Drosophila melanogaster plays an important role in molecular, genetic, and genomic studies of heredity, development, metabolism, behavior, and human disease. The initial reference genome sequence reported more than a decade ago had a profound impact on progress in Drosophila research, and improving the accuracy and completeness of this sequence continues to be important to further progress. We previously described improvement of the 117-Mb sequence in the euchromatic portion of the genome and 21 Mb in the heterochromatic portion, using a whole-genome shotgun assembly, BAC physical mapping, and clone-based finishing. Here, we report an improved reference sequence of the single-copy and middle-repetitive regions of the genome, produced using cytogenetic mapping to mitotic and polytene chromosomes, clone-based finishing and BAC fingerprint verification, ordering of scaffolds by alignment to cDNA sequences, incorporation of other map and sequence data, and validation by whole-genome optical restriction mapping. These data substantially improve the accuracy and completeness of the reference sequence and the order and orientation of sequence scaffolds into chromosome arm assemblies. Representation of the Y chromosome and other heterochromatic regions is particularly improved. The new 143.9-Mb reference sequence, designated Release 6, effectively exhausts clone-based technologies for mapping and sequencing. Highly repeat-rich regions, including large satellite blocks and functional elements such as the ribosomal RNA genes and the centromeres, are largely inaccessible to current sequencing and assembly methods and remain poorly represented. Further significant improvements will require sequencing technologies that do not depend on molecular cloning and that produce very long reads.

Activity in motor cortex predicts specific movements seconds before they occur, but how this preparatory activity relates to upcoming movements is obscure. We dissected the conversion of preparatory activity to movement within a structured motor cortex circuit. An anterior lateral region of the mouse cortex (a possible homologue of premotor cortex in primates) contains equal proportions of intermingled neurons predicting ipsi- or contralateral movements, yet unilateral inactivation of this cortical region during movement planning disrupts contralateral movements. Using cell-type-specific electrophysiology, cellular imaging and optogenetic perturbation, we show that layer 5 neurons projecting within the cortex have unbiased laterality. Activity with a contralateral population bias arises specifically in layer 5 neurons projecting to the brainstem, and only late during movement planning. These results reveal the transformation of distributed preparatory activity into movement commands within hierarchically organized cortical circuits.

"Regulatory evolution," that is, changes in a gene's expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSX(F) in females and DSX(M) in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues.

To date, it has been difficult to reveal physiological Ca(2+) events occurring within the fine astrocytic processes of mature animals. The objective of the study was to explore whether neuronal activity evokes astrocytic Ca(2+) signals at glutamatergic synapses of adult mice. We stimulated the Schaffer collateral/commissural fibers in acute hippocampal slices from adult mice transduced with the genetically encoded Ca(2+) indicator GCaMP5E driven by the glial fibrillary acidic protein promoter. Two-photon imaging revealed global stimulation-evoked astrocytic Ca(2+) signals with distinct latencies, rise rates, and amplitudes in fine processes and somata. Specifically, the Ca(2+) signals in the processes were faster and of higher amplitude than those in the somata. A combination of P2 purinergic and group I/II metabotropic glutamate receptor (mGluR) antagonists reduced the amplitude of the Ca(2+) transients by 30-40% in both astrocytic compartments. Blockage of the mGluRs alone only modestly reduced the magnitude of the stimulation-evoked Ca(2+) signals in processes and failed to affect the somatic Ca(2+) response. Local application of group I or I/II mGluR agonists or adenosine triphosphate (ATP) elicited global astrocytic Ca(2+) signals that mimicked the stimulation-evoked astrocytic Ca(2+) responses. We conclude that stimulation-evoked Ca(2+) signals in astrocytic processes at CA3-CA1 synapses of adult mice (1) differ from those in astrocytic somata and (2) are modulated by glutamate and ATP.

Focused-ion-beam scanning electron microscopy (FIB-SEM) has become an essential tool for studying neural tissue at resolutions below 10 nm × 10 nm × 10 nm, producing data sets optimized for automatic connectome tracing. We present a technical advance, ultrathick sectioning, which reliably subdivides embedded tissue samples into chunks (20 μm thick) optimally sized and mounted for efficient, parallel FIB-SEM imaging. These chunks are imaged separately and then 'volume stitched' back together, producing a final three-dimensional data set suitable for connectome tracing.

