To ensure disjunction to opposite poles during anaphase, sister chromatids must be held together following DNA replication. This is mediated by cohesin, which is thought to entrap sister DNAs inside a tripartite ring composed of its Smc and kleisin (Scc1) subunits. How such structures are created during S phase is poorly understood, in particular whether they are derived from complexes that had entrapped DNAs prior to replication. To address this, we used selective photobleaching to determine whether cohesin associated with chromatin in G1 persists in situ after replication. We developed a non-fluorescent HaloTag ligand to discriminate the fluorescence recovery signal from labeling of newly synthesized Halo-tagged Scc1 protein (pulse-chase or pcFRAP). In cells where cohesin turnover is inactivated by deletion of WAPL, Scc1 can remain associated with chromatin throughout S phase. These findings suggest that cohesion might be generated by cohesin that is already bound to un-replicated DNA.

The contrast between the disruption of genome topology after cohesin loss and the lack of downstream gene expression changes instigates intense debates regarding the structure-function relationship between genome and gene regulation. Here, by analyzing transcriptome and chromatin accessibility at the single-cell level, we discover that, instead of dictating population-wide gene expression levels, cohesin supplies a general function to neutralize stochastic coexpression tendencies of cis-linked genes in single cells. Notably, cohesin loss induces widespread gene coactivation and chromatin co-opening tens of million bases apart in cis. Spatial genome and protein imaging reveals that cohesin prevents gene co-bursting along the chromosome and blocks spatial mixing of transcriptional hubs. Single-molecule imaging shows that cohesin confines the exploration of diverse enhancer and core promoter binding transcriptional regulators. Together, these results support that cohesin arranges nuclear topology to control gene coexpression in single cells.

Recent advances in single-neuron biophysics have enhanced our understanding of information processing on the cellular level, but how the detailed properties of individual neurons give rise to large-scale behavior remains unclear. Here, we present a model of the hippocampal network based on observed biophysical properties of hippocampal and entorhinal cortical neurons. We assembled our model to simulate spatial alternation, a task that requires memory of the previous path through the environment for correct selection of the current path to a reward site. The convergence of inputs from entorhinal cortex and hippocampal region CA3 onto CA1 pyramidal cells make them potentially important for integrating information about place and temporal context on the network level. Our model shows how place and temporal context information might be combined in CA1 pyramidal neurons to give rise to splitter cells, which fire selectively based on a combination of place and temporal context. The model leads to a number of experimentally testable predictions that may lead to a better understanding of the biophysical basis of information processing in the hippocampus.

The ventromedial hypothalamus (VMH) projects to the periaqueductal gray (PAG) and anterior hypothalamic nucleus (AHN), mediating freezing and escape behaviors, respectively. We investigated VMH collateral (VMH-coll) neurons, which innervate both PAG and AHN, to elucidate their role in postsynaptic processing and defensive behavior plasticity. Using all-optical voltage imaging of 22,151 postsynaptic neurons ex vivo, we found that VMH-coll neurons engage inhibitory mechanisms at both synaptic ends and can induce synaptic circuit plasticity. In vivo optogenetic activation of the VMH-coll somas induced escape behaviors. We identified an Esr1-expressing VMH-coll subpopulation with postsynaptic connectome resembling that of wild-type collaterals on the PAG side. Activation of Esr1+VMH-coll neurons evoked freezing and unexpected flattening behavior, previously not linked to the VMH. Neuropeptides such as PACAP and dynorphin modulated both Esr1+VMH-coll connectomes. In vivo κ-opioid receptor antagonism impaired Esr1+VMH-coll-mediated defensive behaviors. These findings unveiled the central role of VMH-coll pathways in innate defensive behavior plasticity.

The GAL4-UAS system has proven its versatility in studying the function and expression patterns of neurons the Drosophila central nervous system. Although the GAL4 system has been used for 25 years, recent genetic intersectional tools have enabled genetic targeting of very small numbers of neurons aiding in the understanding of their function. This split-GAL4 system is extremely powerful for studying neuronal morphology and the neural basis of animal behavior. However, choosing lines to intersect that have overlapping patterns restricted to one to a few neurons has been cumbersome. This challenge is now growing as the collections of GAL4 driver lines has increased. Here we present a new method and software plug-in for Fiji to dramatically improve the speed of querying large databases of potential lines to intersect and aid in the split-GAL4 creation. We also provide pre-computed datasets for the Janelia GAL4 (5,738 lines) and VT GAL4 (7,429 lines) of the Drosophila central nervous system (CNS). The tool reduced our split-GAL4 creation effort dramatically.

