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The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly’s brain.

SummaryNeuronal cell types are the nodes of neural circuits that determine the flow of information within the brain. Neuronal morphology, especially the shape of the axonal arbor, provides an essential descriptor of cell type and reveals how individual neurons route their output across the brain. Despite the importance of morphology, few projection neurons in the mouse brain have been reconstructed in their entirety. Here we present a robust and efficient platform for imaging and reconstructing complete neuronal morphologies, including axonal arbors that span substantial portions of the brain. We used this platform to reconstruct more than 1,000 projection neurons in the motor cortex, thalamus, subiculum, and hypothalamus. Together, the reconstructed neurons comprise more than 75 meters of axonal length and are available in a searchable online database. Axonal shapes revealed previously unknown subtypes of projection neurons and suggest organizational principles of long-range connectivity.

To better understand animal cell plasma membranes, we studied simplified models, namely four-component lipid bilayer mixtures. Here we describe the domain size transition in the region of coexisting liquid-disordered (Ld) + liquid-ordered (Lo) phases. This transition occurs abruptly in composition space with domains increasing in size by two orders of magnitude, from tens of nanometers to microns. We measured the line tension between coexisting Ld and Lo domains close to the domain size transition for a variety of lipid mixtures, finding that in every case the transition occurs at a line tension of ∼0.3 pN. A computational model incorporating line tension and dipole repulsion indicated that even small changes in line tension can result in domains growing in size by several orders of magnitude, consistent with experimental observations. We find that other properties of the coexisting Ld and Lo phases do not change significantly in the vicinity of the abrupt domain size transition.

The presence and the properties of lipid bilayer nanometer-scale domains might be important for understanding the membranes of living cells. We used molecular dynamics (MD) simulations to investigate perturbations of a small patch of fluid-phase DPPC bilayer upon incorporation of fluorescent indocarbocyanine lipid probes commonly used to study membranes (DiI-C12:0, DiI-C18:0, or DiI-C18:2). In simulations containing 1 probe per 64 total lipids in each leaflet, an 8 - 12% decrease in chain order is observed for DPPC molecules in the solvation shell closest to the probe, relative to a pure DPPC bilayer. A ~5% increase in chain order is seen in the next three shells, resulting in a small overall increase in average DPPC chain order. In simulations with 1 probe per 256 total lipids in each leaflet, average DPPC chain order is unaffected by the probe. Thus, these DiI probes cause an oscillatory perturbation of their local environment but do not strongly influence the average properties of even "nanoscopic" lipid phase domains., 30 pages, 12 figures

Fluorescent lipid analogs are a valuable tool for studying membranes, and in recent years a wide variety of fluorescence techniques have contributed significantly to our understanding of lateral heterogeneity in both model and cell membranes. Despite their usefulness, it is often overlooked that these fluorescent molecules are extrinsic to the system of interest, and a meaningful interpretation of data, e.g. properties of nanoscopic domains, local motion and order of the probe environment, or Forster resonance energy transfer, can benefit from understanding probe location within the bilayer, and how the probe itself affects the native membrane state. We have conducted molecular dynamics simulations to investigate perturbations in a DPPC membrane of a family of commonly-used fluorescent lipids: the indocarbocyanine chromophore DiI attached to two alkyl chains, which vary in length and degree of unsaturation. In particular, we report on the order and dynamics of DPPC as a function of distance from the probe molecule, as well as the influence of probe acyl chains on the location and dynamics of the DiI chromophore.