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Brain organoids derived from human pluripotent stem cells provide a highly valuable in vitro model to recapitulate human brain development and neurological diseases. However, the current systems for brain organoid culture require further improvement for the reliable production of high-quality organoids. Here, we demonstrate two engineering elements to improve human brain organoid culture, (1) a human brain extracellular matrix to provide brain-specific cues and (2) a microfluidic device with periodic flow to improve the survival and reduce the variability of organoids. A three-dimensional culture modified with brain extracellular matrix significantly enhanced neurogenesis in developing brain organoids from human induced pluripotent stem cells. Cortical layer development, volumetric augmentation, and electrophysiological function of human brain organoids were further improved in a reproducible manner by dynamic culture in microfluidic chamber devices. Our engineering concept of reconstituting brain-mimetic microenvironments facilitates the development of a reliable culture platform for brain organoids, enabling effective modeling and drug development for human brain diseases. Brain organoids derived from human pluripotent stem cells can model human brain development and disease, though current culture systems fail to ensure reliable production of high-quality organoids. Here the authors combine human brain extracellular matrix and culture in a microfluidic device to promote structural and functional maturation of human brain organoids.

Eating and drinking generate sequential mechanosensory signals along the digestive tract. These signals are communicated to the brain for the timely initiation and regulation of diverse ingestive and digestive processes — ranging from appetite control and tactile perception to gut motility, digestive fluid secretion and defecation — that are vital for the proper intake, breakdown and absorption of nutrients and water. Gut mechanosensation has been investigated for over a century as a common pillar of energy, fluid and gastrointestinal homeostasis, and recent discoveries of specific mechanoreceptors, contributing ion channels and the well-defined circuits underlying gut mechanosensation signalling and function have further expanded our understanding of ingestive and digestive processes at the molecular and cellular levels. In this Review, we discuss our current understanding of the generation of mechanosensory signals from the digestive periphery, the neural afferent pathways that relay these signals to the brain and the neural circuit mechanisms that control ingestive and digestive processes, focusing on the four major digestive tract parts: the oral and pharyngeal cavities, oesophagus, stomach and intestines. We also discuss the clinical implications of gut mechanosensation in ingestive and digestive disorders.Ingestive and digestive processes are initiated and regulated by mechanosensory signals along the digestive tract. In this Review, Kim, Heo and Kim discuss recent discoveries of specific mechanoreceptors, contributing ion channels and well-defined circuits underlying gut mechanosensation, focusing on the oral and pharyngeal cavities, oesophagus, stomach and intestines.

As the principal means of acquiring nutrients, feeding behavior is indispensable to the survival and well-being of animals. In response to energy or nutrient deficits, animals seek and consume food to maintain energy homeostasis. On the other hand, even when animals are calorically replete, non-homeostatic factors, such as the sight, smell, and taste of palatable food, or environmental cues that predict food, can stimulate feeding behavior. These homeostatic and non-homeostatic factors have traditionally been investigated separately, but a growing body of literature highlights that these factors work synergistically to promote feeding behavior. Furthermore, recent breakthroughs in cell type-specific and circuit-specific labeling, recording, and manipulation techniques have markedly accelerated the discovery of well-defined neural populations underlying homeostatic and non-homeostatic appetite control, as well as overlapping circuits that contribute to both types of appetite. This review aims to provide an update on our understanding of the neural circuit mechanisms for promoting homeostatic and non-homeostatic appetites, focusing on the function of recently identified, genetically defined cell types.Appetite: Linking neural circuits to feeding behaviorsResearch on the neural circuit mechanisms underlying feeding behaviors is critical to identifying therapeutic targets for food-related disorders like obesity and anorexia. Sung-Yon Kim and colleagues at Seoul National University, South Korea, reviewed the current understanding of neural circuits promoting feeding behavior, which is regulated by homeostatic and non-homeostatic appetites. In response to deficits in energy (caloric) or nutrients, specific populations of neurons sensitive to hormones leptin and ghrelin generate homeostatic appetite and promote feeding. In addition, diverse neural populations stimulate non-homeostatic appetite in the absence of immediate internal needs and are thought to drive overconsumption in the modern obesogenic environment. These appetites extensively interact through overlapping neural circuits to jointly promote feeding behaviors.

