Current imaging methods such as Magnetic Resonance Imaging (MRI), Confocal microscopy, Electron Microscopy (EM) or Selective Plane Illumination Microscopy (SPIM) yield three-dimensional (3D) data sets in need of appropriate computational methods for their analysis. The reconstruction, segmentation and registration are best approached from the 3D representation of the data set.

The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile. Brains contain a vast number of connections between neurons, termed synapses. The precise patterns of these synaptic contacts form the structural underpinning of electrical microcircuits responsible for animal behavior. Due to their small size, synaptic contacts can be conclusively shown using only high-resolution electron microscopy (EM). Therefore, complete series of ultrathin sections are required to reconstruct neuronal microcircuitry. The acquisition and analysis of EM sections (with 15,000 sections per millimeter of tissue) is practical only by computer-assisted means. In this article, we demonstrate the utility of the software package TrakEM2 to model interconnections of nerve fibers from consecutive EM sections and to efficiently reconstruct the neural networks encountered in different parts of a small brain, the early larval brain of the fruit fly Drosophila melanogaster. Neuronal networks are composed of patterns of axons and dendrites (neuronal extensions that transmit and receive signals, respectively), and using TrakEM2, we describe the most common motifs they form. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect brains.

While the rapid development of personal computers and high-throughput recording systems for circadian rhythms allow chronobiologists to produce huge amounts of data, the software to analyze them often lags behind. Here, we announce newly developed chronobiology software that is easy to use, compatible with many different systems, and freely available. Our system can perform the most frequently used analyses: actogram drawing, periodogram analysis, and waveform analysis. The software is distributed as a pure Java plug-in for ImageJ and so works on the 3 main operating systems: Linux, Macintosh, and Windows. We believe that this free software raises the speed of data analyses and makes studying chronobiology accessible to newcomers.

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