From 1980 to 1992, a series of influential papers reported on the discovery, genetics, and evolution of a periodic cycling of the interval between Drosophila male courtship song pulses. The molecular mechanisms underlying this periodicity were never described. To reinitiate investigation of this phenomenon, we previously performed automated segmentation of songs but failed to detect the proposed rhythm [Arthur BJ, et al. (2013) BMC Biol 11:11; Stern DL (2014) BMC Biol 12:38]. Kyriacou et al. [Kyriacou CP, et al. (2017) Proc Natl Acad Sci USA 114:1970-1975] report that we failed to detect song rhythms because (i) our flies did not sing enough and (ii) our segmenter did not identify many of the song pulses. Kyriacou et al. manually annotated a subset of our recordings and reported that two strains displayed rhythms with genotype-specific periodicity, in agreement with their original reports. We cannot replicate this finding and show that the manually annotated data, the original automatically segmented data, and a new dataset provide no evidence for either the existence of song rhythms or song periodicity differences between genotypes. Furthermore, we have reexamined our methods and analysis and find that our automated segmentation method was not biased to prevent detection of putative song periodicity. We conclude that there is no evidence for the existence of Drosophila courtship song rhythms.

Single-particle electron cryo-microscopy (cryo-EM) has become a popular method for high-resolution study of the structural and functional properties of proteins. However, sufficient expression and purification of membrane proteins holds many challenges. We describe methods to overcome these obstacles using ClC-rm1, a prokaryotic chloride channel (ClC) family protein from Ralstonia metallidurans, overexpressed in Escherichia coli (E. coli) BL21(DE3) strain. Mass spectrometry and electron microscopy analyses of purified samples revealed multiple contaminants that can obfuscate results of subsequent high-resolution structural analysis. Here we describe the systematic optimization of sample preparation procedures, including expression systems, solubilization techniques, purification protocols, and contamination detection. We found that expressing ClC-rm1 in E. coli BL21(DE3) and using n-dodecyl-β-D-maltopyranoside as a detergent for solubilization and purification steps resulted in the highest quality samples of those we tested. However, although protein yield, sample stability, and the resolution of structural detail were improved following these changes, we still detected contaminants including Acriflavine resistant protein AcrB. AcrB was particularly difficult to remove as it co-purified with ClC-rm1 due to four intrinsic histidine residues at its C-terminus that bind to affinity resins. We were able to obtain properly folded pure ClC-rm1 by adding eGFP to the C-terminus and overexpressing the protein in the ΔacrB variant of the JW0451-2 E. coli strain.

Efforts to map neural circuits have been galvanized by the development of genetic technologies that permit the manipulation of targeted sets of neurons in the brains of freely behaving animals. The success of these efforts relies on the experimenter's ability to target arbitrarily small subsets of neurons for manipulation, but such specificity of targeting cannot routinely be achieved using existing methods. In Drosophila melanogaster, a widely used technique for refined cell-type specific manipulation is the Split GAL4 system, which augments the targeting specificity of the binary GAL4-UAS system by making GAL4 transcriptional activity contingent upon two enhancers, rather than one. To permit more refined targeting, we introduce here the "Killer Zipper" (KZip(+)), a suppressor that makes Split GAL4 targeting contingent upon a third enhancer. KZip(+) acts by disrupting both the formation and activity of Split GAL4 heterodimers, and we show how this added layer of control can be used to selectively remove unwanted cells from a Split GAL4 expression pattern or to subtract neurons of interest from a pattern to determine their requirement in generating a given phenotype. To facilitate application of the KZip(+) technology, we have developed a versatile set of LexAop-KZip(+) fly lines that can be used directly with the large number of LexA driver lines with known expression patterns. The Killer Zipper significantly sharpens the precision of neuronal genetic control available in Drosophila and may be extended to other organisms where Split GAL4-like systems are used.

Animals rely on dedicated sensory circuits to extract and encode environmental features. How individual neurons integrate and translate these features into behavioral responses remains a major question. Here, we identify a visual projection neuron type that conveys predator approach information to the Drosophila giant fiber (GF) escape circuit. Genetic removal of this input during looming stimuli reveals that it encodes angular expansion velocity, whereas other input cell type(s) encode angular size. Motor program selection and timing emerge from linear integration of these two features within the GF. Linear integration improves size detection invariance over prior models and appropriately biases motor selection to rapid, GF-mediated escapes during fast looms. Our findings suggest feature integration, and motor control may occur as simultaneous operations within the same neuron and establish the Drosophila escape circuit as a model system in which these computations may be further dissected at the circuit level.

The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. Here we show that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation.

The spatiotemporal fluorescence imaging of biological processes requires effective tools to label intracellular biomolecules in living systems. This review presents a brief overview of recent labeling strategies that permits one to make protein and RNA strongly fluorescent using synthetic fluorogenic probes. Genetically encoded tags selectively binding the exogenously applied molecules ensure high labeling selectivity, while high imaging contrast is achieved using fluorogenic chromophores that are fluorescent only when bound to their cognate tag, and are otherwise dark. Beyond avoiding the need for removal of unbound synthetic dyes, these approaches allow the development of sophisticated imaging assays, and open exciting prospects for advanced imaging, particularly for multiplexed imaging and super-resolution microscopy.

Sensory, motor and cognitive operations involve the coordinated action of large neuronal populations across multiple brain regions in both superficial and deep structures. Existing extracellular probes record neural activity with excellent spatial and temporal (sub-millisecond) resolution, but from only a few dozen neurons per shank. Optical Ca(2+) imaging offers more coverage but lacks the temporal resolution needed to distinguish individual spikes reliably and does not measure local field potentials. Until now, no technology compatible with use in unrestrained animals has combined high spatiotemporal resolution with large volume coverage. Here we design, fabricate and test a new silicon probe known as Neuropixels to meet this need. Each probe has 384 recording channels that can programmably address 960 complementary metal-oxide-semiconductor (CMOS) processing-compatible low-impedance TiN sites that tile a single 10-mm long, 70 × 20-μm cross-section shank. The 6 × 9-mm probe base is fabricated with the shank on a single chip. Voltage signals are filtered, amplified, multiplexed and digitized on the base, allowing the direct transmission of noise-free digital data from the probe. The combination of dense recording sites and high channel count yielded well-isolated spiking activity from hundreds of neurons per probe implanted in mice and rats. Using two probes, more than 700 well-isolated single neurons were recorded simultaneously from five brain structures in an awake mouse. The fully integrated functionality and small size of Neuropixels probes allowed large populations of neurons from several brain structures to be recorded in freely moving animals. This combination of high-performance electrode technology and scalable chip fabrication methods opens a path towards recording of brain-wide neural activity during behaviour.

Transcriptional enhancers are regions of DNA that drive precise patterns of gene expression. While many studies have elucidated how individual enhancers can evolve, most of this work has focused on what are called "minimal" enhancers, the smallest DNA regions that drive expression that approximates an aspect of native gene expression. Here we explore how the Drosophila erecta even-skipped (eve) locus has evolved by testing its activity in the divergent D. melanogaster genome. We found, as has been reported previously, that the D. erecta eve stripe 2 enhancer (eveS2) fails to drive appreciable expression in D. melanogaster (1). However, we found that a large transgene carrying the entire D. erecta eve locus drives normal eve expression, including in stripe 2. We performed a functional dissection of the region upstream of the D. erecta eveS2 region and found multiple Zelda motifs that are required for normal expression. Our results illustrate how sequences outside of minimal enhancer regions can evolve functionally through mechanisms other than changes in transcription factor binding sites that drive patterning.