David G. C. Hildebrand

Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits.

The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly—the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments.

Whole-brain serial-section electron microscopy in larval zebrafish

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure–function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

High-performance prediction of functional residues in proteins with machine learning and computed input features†

One of the major challenges in genomics is to understand the function of gene products from their 3D structures. Computational methods are needed for the high-throughput prediction of the function of proteins from their 3D structure. Methods that identify active sites are important for understanding and annotating the function of proteins. Traditional methods exploiting either sequence similarity or structural similarity can be unreliable and cannot be applied to proteins with novel folds or low homology with other proteins. Here, we present a machine-learning application that combines computed electrostatic, evolutionary, and pocket geometric information for high-performance prediction of catalytic residues. Input features consist of our structure-based theoretical microscopic anomalous titration curve shapes (THEMATICS) electrostatics data, enhanced with sequence-based phylogenetic information from INTREPID and topological pocket information from ConCavity. Our THEMATICS-based input features are augmented with an additional metric, the theoretical buffer range. With the integration of the three different types of input, each of which performs admirably on its own, significantly better performance is achieved than that of any of these methods by itself. This combined method achieves 86.7%, 92.5%, and 93.8% recall of annotated functional residues at 5, 8, and 10% false-positive rates, respectively.

Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish

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.

Vivaldi: A Domain-Specific Language for Volume Processing and Visualization on Distributed Heterogeneous Systems.

As the size of image data from microscopes and telescopes increases, the need for high-throughput processing and visualization of large volumetric data has become more pressing. At the same time, many-core processors and GPU accelerators are commonplace, making high-performance distributed heterogeneous computing systems affordable. However, effectively utilizing GPU clusters is difficult for novice programmers, and even experienced programmers often fail to fully leverage the computing power of new parallel architectures due to their steep learning curve and programming complexity. In this paper, we propose Vivaldi, a new domain-specific language for volume processing and visualization on distributed heterogeneous computing systems. Vivaldis Python-like grammar and parallel processing abstractions provide flexible programming tools for non-experts to easily write high-performance parallel computing code. Vivaldi provides commonly used functions and numerical operators for customized visualization and high-throughput image processing applications. We demonstrate the performance and usability of Vivaldi on several examples ranging from volume rendering to image segmentation.

Sensorimotor computation underlying phototaxis in zebrafish

Animals continuously gather sensory cues to move towards favourable environments. Efficient goal-directed navigation requires sensory perception and motor commands to be intertwined in a feedback loop, yet the neural substrate underlying this sensorimotor task in the vertebrate brain remains elusive. Here, we combine virtual-reality behavioural assays, volumetric calcium imaging, optogenetic stimulation and circuit modelling to reveal the neural mechanisms through which a zebrafish performs phototaxis, i.e. actively orients towards a light source. Key to this process is a self-oscillating hindbrain population (HBO) that acts as a pacemaker for ocular saccades and controls the orientation of successive swim-bouts. It further integrates visual stimuli in a state-dependent manner, i.e. its response to visual inputs varies with the motor context, a mechanism that manifests itself in the phase-locked entrainment of the HBO by periodic stimuli. A rate model is developed that reproduces our observations and demonstrates how this sensorimotor processing eventually biases the animal trajectory towards bright regions.Active locomotion requires closed-loop sensorimotor co ordination between perception and action. Here the authors show using behavioural, imaging and modelling approaches that gaze orientation during phototaxis behaviour in larval zebrafish is related to oscillatory dynamics of a neuronal population in the hindbrain.

Assessing contributions of nucleus accumbens shell subregions to reward-seeking behavior

BACKGROUND The nucleus accumbens (NAc) plays a key role in brain reward processes including drug seeking and reinstatement. Several anatomical, behavioral, and neurochemical studies discriminate between the limbic-associated shell and the motor-associated core regions. Less studied is the fact that the shell can be further subdivided into a dorsomedial shell (NAcDMS) and an intermediate zone (NAcINT) based on differential expression of transient c-Fos and long-acting immediate-early gene ΔFosB upon cocaine sensitization. These disparate expression patterns suggest that NAc shell subregions may play distinct roles in reward-seeking behavior. In this study, we examined potential differences in the contributions of the NAcDMS and the NAcINT to reinstatement of reward-seeking behavior after extinction. METHODS Rats were trained to intravenously self-administer cocaine, extinguished, and subjected to a reinstatement test session consisting of an intracranial microinfusion of either amphetamine or vehicle targeted to the NAcDMS or the NAcINT. RESULTS Small amphetamine microinfusions targeted to the NAcDMS resulted in statistically significant reinstatement of lever pressing, whereas no significant difference was observed for microinfusions targeted to the NAcINT. No significant difference was found for vehicle microinfusions in either case. CONCLUSION These results suggest heterogeneity in the behavioral relevance of NAc shell subregions, a possibility that can be tested in specific neuronal populations in the future with recently developed techniques including optogenetics.

