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Whole-brain imaging of neuronal activity with cellular resolution at almost a brain per second is demonstrated using high-speed light-sheet microscopy in the larval zebrafish brain. Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

Spatial learning in teleost fish requires an intact telencephalon 1 , a brain region that contains putative analogues to components of the mammalian limbic system (for example, hippocampus) 2 – 4 . However, cells fundamental to spatial cognition in mammals—for example, place cells (PCs) 5 , 6 —have yet to be established in any fish species. In this study, using tracking microscopy to record brain-wide calcium activity in freely swimming larval zebrafish 7 , we compute the spatial information content 8 of each neuron across the brain. Strikingly, in every recorded animal, cells with the highest spatial specificity were enriched in the zebrafish telencephalon. These PCs form a population code of space from which we can decode the animal’s spatial location across time. By continuous recording of population-level activity, we found that the activity manifold of PCs refines and untangles over time. Through systematic manipulation of allothetic and idiothetic cues, we demonstrate that zebrafish PCs integrate multiple sources of information and can flexibly remap to form distinct spatial maps. Using analysis of neighbourhood distance between PCs across environments, we found evidence for a weakly preconfigured network in the telencephalon. The discovery of zebrafish PCs represents a step forward in our understanding of spatial cognition across species and the functional role of the early vertebrate telencephalon. Using a tracking microscope for freely moving animals, the authors discover a population of place cells in the zebrafish brain and demonstrate that a non-amniote brain is capable of integrating allothetic and idiothetic information to create a neural map of space.

Multiple scattering and absorption limit the depth at which biological tissues can be imaged with light. In thick unlabeled specimens, multiple scattering randomizes the phase of the field and absorption attenuates light that travels long optical paths. These obstacles limit the performance of transmission imaging. To mitigate these challenges, we developed an epi-illumination gradient light interference microscope (epi-GLIM) as a label-free phase imaging modality applicable to bulk or opaque samples. Epi-GLIM enables studying turbid structures that are hundreds of microns thick and otherwise opaque to transmitted light. We demonstrate this approach with a variety of man-made and biological samples that are incompatible with imaging in a transmission geometry: semiconductors wafers, specimens on opaque and birefringent substrates, cells in microplates, and bulk tissues. We demonstrate that the epi-GLIM data can be used to solve the inverse scattering problem and reconstruct the tomography of single cells and model organisms. Quantitative phase imaging techniques have been limited by multiple scattering of light or its use in transmission mode. Here, the authors show a gradient light interference microscopy method in a reflection geometry which allows for label-free phase imaging of bulk and opaque samples.

Defining the structural and functional changes in the nervous system underlying learning and memory represents a major challenge for modern neuroscience. Although changes in neuronal activity following memory formation have been studied [B. F. Grewe et al., Nature 543, 670–675 (2017); M. T. Rogan, U. V. St€aubli, J. E. LeDoux, Nature 390, 604–607 (1997)], the underlying structural changes at the synapse level remain poorly understood. Here, we capture synaptic changes in the midlarval zebrafish brain that occur during associative memory formation by imaging excitatory synapses labeled with recombinant probes using selective plane illumination microscopy. Imaging the same subjects before and after classical conditioning at single-synapse resolution provides an unbiased mapping of synaptic changes accompanying memory formation. In control animals and animals that failed to learn the task, there were no significant changes in the spatial patterns of synapses in the pallium, which contains the equivalent of the mammalian amygdala and is essential for associative learning in teleost fish [M. Portavella, J. P. Vargas, B. Torres, C. Salas, Brain Res. Bull. 57, 397–399 (2002)]. In zebrafish that formed memories, we saw a dramatic increase in the number of synapses in the ventrolateral pallium, which contains neurons active during memory formation and retrieval. Concurrently, synapse loss predominated in the dorsomedial pallium. Surprisingly, we did not observe significant changes in the intensity of synaptic labeling, a proxy for synaptic strength, with memory formation in any region of the pallium. Our results suggest that memory formation due to classical conditioning is associated with reciprocal changes in synapse numbers in the pallium.