Mechanosensory feedback from the digestive tract to the brain is critical for limiting excessive food and water intake, but the underlying gut–brain communication pathways and mechanisms remain poorly understood 1 – 12 . Here we show that, in mice, neurons in the parabrachial nucleus that express the prodynorphin gene (hereafter, PB Pdyn neurons) monitor the intake of both fluids and solids, using mechanosensory signals that arise from the upper digestive tract. Most individual PB Pdyn neurons are activated by ingestion as well as the stimulation of the mouth and stomach, which indicates the representation of integrated sensory signals across distinct parts of the digestive tract. PB Pdyn neurons are anatomically connected to the digestive periphery via cranial and spinal pathways; we show that, among these pathways, the vagus nerve conveys stomach-distension signals to PB Pdyn neurons. Upon receipt of these signals, these neurons produce aversive and sustained appetite-suppressing signals, which discourages the initiation of feeding and drinking (fully recapitulating the symptoms of gastric distension) in part via signalling to the paraventricular hypothalamus. By contrast, inhibiting the same population of PB Pdyn neurons induces overconsumption only if a drive for ingestion exists, which confirms that these neurons mediate negative feedback signalling. Our findings reveal a neural mechanism that underlies the mechanosensory monitoring of ingestion and negative feedback control of intake behaviours upon distension of the digestive tract. A population of neurons in the parabrachial nucleus that expresses prodynorphin monitors ingestion using mechanosensory signals from the upper digestive tract, and mediates negative feedback control of intake when the digestive tract is distended.

High-speed tracking of effortful responses and neuronal activity in rats during a forced swim test identifies medial prefrontal cortex (mPFC) neurons that respond during escape-related swimming but not normal locomotion, and optogenetics shows that mPFC neurons projecting to the brainstem dorsal raphe nucleus, which is implicated in depression, modulate this behavioural response to challenge The neural circuitry of choice Disruption of the prefrontal cortex (PFC) area in the human brain can lead either to impulsive behaviour or to a lack of motivation. This study explores the role of particular populations of PFC neurons in mice during a challenging behavioural situation — the forced swim test. The authors identify neurons that respond during forced swimming, but not during normal locomotion. Using optogenetic manipulation, they show that only the specific population of PFC neurons projecting to the brainstem dorsal raphe nucleus, a region implicated in depression, induces changes in behaviour during forced swimming. These results throw light on the neural circuitry underlying normal and pathological patterns of action selection and motivation in behaviour. The prefrontal cortex (PFC) is thought to participate in high-level control of the generation of behaviours (including the decision to execute actions 1 ); indeed, imaging and lesion studies in human beings have revealed that PFC dysfunction can lead to either impulsive states with increased tendency to initiate action 2 , or to amotivational states characterized by symptoms such as reduced activity, hopelessness and depressed mood 3 . Considering the opposite valence of these two phenotypes as well as the broad complexity of other tasks attributed to PFC, we sought to elucidate the PFC circuitry that favours effortful behavioural responses to challenging situations. Here we develop and use a quantitative method for the continuous assessment and control of active response to a behavioural challenge, synchronized with single-unit electrophysiology and optogenetics in freely moving rats. In recording from the medial PFC (mPFC), we observed that many neurons were not simply movement-related in their spike-firing patterns but instead were selectively modulated from moment to moment, according to the animal’s decision to act in a challenging situation. Surprisingly, we next found that direct activation of principal neurons in the mPFC had no detectable causal effect on this behaviour. We tested whether this behaviour could be causally mediated by only a subclass of mPFC cells defined by specific downstream wiring. Indeed, by leveraging optogenetic projection-targeting to control cells with specific efferent wiring patterns, we found that selective activation of those mPFC cells projecting to the brainstem dorsal raphe nucleus (DRN), a serotonergic nucleus implicated in major depressive disorder 4 , induced a profound, rapid and reversible effect on selection of the active behavioural state. These results may be of importance in understanding the neural circuitry underlying normal and pathological patterns of action selection and motivation in behaviour.

Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1–3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.

Tissue expansion techniques physically expand swellable gel‐embedded biological specimens to overcome the resolution limit of light microscopy. As the benefits of expansion come at the expense of signal concentration, imaging volume and time, and mechanical integrity of the sample, the optimal expansion ratio may widely differ depending on the experiment. However, existing expansion methods offer only fixed expansion ratios that cannot be easily adjusted to balance the gain and loss associated with expansion. Here, a hydrogel conversion‐based expansion method is presented, that enables easy adjustment of the expansion ratio for individual needs, simply by changing the duration of a heating step. This method, termed ZOOM, isotropically expands samples up to eightfold in a single expansion process. ZOOM preserves biomolecules for post‐processing labelings and supports multi‐round expansion for the imaging of a single sample at multiple zoom factors. ZOOM can be flexibly and scalably applied to nanoscale imaging of diverse samples, ranging from cultured cells to thick tissues, as well as bacteria, exoskeletal Caenorhabditis elegans, and human brain samples. ZOOM, a new tissue expansion technique based on the hydrogel conversion reaction, is developed to enable scalable and isotropic expansion of biological samples with easily tunable expansion ratio (up to eightfold). This method allows for simple and flexible expansion of a wide range of biological samples, from bacteria to human brain tissues, for super‐resolution imaging of samples with ordinary microscopes.

