Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.

A central problem in neuroscience is reconstructing neuronal circuits on the synapse level. Due to a wide range of scales in brain architecture such reconstruction requires imaging that is both high-resolution and high-throughput. Existing electron microscopy (EM) techniques possess required resolution in the lateral plane and either high-throughput or high depth resolution but not both. Here, we exploit recent advances in unsupervised learning and signal processing to obtain high depth-resolution EM images computationally without sacrificing throughput. First, we show that the brain tissue can be represented as a sparse linear combination of localized basis functions that are learned using high-resolution datasets. We then develop compressive sensing-inspired techniques that can reconstruct the brain tissue from very few (typically 5) tomographic views of each section. This enables tracing of neuronal processes and, hence, high throughput reconstruction of neural circuits on the level of individual synapses.

The delivery of tracers into populations of neurons is essential to visualize their anatomy and analyze their function. In some model systems genetically-targeted expression of fluorescent proteins is the method of choice; however, these genetic tools are not available for most organisms and alternative labeling methods are very limited. Here we describe a new method for neuronal labelling by electrophoretic dye delivery from a suction electrode directly through the neuronal sheath of nerves and ganglia in insects. Polar tracer molecules were delivered into the locust auditory nerve without destroying its function, simultaneously staining peripheral sensory structures and central axonal projections. Local neuron populations could be labelled directly through the surface of the brain, and in-vivo optical imaging of sound-evoked activity was achieved through the electrophoretic delivery of calcium indicators. The method provides a new tool for studying how stimuli are processed in peripheral and central sensory pathways and is a significant advance for the study of nervous systems in non-model organisms.

DNA microarrays are used for gene-expression profiling, single-nucleotide polymorphism detection and disease diagnosis. A persistent challenge in this area is the lack of microarray screening technology suitable for integration into routine clinical care. Here, we describe a method for sensitive and label-free electrostatic readout of DNA or RNA hybridization on microarrays. The electrostatic properties of the microarray are measured from the position and motion of charged microspheres randomly dispersed over the surface. We demonstrate nondestructive electrostatic imaging with 10-mum lateral resolution over centimeter-length scales, which is four-orders of magnitude larger than that achievable with conventional scanning electrostatic force microscopy. Changes in surface charge density as a result of specific hybridization can be detected and quantified with 50-pM sensitivity, single base-pair mismatch selectivity and in the presence of complex background. Because the naked eye is sufficient to read out hybridization, this approach may facilitate broad application of multiplexed assays.

Psychological stress and its sequelae pose a major challenge to public health. Immune activation is conventionally thought to aggravate stress-related mental diseases such as anxiety disorders and depression. Here, we sought to identify potentially beneficial consequences of immune activation in response to stress. We showed that stress led to increased interleukin (IL)-22 production in the intestine as a result of stress-induced gut leakage. IL-22 was both necessary and sufficient to attenuate stress-induced anxiety behaviors in mice. More specifically, IL-22 gained access to the septal area of the brain and directly suppressed neuron activation. Furthermore, human patients with clinical depression displayed reduced IL-22 levels, and exogenous IL-22 treatment ameliorated depressive-like behavior elicited by chronic stress in mice. Our study thus identifies a gut-brain axis in response to stress, whereby IL-22 reduces neuronal activation and concomitant anxiety behavior, suggesting that early immune activation can provide protection against psychological stress.

Photoactivatable or "caged" pharmacological agents combine the high spatiotemporal specificity of light application with the molecular specificity of drugs. A key factor in all optopharmacology experiments is the mechanism of uncaging, which dictates the photochemical quantum yield and determines the byproducts produced by the light-driven chemical reaction. In previous work, we demonstrated that coumarin-based photolabile groups could be used to cage tertiary amine drugs as quaternary ammonium salts. Although stable, water-soluble, and useful for experiments in brain tissue, these first-generation compounds exhibit relatively low uncaging quantum yield (Φ < 1%) and release the toxic byproduct formaldehyde upon photolysis. Here, we elucidate the photochemical mechanisms of coumarin-caged tertiary amines and then optimize the major pathway using chemical modification. We discovered that the combination of 3,3-dicarboxyazetidine and bromine substituents shift the mechanism of release to heterolysis, eliminating the formaldehyde byproduct and giving photolabile tertiary amine drugs with Φ > 20%─a 35-fold increase in uncaging efficiency. This new "ABC" cage allows synthesis of improved photoactivatable derivatives of escitalopram and nicotine along with a novel caged agonist of the oxytocin receptor.

