Expansion microscopy (ExM) is a physical form of magnification that increases the effective resolving power of any microscope. Here, we describe the fundamental principles of ExM, as well as how recently developed ExM variants build upon and apply those principles. We examine applications of ExM in cell and developmental biology for the study of nanoscale structures as well as ExM's potential for scalable mapping of nanoscale structures across large sample volumes. Finally, we explore how the unique anchoring and hydrogel embedding properties enable postexpansion molecular interrogation in a purified chemical environment. ExM promises to play an important role complementary to emerging live-cell imaging techniques, because of its relative ease of adoption and modification and its compatibility with tissue specimens up to at least 200 μm thick. Expected final online publication date for the , Volume 35 is October 7, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Imaging changes in membrane potential using genetically encoded fluorescent voltage indicators (GEVIs) has great potential for monitoring neuronal activity with high spatial and temporal resolution. Brightness and photostability of fluorescent proteins and rhodopsins have limited the utility of existing GEVIs. We engineered a novel GEVI, "Voltron", that utilizes bright and photostable synthetic dyes instead of protein-based fluorophores, extending the combined duration of imaging and number of neurons imaged simultaneously by more than tenfold relative to existing GEVIs. We used Voltron for in vivo voltage imaging in mice, zebrafish, and fruit flies. In mouse cortex, Voltron allowed single-trial recording of spikes and subthreshold voltage signals from dozens of neurons simultaneously, over 15 min of continuous imaging. In larval zebrafish, Voltron enabled the precise correlation of spike timing with behavior.

In the current model of endothelial barrier regulation, the tyrosine kinase SRC is purported to induce disassembly of endothelial adherens junctions (AJs) via phosphorylation of VE cadherin, and thereby increase junctional permeability. Here, using a chemical biology approach to temporally control SRC activation, we show that SRC exerts distinct time-variant effects on the endothelial barrier. We discovered that the immediate effect of SRC activation was to transiently enhance endothelial barrier function as the result of accumulation of VE cadherin at AJs and formation of morphologically distinct reticular AJs. Endothelial barrier enhancement via SRC required phosphorylation of VE cadherin at Y731. In contrast, prolonged SRC activation induced VE cadherin phosphorylation at Y685, resulting in increased endothelial permeability. Thus, time-variant SRC activation differentially phosphorylates VE cadherin and shapes AJs to fine-tune endothelial barrier function. Our work demonstrates important advantages of synthetic biology tools in dissecting complex signaling systems.

Neural computation involves diverse types of GABAergic inhibitory interneurons that are integrated with excitatory (E) neurons into precisely structured circuits. To understand how each neuron type shapes sensory representations, we measured firing patterns of defined types of neurons in the barrel cortex while mice performed an active, whisker-dependent object localization task. Touch excited fast-spiking (FS) interneurons at short latency, followed by activation of E neurons and somatostatin-expressing (SST) interneurons. Touch only weakly modulated vasoactive intestinal polypeptide-expressing (VIP) interneurons. Voluntary whisker movement activated FS neurons in the ventral posteromedial nucleus (VPM) target layers, a subset of SST neurons and a majority of VIP neurons. Together, FS neurons track thalamic input, mediating feedforward inhibition. SST neurons monitor local excitation, providing feedback inhibition. VIP neurons are activated by non-sensory inputs, disinhibiting E and FS neurons. Our data reveal rules of recruitment for interneuron types during behavior, providing foundations for understanding computation in cortical microcircuits.

Animals can perform complex and purposeful behaviors by executing simpler movements in flexible sequences. It is particularly challenging to analyze behavior sequences when they are highly variable, as is the case in language production, certain types of birdsong and, as in our experiments, flies grooming. High sequence variability necessitates rigorous quantification of large amounts of data to identify organizational principles and temporal structure of such behavior. To cope with large amounts of data, and minimize human effort and subjective bias, researchers often use automatic behavior recognition software. Our standard grooming assay involves coating flies in dust and videotaping them as they groom to remove it. The flies move freely and so perform the same movements in various orientations. As the dust is removed, their appearance changes. These conditions make it difficult to rely on precise body alignment and anatomical landmarks such as eyes or legs and thus present challenges to existing behavior classification software. Human observers use speed, location, and shape of the movements as the diagnostic features of particular grooming actions. We applied this intuition to design a new automatic behavior recognition system (ABRS) based on spatiotemporal features in the video data, heavily weighted for temporal dynamics and invariant to the animal’s position and orientation in the scene. We use these spatiotemporal features in two steps of supervised classification that reflect two time-scales at which the behavior is structured. As a proof of principle, we show results from quantification and analysis of a large data set of stimulus-induced fly grooming behaviors that would have been difficult to assess in a smaller dataset of human-annotated ethograms. While we developed and validated this approach to analyze fly grooming behavior, we propose that the strategy of combining alignment-invariant features and multi-timescale analysis may be generally useful for movement-based classification of behavior from video data.