Schwann cells (SCs) constitute a crucial element of the peripheral nervous system, by structurally supporting the formation of myelin and conveying vital trophic factors to the nervous system. However, the functions of SCs in developmental and regenerative stages remain unclear. Here, we investigated how optogenetic stimulation (OS) of SCs regulates their development. In SC monoculture, OS substantially enhanced SC proliferation and the number of BrdU + -S100ß + -SCs over time. In addition, OS also markedly promoted the expression of both Krox20 and myelin basic protein (MBP) in SC culture medium containing dBcAMP/NRG1, which induced differentiation. We found that the effects of OS are dependent on the intracellular Ca 2+ level. OS induces elevated intracellular Ca 2+ levels through the T-type voltage-gated calcium channel (VGCC) and mobilization of Ca 2+ from both inositol 1,4,5-trisphosphate (IP 3 )-sensitive stores and caffeine/ryanodine-sensitive stores. Furthermore, we confirmed that OS significantly increased expression levels of both Krox20 and MBP in SC-motor neuron (MN) coculture, which was notably prevented by pharmacological intervention with Ca 2+ . Taken together, our results demonstrate that OS of SCs increases the intracellular Ca 2+ level and can regulate proliferation, differentiation, and myelination, suggesting that OS of SCs may offer a new approach to the treatment of neurodegenerative disorders.

MicroRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes. Therefore, control over the disease-related miRNA expression is important both for basic research and for a new class of therapeutic modality to treat serious diseases such as cancer. Here, we present a high-throughput screening strategy to identify small molecules that modulate miRNA expression in living cells. The screen enables simultaneous monitoring of the phenotypic cellular changes associated with the miRNA expression by measuring quantitative fluorescent signals corresponding to target miRNA level in living cells based on a novel biosensor composed of peptide nucleic acid and nano-sized graphene oxide. In this study, the biosensor based cellular screening of 967 compounds (including FDA-approved drugs, enzyme inhibitors, agonists, and antagonists) in cells identified four different classes of small molecules consisting of (i) 70 compounds that suppress both miRNA-21 (miR-21) expression and cell proliferation, (ii) 65 compounds that enhance miR-21 expression and reduce cell proliferation, (iii) 2 compounds that suppress miR-21 expression and increase cell proliferation, and (iv) 21 compounds that enhance both miR-21 expression and cell proliferation. We further investigated the hit compounds to correlate cell morphology changes and cell migration ability with decreased expression of miR-21.

Obtaining high-resolution information from a complex system, while maintaining the global perspective needed to understand system function, represents a key challenge in biology. Here we address this challenge with a method (termed CLARITY) for the transformation of intact tissue into a nanoporous hydrogel-hybridized form (crosslinked to a three-dimensional network of hydrophilic polymers) that is fully assembled but optically transparent and macromolecule-permeable. Using mouse brains, we show intact-tissue imaging of long-range projections, local circuit wiring, cellular relationships, subcellular structures, protein complexes, nucleic acids and neurotransmitters. CLARITY also enables intact-tissue in situ hybridization, immunohistochemistry with multiple rounds of staining and de-staining in non-sectioned tissue, and antibody labelling throughout the intact adult mouse brain. Finally, we show that CLARITY enables fine structural analysis of clinical samples, including non-sectioned human tissue from a neuropsychiatric-disease setting, establishing a path for the transmutation of human tissue into a stable, intact and accessible form suitable for probing structural and molecular underpinnings of physiological function and disease. High-resolution imaging has traditionally required thin sectioning, a process that disrupts long-range connectivity in the case of brains: here, intact mouse brains and human brain samples have been made fully transparent and macromolecule permeable using a new method termed CLARITY, which allows for intact-tissue imaging as well as repeated antibody labelling and in situ hybridization of non-sectioned tissue. Structure in a see-through brain High-resolution imaging of biological tissue has traditionally required sectioning, which for tissues like the brain means the loss of long-range connectivity. Now Karl Deisseroth and colleagues have developed a way of making full, intact organs optically transparent and macromolecule-permeable by building a hydrogel-based infrastructure from within the tissue that allows subsequent removal of light-scattering lipids, resulting in a transparent brain. The method, termed CLARITY, also allows repeated antibody labelling of proteins, and in situ hybridization of nucleic acids in non-sectioned tissue, such as full mouse brains or human clinical samples stored in formalin for many years.