To image the accessible genome at nanometer scale in situ, we developed three-dimensional assay for transposase-accessible chromatin-photoactivated localization microscopy (3D ATAC-PALM) that integrates an assay for transposase-accessible chromatin with visualization, PALM super-resolution imaging and lattice light-sheet microscopy. Multiplexed with oligopaint DNA–fluorescence in situ hybridization (FISH), RNA–FISH and protein fluorescence, 3D ATAC-PALM connected microscopy and genomic data, revealing spatially segregated accessible chromatin domains (ACDs) that enclose active chromatin and transcribed genes. Using these methods to analyze genetically perturbed cells, we demonstrated that genome architectural protein CTCF prevents excessive clustering of accessible chromatin and decompacts ACDs. These results highlight 3D ATAC-PALM as a useful tool to probe the structure and organizing mechanism of the genome.

Calcium imaging with fluorescent protein sensors is widely used to record activity in neuronal populations. The transform between neural activity and calcium-related fluorescence involves nonlinearities and low-pass filtering, but the effects of the transformation on analyses of neural populations are not well understood. We compared neuronal spikes and fluorescence in matched neural populations in behaving mice. We report multiple discrepancies between analyses performed on the two types of data, including changes in single-neuron selectivity and population decoding. These were only partially resolved by spike inference algorithms applied to fluorescence. To model the relation between spiking and fluorescence we simultaneously recorded spikes and fluorescence from individual neurons. Using these recordings we developed a model transforming spike trains to synthetic-imaging data. The model recapitulated the differences in analyses. Our analysis highlights challenges in relating electrophysiology and imaging data, and suggests forward modeling as an effective way to understand differences between these data.

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly . Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the fly's brain.

When used in combination, self-labelling protein tags such as Halo, SNAP, and CLIP allow for the simultaneous visualization of proteins across a wide fluorescence spectrum. However, the combination of cell type, ligand binding and fluorescent dye chemistry introduces several variables that need to be determined to achieve orthogonal labelling. The Janelia Cell Culture Shared Resource in collaboration with a Research Scientist, and the Lavis Lab have developed a high throughput cytometry-based assay to determine optimal conditions for various combinations of cell type, ligand and JF dyes.

Here we design and optimize a genetically encoded fluorescent indicator, iAChSnFR, for the ubiquitous neurotransmitter acetylcholine, based on a bacterial periplasmic binding protein. iAChSnFR shows large fluorescence changes, rapid rise and decay kinetics, and insensitivity to most cholinergic drugs. iAChSnFR revealed large transients in a variety of slice and in vivo preparations in mouse, fish, fly and worm. iAChSnFR will be useful for the study of acetylcholine in all animals.

We engineered electrochromic fluorescence resonance energy transfer (eFRET) genetically encoded voltage indicators (GEVIs) with “positive-going” fluorescence response to membrane depolarization through rational manipulation of the native proton transport pathway in microbial rhodopsins. We transformed the state-of-the-art eFRET GEVI Voltron into Positron, with kinetics and sensitivity equivalent to Voltron but flipped fluorescence signal polarity. We further applied this general approach to GEVIs containing different voltage sensitive rhodopsin domains and various fluorescent dye and fluorescent protein reporters.

Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted 'Janelia Fluor' (JF) dyes useful for biological imaging experiments in cells and in vivo.

The anatomy of many neural circuits is being characterized with increasing resolution, but their molecular properties remain mostly unknown. Here, we characterize gene expression patterns in distinct neural cell types of the visual system using genetic lines to access individual cell types, the TAPIN-seq method to measure their transcriptomes, and a probabilistic method to interpret these measurements. We used these tools to build a resource of high-resolution transcriptomes for 100 driver lines covering 67 cell types, available at http://www.opticlobe.com. Combining these transcriptomes with recently reported connectomes helps characterize how information is transmitted and processed across a range of scales, from individual synapses to circuit pathways. We describe examples that include identifying neurotransmitters, including cases of apparent co-release, generating functional hypotheses based on receptor expression, as well as identifying strong commonalities between different cell types.

The striosome compartment within the dorsal striatum has been implicated in reinforcement learning and regulation of motivation, but how striosomal neurons contribute to these functions remains elusive. Here, we show that a genetically identified striosomal population, which expresses the Teashirt family zinc finger 1 (Tshz1) and belongs to the direct pathway, drives negative reinforcement and is essential for aversive learning in mice. Contrasting a "conventional" striosomal direct pathway, the Tshz1 neurons cause aversion, movement suppression, and negative reinforcement once activated, and they receive a distinct set of synaptic inputs. These neurons are predominantly excited by punishment rather than reward and represent the anticipation of punishment or the motivation for avoidance. Furthermore, inhibiting these neurons impairs punishment-based learning without affecting reward learning or movement. These results establish a major role of striosomal neurons in behaviors reinforced by punishment and moreover uncover functions of the direct pathway unaccounted for in classic models.