Eyes may be 'the window to the soul' in humans, but whiskers provide a better path to the inner lives of rodents. The brain has remarkable abilities to focus its limited resources on information that matters, while ignoring a cacophony of distractions. While inspecting a visual scene, primates foveate to multiple salient locations, for example mouths and eyes in images of people, and ignore the rest. Similar processes have now been observed and studied in rodents in the context of whisker-based tactile sensation. Rodents use their mechanosensitive whiskers for a diverse range of tactile behaviors such as navigation, object recognition and social interactions. These animals move their whiskers in a purposive manner to locations of interest. The shapes of whiskers, as well as their movements, are exquisitely adapted for tactile exploration in the dark tight burrows where many rodents live. By studying whisker movements during tactile behaviors, we can learn about the tactile information available to rodents through their whiskers and how rodents direct their attention. In this primer, we focus on how the whisker movements of rats and mice are providing clues about the logic of active sensation and the underlying neural mechanisms.

The identification of active neurons and circuits in vivo is a fundamental challenge in understanding the neural basis of behavior. Genetically encoded calcium (Ca(2+)) indicators (GECIs) enable quantitative monitoring of cellular-resolution activity during behavior. However, such indicators require online monitoring within a limited field of view. Alternatively, post hoc staining of immediate early genes (IEGs) indicates highly active cells within the entire brain, albeit with poor temporal resolution. We designed a fluorescent sensor, CaMPARI, that combines the genetic targetability and quantitative link to neural activity of GECIs with the permanent, large-scale labeling of IEGs, allowing a temporally precise "activity snapshot" of a large tissue volume. CaMPARI undergoes efficient and irreversible green-to-red conversion only when elevated intracellular Ca(2+) and experimenter-controlled illumination coincide. We demonstrate the utility of CaMPARI in freely moving larvae of zebrafish and flies, and in head-fixed mice and adult flies.

Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein variants is determined in parallel using a microplate reader, multichannel pipets, serial dilutions, and fluorogenic ester substrates. The utility of this methodology is illustrated by the measurement of differential enzyme activity in microplate volumes in triplicate with small protein samples and low activity enzyme variants. Enzyme kinetic measurements using fluorogenic substrates are, thus, adaptable for use with student-purified enzyme variants and for comparative enzyme kinetics studies. The rapid setup and analysis of these kinetic experiments not only provides advanced undergraduates with experience in a fundamental biochemical technique, but also provides the adaptability for use in inquiry-based laboratories.

Dendrites are the predominant entry site for excitatory synaptic potentials in most types of central neurons. There is increasing evidence that dendrites are not just passive transmitting devices but play active roles in synaptic integration through linear and non-linear mechanisms. Frequently, excitatory synapses are formed on dendritic spines. In addition to relaying incoming electrical signals, spines can play important roles in modifying these signals through complex biochemical processes and, thereby, determine learning and memory formation. Here, we review recent advances in our understanding of the function of spines and dendrites in central mammalian neurons in vivo by focusing particularly on insights obtained from Ca(2+) imaging studies.

Genomics revolutionized the field of social insect research by providing powerful tools to understand the relationship between genes, physiology and behaviour of social insects. Notably, analysis of gene expression and methylation patterns in the different castes of insect colonies highlighted many genes that likely underlie caste-specific physiological and behavioural phenotypes. However, earlier studies of social insect genomes lacked an ‘evolutionary’ context. Out of the millions of DNA bases found in the genome of a social insect, which pieces were most important to fitness over the timescale of social evolution? Here, we review a burgeoning body of literature that utilizes between-species or within-species genomic comparisons to highlight the evolutionary forces that have shaped social insect genomes. These pioneering phylogenetic and population genomic studies provide a critically needed evolutionary context to social insect genomes and underscore the importance of adaptive changes in physiology and behaviour in social evolution.