Branch Dynamics in the Developing Nervous System.

Branching is an important mechanism by which axons navigate to their targets during neural development. For instance, in the developing zebrafish retinotectal system, selective branching plays a critical role during both initial pathfinding and subsequent arborisation once the target zone has been reached. Here we show how quantitative methods can help extract new information from time-lapse imaging about the nature of the underlying branch dynamics. First, we introduce Dynamic Time Warping to this domain as a method for automatically matching branches between frames, replacing the effort required for manual matching. Second, we model branch dynamics as a birth-death process, i.e. a special case of a continuous-time Markov process. This reveals that the birth rate for branches from zebrafish retinotectal axons, as they navigate across the tectum, increased over time. We observed no significant change in the death rate for branches over this time period. However, blocking neuronal activity with TTX slightly increased the death rate, without a detectable change in the birth rate. Third, we show how the extraction of these rates allows computational simulations of branch dynamics whose statistics closely match the data. Together these results reveal new aspects of the biology of retinotectal pathfinding, and introduce computational techniques which are applicable to the study of axon branching more generally.

The dynamics of growth cone morphology.

BACKGROUND: Normal brain function depends on the development of appropriate patterns of neural connections. A critical role in guiding axons to their targets during neural development is played by neuronal growth cones. These have a complex and rapidly changing morphology; however, a quantitative understanding of this morphology, its dynamics and how these are related to growth cone movement, is lacking. RESULTS: Here we use eigenshape analysis (principal components analysis in shape space) to uncover the set of five to six basic shape modes that capture the most variance in growth cone form. By analysing how the projections of growth cones onto these principal modes evolve in time, we found that growth cone shape oscillates with a mean period of 30 min. The variability of oscillation periods and strengths between different growth cones was correlated with their forward movement, such that growth cones with strong, fast shape oscillations tended to extend faster. A simple computational model of growth cone shape dynamics based on dynamic microtubule instability was able to reproduce quantitatively both the mean and variance of oscillation periods seen experimentally, suggesting that the principal driver of growth cone shape oscillations may be intrinsic periodicity in cytoskeletal rearrangements. CONCLUSIONS: Intrinsically driven shape oscillations are an important component of growth cone shape dynamics. More generally, eigenshape analysis has the potential to provide new quantitative information about differences in growth cone behaviour in different conditions.

Optogenetic dissection of a behavioural module in the vertebrate spinal cord.

Locomotion relies on neural networks called central pattern generators (CPGs) that generate periodic motor commands for rhythmic movements. In vertebrates, the excitatory synaptic drive for inducing the spinal CPG can originate from either supraspinal glutamatergic inputs or from within the spinal cord. Here we identify a spinal input to the CPG that drives spontaneous locomotion using a combination of intersectional gene expression and optogenetics in zebrafish larvae. The photo-stimulation of one specific cell type was sufficient to induce a symmetrical tail beating sequence that mimics spontaneous slow forward swimming. This neuron is the Kolmer-Agduhr cell, which extends cilia into the central cerebrospinal-fluid-containing canal of the spinal cord and has an ipsilateral ascending axon that terminates in a series of consecutive segments. Genetically silencing Kolmer-Agduhr cells reduced the frequency of spontaneous free swimming, indicating that activity of Kolmer-Agduhr cells provides necessary tone for spontaneous forward swimming. Kolmer-Agduhr cells have been known for over 75 years, but their function has been mysterious. Our results reveal that during early development in zebrafish these cells provide a positive drive to the spinal CPG for spontaneous locomotion.

Brain-wide impacts of sedation on spontaneous activity and auditory processing in larval zebrafish.