In pursuit of food, hungry animals mobilize significant energy resources and overcome exhaustion and fear. How need and motivation control the decision to continue or change behavior is not understood. Using a single fly treadmill, we show that hungry flies persistently track a food odor and increase their effort over repeated trials in the absence of reward suggesting that need dominates negative experience. We further show that odor tracking is regulated by two mushroom body output neurons (MBONs) connecting the MB to the lateral horn. These MBONs, together with dopaminergic neurons and Dop1R2 signaling, control behavioral persistence. Conversely, an octopaminergic neuron, VPM4, which directly innervates one of the MBONs, acts as a brake on odor tracking by connecting feeding and olfaction. Together, our data suggest a function for the MB in internal state-dependent expression of behavior that can be suppressed by external inputs conveying a competing behavioral drive.

In the Drosophila model of aggression, males and females fight in same-sex pairings, but a wide disparity exists in the levels of aggression displayed by the 2 sexes. A screen of Drosophila Flylight Gal4 lines by driving expression of the gene coding for the temperature sensitive dTRPA1 channel, yielded a single line (GMR26E01-Gal4) displaying greatly enhanced aggression when thermoactivated. Targeted neurons were widely distributed throughout male and female nervous systems, but the enhanced aggression was seen only in females. No effects were seen on female mating behavior, general arousal, or male aggression. We quantified the enhancement by measuring fight patterns characteristic of female and male aggression and confirmed that the effect was female-specific. To reduce the numbers of neurons involved, we used an intersectional approach with our library of enhancer trap flp-recombinase lines. Several crosses reduced the populations of labeled neurons, but only 1 cross yielded a large reduction while maintaining the phenotype. Of particular interest was a small group (2 to 4 pairs) of neurons in the approximate position of the pC1 cluster important in governing male and female social behavior. Female brains have approximately 20 doublesex (dsx)-expressing neurons within pC1 clusters. Using dsxFLP instead of 357FLP for the intersectional studies, we found that the same 2 to 4 pairs of neurons likely were identified with both. These neurons were cholinergic and showed no immunostaining for other transmitter compounds. Blocking the activation of these neurons blocked the enhancement of aggression.

Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.

In a recent Editorial, De Schutter commented on our recent study on the roles of a cortico-cerebellar loop in motor planning in mice (De Schutter 2019, Neuroinformatics, 17, 181-183, Gao et al. 2018, Nature, 563, 113-116). Two issues were raised. First, De Schutter questions the involvement of the fastigial nucleus in motor planning, rather than the dentate nucleus, given previous anatomical studies in non-human primates. Second, De Schutter suggests that our study design did not delineate different components of the behavior and the fastigial nucleus might play roles in sensory discrimination rather than motor planning. These comments are based on anatomical studies in other species and homology-based arguments and ignore key anatomical data and neurophysiological experiments from our study. Here we outline our interpretation of existing data and point out gaps in knowledge where future studies are needed.

The palette of tools for stimulation and regulation of neural activity is continually expanding. One of the new methods being introduced is magnetogenetics, where mechano-sensitive and thermo-sensitive ion channels are genetically engineered to be closely coupled to the iron-storage protein ferritin. Such genetic constructs could provide a powerful new way of non-invasively activating ion channels in-vivo using external magnetic fields that easily penetrate biological tissue. Initial reports that introduced this new technology have sparked a vigorous debate on the plausibility of physical mechanisms of ion channel activation by means of external magnetic fields. I argue that the initial criticisms leveled against magnetogenetics as being physically implausible were possibly based on the overly simplistic and unnecessarily pessimistic assumptions about the magnetic spin configurations of iron in ferritin protein. Additionally, all the possible magnetic-field-based mechanisms of ion channel activation in magnetogenetics might not have been fully considered. I present and propose several new magneto-mechanical and magneto-thermal mechanisms of ion channel activation by iron-loaded ferritin protein that may elucidate and clarify some of the mysteries that presently challenge our understanding of the reported biological experiments. Finally, I present some additional puzzles that will require further theoretical and experimental investigation.