Intracellular recording allows measurement and perturbation of the membrane potential of identified neurons with sub-millisecond and sub-millivolt precision. This gives intracellular recordings a unique capacity to provide rich information about individual cells (e.g., high-resolution characterization of inputs, outputs, excitability, and structure). Hence, such recordings can elucidate the mechanisms that underlie fundamental phenomena, such as brain state, sparse coding, gating, gain modulation, and learning. Technical developments have increased the range of behaviors during which intracellular recording methods can be employed, such as in freely moving animals and head-fixed animals actively performing tasks, including in virtual environments. Such advances, and the combination of intracellular recordings with genetic and imaging techniques, have enabled investigation of the mechanisms that underlie neural computations during natural and trained behaviors.

Broad (BR), an ecdysone-inducible transcription factor, is a major determinant of the pupal stage. The misexpression of BR-Z1 isoform (BR-Z1) during adult development of Drosophila melanogaster prevents the expression of the adult cuticle protein 65A gene (Acp65A). We found that the proximal 237 bp of the 5’ flanking region of Acp65A were sufficient to mediate this suppression. A targeted point mutation of a putative BR-Z1 response element (BRE) within this region showed that it was not involved. Drosophila hormone receptor-like 38 (DHR38) is required for Acp65A expression. We found that BR-Z1 repressed DHR38 expression and that BR’s inhibition of Acp65A expression was rescued by exogenous expression of DHR38. Thus, BR-Z1 suppresses Acp65A expression by preventing the normal up-regulation of DHR38 at the time of adult cuticle formation.

Embryonic stem (ES) cells can undergo many aspects of mammalian embryogenesis in vitro, but their developmental potential is substantially extended by interactions with extraembryonic stem cells, including trophoblast stem (TS) cells, extraembryonic endoderm stem (XEN) cells and inducible XEN (iXEN) cells. Here we assembled stem cell-derived embryos in vitro from mouse ES cells, TS cells and iXEN cells and showed that they recapitulate the development of whole natural mouse embryo in utero up to day 8.5 post-fertilization. Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells. This complete embryo model develops within an extraembryonic yolk sac that initiates blood island development. Notably, we demonstrate that the neurulating embryo model assembled from Pax6-knockout ES cells aggregated with wild-type TS cells and iXEN cells recapitulates the ventral domain expansion of the neural tube that occurs in natural, ubiquitous Pax6-knockout embryos. Thus, these complete embryoids are a powerful in vitro model for dissecting the roles of diverse cell lineages and genes in development. Our results demonstrate the self-organization ability of ES cells and two types of extraembryonic stem cells to reconstitute mammalian development through and beyond gastrulation to neurulation and early organogenesis.

The early formation of sensorimotor circuits is essential for survival. While the development and function of exteroceptive circuits and their associated motor pathways are well characterized, far less is known about the circuits that convey viscerosensory inputs to the brain and transmit visceromotor commands from the central nervous system to internal organs. Technical limitations, such as the in utero development of viscerosensory and visceromotor circuits and the invasiveness of procedures required to access them, have hindered studies of their functional development in mammals. Using larval zebrafish—which are genetically accessible and optically transparent—we tracked, in vivo, how cardiosensory and cardiomotor neural circuits assemble and begin to function. We uncovered a staged program. First, a minimal efferent circuit suffices for heart-rate control: direct brain-to-heart vagal motor innervation is required, intracardiac neurons are not, and heart rate is governed exclusively by the motor vagus nerve. Within the hindbrain, we functionally localize a vagal premotor population that drives this early efferent control. Second, sympathetic innervation arrives and enhances the dynamics and amplitude of cardiac responses, as neurons in the most anterior sympathetic ganglia acquire the ability to drive cardiac acceleration. These neurons exhibit proportional, integral, and derivative–like relationships to heart rate, consistent with controller motifs that shape gain and dynamics. Third, vagal sensory neurons innervate the heart. Distinct subsets increase activity when heart rate falls or rises, and across spontaneous fluctuations, responses to aversive stimuli, and optogenetically evoked cardiac perturbations, their dynamics are captured by a single canonical temporal kernel with neuron-specific phase offsets, supporting a population code for heart rate. This temporally segregated maturation isolates three experimentally tractable regimes—unidirectional brain-to-heart communication, dual efferent control, and closed-loop control after sensory feedback engages—providing a framework for mechanistic dissection of organism-wide heart–brain circuits.