Despite their widespread use, we have limited knowledge of the mechanisms by which sedatives mediate their effects on brain-wide networks. This is, in part, due to the technical challenge of observing activity across large populations of neurons in normal and sedated brains. In this study, we examined the effects of the sedative dexmedetomidine, and its antagonist atipamezole, on spontaneous brain dynamics and auditory processing in zebrafish larvae. Our brain-wide, cellular-resolution calcium imaging reveals, for the first time, the brain regions involved in these network-scale dynamics and the individual neurons that are affected within those regions. Further analysis reveals a variety of dynamic changes in the brain at baseline, including marked reductions in spontaneous activity, correlation, and variance. The reductions in activity and variance represent a "quieter" brain state during sedation, an effect that causes highly correlated evoked activity in the auditory system to stand out more than it does in un-sedated brains. We also observe a reduction in auditory response latencies across the brain during sedation, suggesting that the removal of spontaneous activity leaves the core auditory pathway free of impingement from other non-auditory information. Finally, we describe a less dynamic brain-wide network during sedation, with a higher energy barrier and a lower probability of brain state transitions during sedation. In total, our brain-wide, cellular-resolution analysis shows that sedation leads to quieter, more stable, and less dynamic brain, and that against this background, responses across the auditory processing pathway become sharper and more prominent. Significance Statement: Animals' brain states constantly fluctuate in response to their environment and context, leading to changes in perception and behavioral choices. Alterations in perception, sensorimotor gating, and behavioral selection are hallmarks of numerous neuropsychiatric disorders, but the circuit- and network-level underpinnings of these alterations are poorly understood.Pharmacological sedation alters perception and responsiveness and provides a controlled and repeatable manipulation for studying brain states and their underlying circuitry. Here, we show that sedation of larval zebrafish with dexmedetomidine reduces brain-wide spontaneous activity and locomotion but leaves portions of brain-wide auditory processing and behavior intact. We describe and computationally model changes at the levels of individual neurons, local circuits, and brain-wide networks that lead to altered brain states and sensory processing during sedation.

EVIDENCE FOR AUDITORY STIMULUS-SPECIFIC ADAPTATION BUT NOT DEVIANCE DETECTION IN LARVAL ZEBRAFISH BRAINS.

Animals receive a constant stream of sensory input, and detecting changes in this sensory landscape is critical to their survival. One signature of change detection in humans is the auditory mismatch negativity (MMN), a neural response to unexpected stimuli that deviate from a predictable sequence. This process requires the auditory system to adapt to specific repeated stimuli while remaining sensitive to novel input (stimulus-specific adaptation). MMN was originally described in humans, and equivalent responses have been found in other mammals and birds, but it is not known to what extent this deviance detection circuitry is evolutionarily conserved. Here we present the first evidence for stimulus-specific adaptation in the brain of a teleost fish, using whole-brain calcium imaging of larval zebrafish at single-neuron resolution with selective plane illumination microscopy. We found frequency-specific responses across the brain with variable response amplitudes for frequencies of the same volume, and created a loudness curve to model this effect. We presented an auditory 'oddball' stimulus in an otherwise predictable train of pure tone stimuli, and did not find a population of neurons with specific responses to deviant tones that were not otherwise explained by stimulus-specific adaptation. Further, we observed no deviance responses to an unexpected omission of a sound in a repetitive sequence of white noise bursts. These findings extend the known scope of auditory adaptation and deviance responses across the evolutionary tree, and lay groundwork for future studies to describe the circuitry underlying auditory adaptation at the level of individual neurons.

Optical tweezers across scales in cell biology.

Optical tweezers (OT) provide a noninvasive approach for delivering minute physical forces to targeted objects. Controlling such forces in living cells or in vitro preparations allows for the measurement and manipulation of numerous processes relevant to the form and function of cells. As such, OT have made important contributions to our understanding of the structures of proteins and nucleic acids, the interactions that occur between microscopic structures within cells, the choreography of complex processes such as mitosis, and the ways in which cells interact with each other. In this review, we highlight recent contributions made to the field of cell biology using OT and provide basic descriptions of the physics, the methods, and the equipment that made these studies possible.

Auditory processing in rodent models of autism: a systematic review.

Autism is a complex condition with many traits, including differences in auditory sensitivity. Studies in human autism are plagued by the difficulty of controlling for aetiology, whereas studies in individual rodent models cannot represent the full spectrum of human autism. This systematic review compares results in auditory studies across a wide range of established rodent models of autism to mimic the wide range of aetiologies in the human population. A search was conducted in the PubMed and Web of Science databases to find primary research articles in mouse or rat models of autism which investigate central auditory processing. A total of 88 studies were included. These used non-invasive measures of auditory function, such as auditory brainstem response recordings, cortical event-related potentials, electroencephalography, and behavioural tests, which are translatable to human studies. They also included invasive measures, such as electrophysiology and histology, which shed insight on the origins of the phenotypes found in the non-invasive studies. The most consistent results across these studies were increased latency of the N1 peak of event-related potentials, decreased power and coherence of gamma activity in the auditory cortex, and increased auditory startle responses to high sound levels. Invasive studies indicated loss of subcortical inhibitory neurons, hyperactivity in the lateral superior olive and auditory thalamus, and reduced specificity of responses in the auditory cortex. This review compares the auditory phenotypes across rodent models and highlights those that mimic findings in human studies, providing a framework and avenues for future studies to inform understanding of the auditory system in autism.

