Reconstructing neuronal circuits at the level of synapses is a central problem in neuroscience and becoming a focus of the emerging field of connectomics. To date, electron microscopy (EM) is the most proven technique for identifying and quantifying synaptic connections. As advances in EM make acquiring larger datasets possible, subsequent manual synapse identification ({\em i.e.}, proofreading) for deciphering a connectome becomes a major time bottleneck. Here we introduce a large-scale, high-throughput, and semi-automated methodology to efficiently identify synapses. We successfully applied our methodology to the Drosophila medulla optic lobe, annotating many more synapses than previous connectome efforts. Our approaches are extensible and will make the often complicated process of synapse identification accessible to a wider-community of potential proofreaders.

Mapping the connectivity of neurons in the brain (i.e., connectomics) is a challenging problem due to both the number of connections in even the smallest organisms and the nanometer resolution required to resolve them. Because of this, previous connectomes contain only hundreds of neurons, such as in the C.elegans connectome. Recent technological advances will unlock the mysteries of increasingly large connectomes (or partial connectomes). However, the value of these maps is limited by our ability to reason with this data and understand any underlying motifs. To aid connectome analysis, we introduce algorithms to cluster similarly-shaped neurons, where 3D neuronal shapes are represented as skeletons. In particular, we propose a novel location-sensitive clustering algorithm. We show clustering results on neurons reconstructed from the Drosophila medulla that show high-accuracy.

In this work, we propose a learning framework for identifying synapses using a deep and wide multi-scale recursive (DAWMR) network, previously considered in image segmentation applications. We apply this approach on electron microscopy data from invertebrate fly brain tissue. By learning features directly from the data, we are able to achieve considerable improvements over existing techniques that rely on a small set of hand-designed features. We show that this system can reduce the amount of manual annotation required, in both acquisition of training data as well as verification of inferred detections.

Pixel and superpixel classifiers have become essential tools for EM segmentation algorithms. Training these classifiers remains a major bottleneck primarily due to the requirement of completely annotating the dataset which is tedious, error-prone and costly. In this paper, we propose an interactive learning scheme for the superpixel classifier for EM segmentation. Our algorithm is "active semi-supervised" because it requests the labels of a small number of examples from user and applies label propagation technique to generate these queries. Using only a small set (<20%) of all datapoints, the proposed algorithm consistently generates a classifier almost as accurate as that estimated from a complete groundtruth. We provide segmentation results on multiple datasets to show the strength of these classifiers.

The basal ganglia plays a significant role in transforming activity in the cerebral cortex into directed behavior, involving motor learning, habit formation and the selection of actions based on desirable outcomes, and the organization of the basal ganglia is intimately linked to that of the cerebral cortex. In this chapter, we focus primarily on the neocortical part of the basal ganglia. A general canonical organizational plan of the neocortical-related basal ganglia is described. An understanding of the canonical organization of the neostriatal part of the basal ganglia, provides a framework for determining the general organizational principles of the parts of the basal ganglia connected with allocortical areas and the amygdala, and this is discussed. While it has been proposed that the basal ganglia provide interactions between disparate functional circuits, another approach might be that there are parallel functional circuits, in which distinct functions are for the most part maintained, or segregated, one from the other. This chapter, however, is biased toward the view that there is maintenance of functional parallel circuits in the organization of the basal ganglia, but that the circuit contains neuroanatomical features that provide for considerable interaction between adjacent circuits.

MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.

The fruit fly is an excellent model system for investigating the sequence of epithelial tissue invaginations constituting the process of gastrulation. By combining recent advancements in light sheet fluorescence microscopy (LSFM) and image processing, the three-dimensional fly embryo morphology and relevant gene expression patterns can be accurately recorded throughout the entire process of embryogenesis. LSFM provides exceptionally high imaging speed, high signal-to-noise ratio, low level of photoinduced damage, and good optical penetration depth. This powerful combination of capabilities makes LSFM particularly suitable for live imaging of the fly embryo.The resulting high-information-content image data are subsequently processed to obtain the outlines of cells and cell nuclei, as well as the geometry of the whole embryo tissue by image segmentation. Furthermore, morphodynamics information is extracted by computationally tracking objects in the image. Towards that goal we describe the successful implementation of a fast fitting strategy of Gaussian mixture models.The data obtained by image processing is well-suited for hypothesis testing of the detailed biomechanics of the gastrulating embryo. Typically this involves constructing computational mechanics models that consist of an objective function providing an estimate of strain energy for a given morphological configuration of the tissue, and a numerical minimization mechanism of this energy, achieved by varying morphological parameters.In this chapter, we provide an overview of in vivo imaging of fruit fly embryos using LSFM, computational tools suitable for processing the resulting images, and examples of computational biomechanical simulations of fly embryo gastrulation.

We describe an adaptive optics method that modulates the intensity or phase of light rays at multiple pupil segments in parallel to determine the sample-induced aberration. Applicable to fluorescent protein-labeled structures of arbitrary complexity, it allowed us to obtain diffraction-limited resolution in various samples in vivo. For the strongly scattering mouse brain, a single aberration correction improved structural and functional imaging of fine neuronal processes over a large imaging volume.

Chromatin immunoprecipitation (ChIP) is a technique that reveals in vivo location of a protein bound to DNA. ChIP coupled with DNA microarrays (ChIP-chip) or next-generation sequencing (ChIP-seq) allows for identification of binding sites of transcription factors on a global scale. Here we describe a protocol for ChIP to identify binding of the Ultrabithorax (Ubx) Hox transcription factors from imaginal discs of Drosophila larvae. The protocol can be extended to other model organisms and transcription factors.

The rules governing the formation of spatial maps in the hippocampus have not been determined. We investigated the large-scale structure of place field activity by recording hippocampal neurons in rats exploring a previously unencountered 48-meter-long track. Single-cell and population activities were well described by a two-parameter stochastic model. Individual neurons had their own characteristic propensity for forming fields randomly along the track, with some cells expressing many fields and many exhibiting few or none. Because of the particular distribution of propensities across cells, the number of neurons with fields scaled logarithmically with track length over a wide, ethological range. These features constrain hippocampal memory mechanisms, may allow efficient encoding of environments and experiences of vastly different extents and durations, and could reflect general principles of population coding.