Wolbachia are intracellular bacteria found in the reproductive tissue of all major groups of arthropods. They are transmitted vertically from the female hosts to their offspring, in a pattern analogous to mitochondria inheritance. But Wolbachia phylogeny does not parallel that of the host, indicating that horizontal infectious transmission must also occur. Insect parasitoids are considered the most likely vectors, but the mechanism for horizontal transfer is largely unknown. Here we show that newly introduced Wolbachia cross several tissues and infect the germline of the adult Drosophila melanogaster female. Through investigation of bacterial migration patterns during the course of infection, we found that Wolbachia reach the germline through the somatic stem cell niche in the D. melanogaster germarium. In addition, our data suggest that Wolbachia are highly abundant in the somatic stem cell niche of long-term infected hosts, implying that this location may also contribute to efficient vertical transmission. This is, to our knowledge, the first report of an intracellular parasite displaying tropism for a stem cell niche.

The brain has persistent internal states that can modulate every aspect of an animal’s mental experience 1 – 4 . In complex tasks such as foraging, the internal state is dynamic 5 – 8 . Caenorhabditis elegans alternate between local search and global dispersal 5 . Rodents and primates exhibit trade-offs between exploitation and exploration 6 , 7 . However, fundamental questions remain about how persistent states are maintained in the brain, which upstream networks drive state transitions and how state-encoding neurons exert neuromodulatory effects on sensory perception and decision-making to govern appropriate behaviour. Here, using tracking microscopy to monitor whole-brain neuronal activity at cellular resolution in freely moving zebrafish larvae 9 , we show that zebrafish spontaneously alternate between two persistent internal states during foraging for live prey ( Paramecia ). In the exploitation state, the animal inhibits locomotion and promotes hunting, generating small, localized trajectories. In the exploration state, the animal promotes locomotion and suppresses hunting, generating long-ranging trajectories that enhance spatial dispersion. We uncover a dorsal raphe subpopulation with persistent activity that robustly encodes the exploitation state. The exploitation-state-encoding neurons, together with a multimodal trigger network that is associated with state transitions, form a stochastically activated nonlinear dynamical system. The activity of this oscillatory network correlates with a global retuning of sensorimotor transformations during foraging that leads to marked changes in both the motivation to hunt for prey and the accuracy of motor sequences during hunting. This work reveals an important hidden variable that shapes the temporal structure of motivation and decision-making. During foraging for live prey, zebrafish larvae alternate between persistent exploitation and exploration behavioural states that correlate with distinct patterns of neuronal activation.

A tracking microscope for imaging neuronal activity at single-cell resolution in freely swimming zebrafish larvae is described. This technology allows functional imaging during behaviors that have previously been inaccessible to real-time analysis. Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour. Neural activity is recorded at the cellular level, throughout the brain of larval zebrafish, while the animals interact with a virtual environment and adapt their motor output to changes in visual feedback; this is used to derive candidates of functional elements driving motor learning. Brain activity at the single-cell level Improvements in technology have allowed neuroscientists to dissect the relationship between behaviour and activity in specific neural circuits. However, in a model system such as the zebrafish, it is potentially possible to track neural activity in circuits on a much broader scale. Using a virtual-reality-based imaging system, Florian Engert and colleagues track the dynamics of adaptive locomotion at single-neuron resolution in navigating fish to derive candidates for functional elements that drive motor learning.

Spatial learning in teleost fish requires an intact telencephalon1, a brain region that contains putative analogues to components of the mammalian limbic system (for example, hippocampus)2 4. However, cells fundamental to spatial cognition in mammals-for example, place cells (PCs)5 6-have yet to be established in any fish species. In this study, using tracking microscopy to record brain-wide calcium activity in freely swimming larval zebrafish7, we compute the spatial information content8 of each neuron across the brain. Strikingly, in every recorded animal, cells with the highest spatial specificity were enriched in the zebrafish telencephalon. These PCs form a population code of space from which we can decode the animal's spatial location across time. By continuous recording of population-level activity, we found that the activity manifold of PCs refines and untangles over time. Through systematic manipulation of allothetic and idiothetic cues, we demonstrate that zebrafish PCs integrate multiple sources of information and can flexibly remap to form distinct spatial maps. Using analysis of neighbourhood distance between PCs across environments, we found evidence for a weakly preconfigured network in the telencephalon. The discovery of zebrafish PCs represents a step forward in our understanding of spatial cognition across species and the functional role of the early vertebrate telencephalon.