A recent study reports a novel form of lateral inhibition between photoreceptors supporting colour vision in the vinegar fly, Drosophila melanogaster.

Wide-field motion-sensitive neurons in the lobula plate (lobula plate tangential cells, LPTCs) of the fly have been studied for decades. However, it has never been conclusively shown which cells constitute their major presynaptic elements. LPTCs are supposed to be rendered directionally selective by integrating excitatory as well as inhibitory input from many local motion detectors. Based on their stratification in the different layers of the lobula plate, the columnar cells T4 and T5 are likely candidates to provide some of this input. To study their role in motion detection, we performed whole-cell recordings from LPTCs in Drosophila with T4 and T5 cells blocked using two different genetically encoded tools. In these flies, motion responses were abolished, while flicker responses largely remained. We thus demonstrate that T4 and T5 cells indeed represent those columnar cells that provide directionally selective motion information to LPTCs. Contrary to previous assumptions, flicker responses seem to be largely mediated by a third, independent pathway. This work thus represents a further step towards elucidating the complete motion detection circuitry of the fly.

Motor systems flexibly implement diverse motor programs to pattern behavioral sequences, yet their neural underpinnings remain unclear. Here, we investigated the neural circuit mechanisms of flexible courtship behavior in Drosophila. Courting males alternately produce two types of courtship song. By recording calcium signals in the ventral nerve cord (VNC) in behaving flies, we found that different songs are produced by activating overlapping neural populations with distinct motor functions in a combinatorial manner. Recordings from the brain suggest that song is driven by two descending pathways – one defines when to sing and the other specifies what song to sing. Connectomic analysis reveals that these “when” and “what” descending pathways provide structured input to VNC neurons with different motor functions. These results suggest that dynamic changes in the activation patterns of descending pathways drive different combinations of motor modules, thereby flexibly switching between different motor actions.

Programmed genome rearrangements in the unicellular eukaryote Oxytricha trifallax produce a transcriptionally active somatic nucleus from a copy of its germline nucleus during development. This process eliminates noncoding sequences that interrupt coding regions in the germline genome, and joins over 225,000 remaining DNA segments, some of which require inversion or complex permutation to build functional genes. This dynamic genomic organization permits some single DNA segments in the germline to contribute to multiple, distinct somatic genes via alternative processing. Like alternative mRNA splicing, the combinatorial assembly of DNA segments contributes to genetic variation and facilitates the evolution of new genes. In this study, we use comparative genomic analysis to demonstrate that the emergence of alternative DNA splicing is associated with the origin of new genes. Short duplications give rise to alternative gene segments that are spliced to the shared gene segments. Alternative gene segments evolve faster than shared, constitutive segments. Genes with shared segments frequently have different expression profiles, permitting functional divergence. This study reports alternative DNA splicing as a mechanism of new gene origination, illustrating how the process of programmed genome rearrangement gives rise to evolutionary innovation.

Mapping nanoscale neuronal morphology with molecular annotations is critical for understanding healthy and dysfunctional brain circuits. Current methods are constrained by image segmentation errors and by sample defects (e.g., signal gaps, section loss). Genetic strategies promise to overcome these challenges by using easily distinguishable cell identity labels. However, multicolor approaches are spectrally limited in diversity, whereas nucleic acid barcoding lacks a cell-filling morphology signal for segmentation. Here, we introduce PRISM (Protein-barcode Reconstruction via Iterative Staining with Molecular annotations), a platform that integrates combinatorial delivery of antigenically distinct, cell-filling proteins with tissue expansion, multi-cycle imaging, barcode-augmented reconstruction, and molecular annotation. Protein barcodes increase label diversity by >750-fold over multicolor labeling and enable morphology reconstruction with intrinsic error correction. We acquired a \~10 million μm3 volume of mouse hippocampal area CA2/3, multiplexed across 23 barcode antigen and synaptic marker channels. By combining barcodes with shape information we achieve an 8x increase in automatic tracing accuracy of genetically labelled neurons. We demonstrate PRISM supports automatic proofreading across micron-scale spatial gaps and reconnects neurites across discontinuities spanning hundreds of microns. Using PRISM’s molecular annotation capability, we map the distribution of synapses onto traced neural morphology, characterizing challenging synaptic structures such as thorny excrescences (TEs), and discovering a size correlation among spatially proximal TEs on the same dendrite. PRISM thus supports self-correcting neuron reconstruction with molecular context.