The thalamus is a gateway for stimulus-evoked activity in the habenula

Neural activity in the vertebrate habenula is affected by changes in ambient illumination. The nucleus that links photoreceptors with the habenula is not well characterized. Here, we describe the location, inputs and potential function of this nucleus in larval zebrafish. High-speed calcium imaging shows that onset and offset of light evokes a rapid response in the dorsal left neuropil of the habenula, indicating preferential targeting of this neuropil by afferents mediating response to change in irradiance. Injection of a lipophilic dye into this neuropil led to bilateral labeling of a nucleus in the anterior thalamus that responds to onset and offset of light, and that receives innervation from the retina and pineal organ. Lesioning the neuropil of this thalamic nucleus reduced the habenula response to light. Optogenetic stimulation of the thalamus with channelrhodopsin-2 caused depolarization in the habenula, while manipulation with anion channelrhodopsins inhibited habenula response to light and disrupted climbing and diving that is evoked by irradiance change. A nucleus in the anterior thalamus of larval zebrafish innervates the dorsal left habenula. This nucleus receives input from the retina and pineal, responds to increase and decrease in irradiance, enables habenula responses to change in irradiance, and may function in light-evoked vertical migration.The habenula integrates sensory stimuli and reward information to regulate the release of neuromodulators with broad effects on brain state and behavior. One stimulus that affects habenula activity is light, but how it does so is unknown. Here, we address this question using larval zebrafish. Calcium imaging shows that light evokes widespread activity in habenula neurons, coupled with a prominent early response in the dorsal left neuropil. Injection of a lipophilic dye into this region retrogradely labels a retino-recipient thalamic nucleus. Anterograde tracing of the thalamus demonstrates a projection to the habenula, while optogenetic and lesion experiments confirm functional connectivity. An analysis of the mouse mesoscale connectome indicates that a visual nucleus in the thalamus, the ventral lateral geniculate nucleus, projects to the habenula in this species also. Together, these data suggest the existence of a conserved thalamo-habenula projection that enables light to affect habenula activity in vertebrates.The thalamus receives input from multiple brain systems and has an essential role in controlling brain state. This is thought to occur primarily because of its connectivity with the forebrain. Here, we provide evidence for an additional mechanism. By calcium imaging of larval zebrafish, we show that two stimuli, light and darkness, trigger distinct activity patterns in the habenula. Responses appear first in a neuropil that is innervated by retino-recipient thalamic nuclei. Thalamic responses to light and darkness resemble habenula responses, and the thalamus appears to be the only source of GABAergic afferents that would underlie the inhibitory component of light-evoked activity. These data establish that the thalamus directly controls the habenula, a regulator of many broadly acting neuromodulators. We thus propose that the thalamus influences brain state via a pathway to the habenula, which can act in parallel with projections to the forebrain.

Registering large volume serial-section electron microscopy image sets for neural circuit reconstruction using FFT signal whitening

The detailed reconstruction of neural anatomy for connectomics studies requires a combination of resolution and large three-dimensional data capture provided by serial section electron microscopy (ssEM). The convergence of high throughput ssEM imaging and improved tissue preparation methods now allows ssEM capture of complete specimen volumes up to cubic millimeter scale. The resulting multi-terabyte image sets span thousands of serial sections and must be precisely registered into coherent volumetric forms in which neural circuits can be traced and segmented. This paper introduces a Signal Whitening Fourier Transform Image Registration approach (SWiFT-IR) under development at the Pittsburgh Supercomputing Center and its use to align mouse and zebrafish brain datasets acquired using the wafer mapper ssEM imaging technology recently developed at Harvard University. Unlike other methods now used for ssEM registration, SWiFT-IR modifies its spatial frequency response during image matching to maximize a signal-to-noise measure used as its primary indicator of alignment quality. This alignment signal is more robust to rapid variations in biological content and unavoidable data distortions than either phase-only or standard Pearson correlation, thus allowing more precise alignment and statistical confidence. These improvements in turn enable an iterative registration procedure based on projections through multiple sections rather than more typical adjacent-pair matching methods. This projection approach, when coupled with known anatomical constraints and iteratively applied in a multi-resolution pyramid fashion, drives the alignment into a smooth form that properly represents complex and widely varying anatomical content such as the full crosssection zebrafish data.

A flexible system for hands-free intracranial microinjection.

Neuroscience research projects often use intracranial (IC) microinfusions to target drug delivery to specific brain areas during behavioral testing. These experiments require accurate and precisely-timed delivery of small volumes. We present here a stepper motor-powered micropump assembly for such delivery. This system is hands-free, does not use a potentially leaky fluid swivel or use long delivery tubes that are subject to peristaltic forces during animal movements, and has been applied in combination with other paradigms. This micropump system reliably delivers a wide range of fluid volumes (e.g. 50 nL to 1 microL in tissue or greater for intraventricular injections) bilaterally from two independent, commercially available microsyringes through standard surgically implanted guide cannulae. It is easy to build and disassemble for cleaning or changing microsyringes. This system can also be used for a variety of purposes, e.g. intracranial self-administration, place conditioning, and many more, with the advantage that it provides a way to gather important data in the seconds and minutes following IC microinfusion without disruption of the animals behavior by handling.