From calcium imaging to graph topology.

Systems neuroscience is facing an ever-growing mountain of data. Recent advances in protein engineering and microscopy have together led to a paradigm shift in neuroscience; using fluorescence, we can now image the activity of every neuron through the whole brain of behaving animals. Even in larger organisms, the number of neurons that we can record simultaneously is increasing exponentially with time. This increase in the dimensionality of the data is being met with an explosion of computational and mathematical methods, each using disparate terminology, distinct approaches, and diverse mathematical concepts. Here we collect, organize, and explain multiple data analysis techniques that have been, or could be, applied to whole-brain imaging, using larval zebrafish as an example model. We begin with methods such as linear regression that are designed to detect relations between two variables. Next, we progress through network science and applied topological methods, which focus on the patterns of relations among many variables. Finally, we highlight the potential of generative models that could provide testable hypotheses on wiring rules and network progression through time, or disease progression. While we use examples of imaging from larval zebrafish, these approaches are suitable for any population-scale neural network modeling, and indeed, to applications beyond systems neuroscience. Computational approaches from network science and applied topology are not limited to larval zebrafish, or even to systems neuroscience, and we therefore conclude with a discussion of how such methods can be applied to diverse problems across the biological sciences.

Brain-wide visual habituation networks in wild type and fmr1 zebrafish.

Habituation is a form of learning during which animals stop responding to repetitive stimuli, and deficits in habituation are characteristic of several psychiatric disorders. Due to technical challenges, the brain-wide networks mediating habituation are poorly understood. Here we report brain-wide calcium imaging during larval zebrafish habituation to repeated visual looming stimuli. We show that different functional categories of loom-sensitive neurons are located in characteristic locations throughout the brain, and that both the functional properties of their networks and the resulting behavior can be modulated by stimulus saliency and timing. Using graph theory, we identify a visual circuit that habituates minimally, a moderately habituating midbrain population proposed to mediate the sensorimotor transformation, and downstream circuit elements responsible for higher order representations and the delivery of behavior. Zebrafish larvae carrying a mutation in the fmr1 gene have a systematic shift toward sustained premotor activity in this network, and show slower behavioral habituation.

Remote control of neuronal activity with a light-gated glutamate receptor.

The ability to stimulate select neurons in isolated tissue and in living animals is important for investigating their role in circuits and behavior. We show that the engineered light-gated ionotropic glutamate receptor (LiGluR), when introduced into neurons, enables remote control of their activity. Trains of action potentials are optimally evoked and extinguished by 380 nm and 500 nm light, respectively, while intermediate wavelengths provide graded control over the amplitude of depolarization. Light pulses of 1-5 ms in duration at approximately 380 nm trigger precisely timed action potentials and EPSP-like responses or can evoke sustained depolarizations that persist for minutes in the dark until extinguished by a short pulse of approximately 500 nm light. When introduced into sensory neurons in zebrafish larvae, activation of LiGluR reversibly blocks the escape response to touch. Our studies show that LiGluR provides robust control over neuronal activity, enabling the dissection and manipulation of neural circuitry in vivo.

Focusing on optic tectum circuitry through the lens of genetics.

The visual pathway is tasked with processing incoming signals from the retina and converting this information into adaptive behavior. Recent studies of the larval zebrafish tectum have begun to clarify how the 'micro-circuitry' of this highly organized midbrain structure filters visual input, which arrives in the superficial layers and directs motor output through efferent projections from its deep layers. The new emphasis has been on the specific function of neuronal cell types, which can now be reproducibly labeled, imaged and manipulated using genetic and optical techniques. Here, we discuss recent advances and emerging experimental approaches for studying tectal circuits as models for visual processing and sensorimotor transformation by the vertebrate brain.

