The spatial and temporal structure of neural activity across the fly brain.

What are the spatial and temporal scales of brainwide neuronal activity? We used swept, confocally-aligned planar excitation (SCAPE) microscopy to image all cells in a large volume of the brain of adult Drosophila with high spatiotemporal resolution while flies engaged in a variety of spontaneous behaviors. This revealed neural representations of behavior on multiple spatial and temporal scales. The activity of most neurons correlated (or anticorrelated) with running and flailing over timescales that ranged from seconds to a minute. Grooming elicited a weaker global response. Significant residual activity not directly correlated with behavior was high dimensional and reflected the activity of small clusters of spatially organized neurons that may correspond to genetically defined cell types. These clusters participate in the global dynamics, indicating that neural activity reflects a combination of local and broadly distributed components. This suggests that microcircuits with highly specified functions are provided with knowledge of the larger context in which they operate.

Optogenetic control of Drosophila using a red-shifted channelrhodopsin reveals experience-dependent influences on courtship.

Optogenetics allows the manipulation of neural activity in freely moving animals with millisecond precision, but its application in Drosophila melanogaster has been limited. Here we show that a recently described red activatable channelrhodopsin (ReaChR) permits control of complex behavior in freely moving adult flies, at wavelengths that are not thought to interfere with normal visual function. This tool affords the opportunity to control neural activity over a broad dynamic range of stimulation intensities. Using time-resolved activation, we show that the neural control of male courtship song can be separated into (i) probabilistic, persistent and (ii) deterministic, command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, which supports the idea that they constitute a locus of state-dependent influence. This separation is not evident using thermogenetic tools, a result underscoring the importance of temporally precise control of neuronal activation in the functional dissection of neural circuits in Drosophila.

Collective mass spectrometry approaches reveal broad and combinatorial modification of high mobility group protein A1a.

Transcriptional states are formed and maintained by the interaction and post-translational modification (PTM) of several chromatin proteins, such as histones and high mobility group (HMG) proteins. Among these, HMGA1a, a small heterochromatin-associated nuclear protein has been shown to be post-translationally modified, and some of these PTMs have been linked to apoptosis and cancer. In cancerous cells, HMGA1a PTMs differ between metastatic and nonmetastatic cells, suggesting the existence of an HMGA1a PTM code analogous to the "histone code." In this study, we expand on current knowledge by comprehensively characterizing PTMs on HMGA1a purified from human cells using both nanoflow liquid chromatography collision activated dissociation mediated Bottom Up and electron-transfer dissociation facilitated middle and Top Down mass spectrometry (MS). We find HMGA1a to be pervasively modified with many types of modifications such as methylation, acetylation, and phosphorylation, including finding novel sites. While Bottom Up MS identified lower level modification sites, Top and Middle Down MS were utilized to identify the most commonly occurring combinatorially modified forms. Remarkably, although we identify several individual modification sites through our Bottom Up and Middle Down MS analyses, we find relatively few combinatorially modified forms dominate the population through Top Down proteomics. The main combinatorial PTMs we find through the Top Down approach are N-terminal acetylation, Arg25 methylation along with phosphorylation of the three most C-terminal serine residues in primarily a diphosphorylated form. This report presents one of the most detailed analyses of HMGA1a to date and illustrates the strength of using a combined MS effort.