The tectum/superior colliculus as the vertebrate solution for spatial sensory integration and action.

The superior colliculus, or tectum in the case of non-mammalian vertebrates, is a part of the brain that registers events in the surrounding space, often through vision and hearing, but also through electrosensation, infrared detection, and other sensory modalities in diverse vertebrate lineages. This information is used to form maps of the surrounding space and the positions of different salient stimuli in relation to the individual. The sensory maps are arranged in layers with visual input in the uppermost layer, other senses in deeper positions, and a spatially aligned motor map in the deepest layer. Here, we will review the organization and intrinsic function of the tectum/superior colliculus and the information that is processed within tectal circuits. We will also discuss tectal/superior colliculus outputs that are conveyed directly to downstream motor circuits or via the thalamus to cortical areas to control various aspects of behavior. The tectum/superior colliculus is evolutionarily conserved among all vertebrates, but tailored to the sensory specialties of each lineage, and its roles have shifted with the emergence of the cerebral cortex in mammals. We will illustrate both the conserved and divergent properties of the tectum/superior colliculus through vertebrate evolution by comparing tectal processing in lampreys belonging to the oldest group of extant vertebrates, larval zebrafish, rodents, and other vertebrates including primates.

Contributions of Luminance and Motion to Visual Escape and Habituation in Larval Zebrafish.

Animals from insects to humans perform visual escape behavior in response to looming stimuli, and these responses habituate if looms are presented repeatedly without consequence. While the basic visual processing and motor pathways involved in this behavior have been described, many of the nuances of predator perception and sensorimotor gating have not. Here, we have performed both behavioral analyses and brain-wide cellular-resolution calcium imaging in larval zebrafish while presenting them with visual loom stimuli or stimuli that selectively deliver either the movement or the dimming properties of full loom stimuli. Behaviorally, we find that, while responses to repeated loom stimuli habituate, no such habituation occurs when repeated movement stimuli (in the absence of luminance changes) are presented. Dim stimuli seldom elicit escape responses, and therefore cannot habituate. Neither repeated movement stimuli nor repeated dimming stimuli habituate the responses to subsequent full loom stimuli, suggesting that full looms are required for habituation. Our calcium imaging reveals that motion-sensitive neurons are abundant in the brain, that dim-sensitive neurons are present but more rare, and that neurons responsive to both stimuli (and to full loom stimuli) are concentrated in the tectum. Neurons selective to full loom stimuli (but not to movement or dimming) were not evident. Finally, we explored whether movement- or dim-sensitive neurons have characteristic response profiles during habituation to full looms. Such functional links between baseline responsiveness and habituation rate could suggest a specific role in the brain-wide habituation network, but no such relationships were found in our data. Overall, our results suggest that, while both movement- and dim-sensitive neurons contribute to predator escape behavior, neither plays a specific role in brain-wide visual habituation networks or in behavioral habituation.

Broad frequency sensitivity and complex neural coding in the larval zebrafish auditory system.

Most animals have complex auditory systems that identify salient features of the acoustic landscape to direct appropriate responses. In fish, these features include the volume, frequency, complexity, and temporal structure of acoustic stimuli transmitted through water. Larval fish have simple brains compared to adults but swim freely and depend on sophisticated sensory processing for survival.1-5 Zebrafish larvae, an important model for studying brain-wide neural networks, have thus far been found to possess a rudimentary auditory system, sensitive to a narrow range of frequencies and without evident sensitivity to acoustic features that are salient and ethologically important to adult fish.6,7 Here, we have combined a novel method for delivering water-borne sounds, a diverse assembly of acoustic stimuli, and whole-brain calcium imaging to describe the responses of individual auditory-responsive neurons across the brains of zebrafish larvae. Our results reveal responses to frequencies ranging from 100 Hz to 4 kHz, with evidence of frequency discrimination from 100 Hz to 2.5 kHz. Frequency-selective neurons are located in numerous regions of the brain, and neurons responsive to the same frequency are spatially grouped in some regions. Using functional clustering, we identified categories of neurons that are selective for a single pure-tone frequency, white noise, the sharp onset of acoustic stimuli, and stimuli involving a gradual crescendo. These results suggest a more nuanced auditory system than has previously been described in larval fish and provide insights into how a young animal's auditory system can both function acutely and serve as the scaffold for a more complex adult system.