Divergent opioid-mediated suppression of inhibition between hippocampus and neocortex across species and development.

Opioid receptors within the CNS regulate pain sensation and mood and are key targets for drugs of abuse. Within the adult rodent hippocampus (HPC), µ-opioid receptor agonists suppress inhibitory parvalbumin-expressing interneurons (PV-INs), thus disinhibiting the circuit. However, it is uncertain if this disinhibitory motif is conserved in other cortical regions, species, or across development. We observed that PV-IN mediated inhibition is robustly suppressed by opioids in HPC but not neocortex in mice and nonhuman primates, with spontaneous inhibitory tone in resected human tissue also following a consistent dichotomy. This hippocampal disinhibitory motif was established in early development when immature PV-INs and opioids already influence primordial network rhythmogenesis. Acute opioid-mediated modulation was partially occluded with morphine pretreatment, with implications for the effects of opioids on hippocampal network activity during circuit maturation as well as learning and memory. Together, these findings demonstrate that PV-INs exhibit a divergence in opioid sensitivity across brain regions that is remarkably conserved across evolution and highlights the underappreciated role of opioids acting through immature PV-INs in shaping hippocampal development.

Symmetry Breaking of Human Pluripotent Stem Cells (hPSCs) in Micropattern Generates a Polarized Spinal Cord-Like Organoid (pSCO) with Dorsoventral Organization.

Axis formation and related spatial patterning are initiated by symmetry breaking during development. A geometrically confined culture of human pluripotent stem cells (hPSCs) mimics symmetry breaking and cell patterning. Using this, polarized spinal cord organoids (pSCOs) with a self-organized dorsoventral (DV) organization are generated. The application of caudalization signals promoted regionalized cell differentiation along the radial axis and protrusion morphogenesis in confined hPSC colonies. These detached colonies grew into extended spinal cord-like organoids, which established self-ordered DV patterning along the long axis through the spontaneous expression of polarized DV patterning morphogens. The proportions of dorsal/ventral domains in the pSCOs can be controlled by the changes in the initial size of micropatterns, which altered the ratio of center-edge cells in 2D. In mature pSCOs, highly synchronized neural activity is separately detected in the dorsal and ventral side, indicating functional as well as structural patterning established in the organoids. This study provides a simple and precisely controllable method to generate spatially ordered organoids for the understanding of the biological principles of cell patterning and axis formation during neural development.

Whole structural reconstruction and quantification of epidermal innervation through the suction blister method and skin-clearing technique.

Three-dimensional (3-D) analysis of intraepidermal nerve fibers (IENFs) is conducted to advance assessment methods for peripheral neuropathies and pruritic skin disorders. The skin-clearing technique was proven to be a reliable method for 3-D imaging of IENFs. Nonetheless, it still requires further improvement in the imaging process. The aim of this study was to standardize the 3-D evaluation method of IENFs and to suggest promising 3-D biomarkers for clinical application. A total of nine healthy individuals were prospectively enrolled. The newly adopted suction blister method was combined with the tissue-clearing technique. The detailed structure of the IENFs was reconstructed and quantified using the neuron tracing software. The suction blister method showed improved linear integrity of IENFs compared with those obtained from the previously used salt-split skin test. The 3-D parameters most significantly related to natural aging were the convex hull two-dimensional perimeter and the total length (both p = 0.020). The meaningful correlations were followed by total volume (p = 0.025), ends (p = 0.026), convex hull 3-D surface, and complexity (both p = 0.030). Thus, the 3-D parameters could be utilized as possible biomarkers to identify ambiguous pathologies of peripheral neuropathies and pruritic skin disorders.

Production of human spinal-cord organoids recapitulating neural-tube morphogenesis.

Human spinal-cord-like tissues induced from human pluripotent stem cells are typically insufficiently mature and do not mimic the morphological features of neurulation. Here, we report a three-dimensional culture system and protocol for the production of human spinal-cord-like organoids (hSCOs) recapitulating the neurulation-like tube-forming morphogenesis of the early spinal cord. The hSCOs exhibited neurulation-like tube-forming morphogenesis, cellular differentiation into the major types of spinal-cord neurons as well as glial cells, and mature synaptic functional activities, among other features of the development of the spinal cord. We used the hSCOs to screen for antiepileptic drugs that can cause neural-tube defects. hSCOs may also facilitate the study of the development of the human spinal cord and the modelling of diseases associated with neural-tube defects.

Cell position within human pluripotent stem cell colonies determines apical specialization via an actin cytoskeleton-based mechanism.

Human pluripotent stem cells (hPSCs) grow as colonies with epithelial-like features including cell polarity and position-dependent features that contribute to symmetry breaking during development. Our study provides evidence that hPSC colonies exhibit position-dependent differences in apical structures and functions. With this apical difference, edge cells were preferentially labeled with amphipathic dyes, which enabled separation of edge and center cells by fluorescence-activated cell sorting. Transcriptome comparison between center and edge cells showed differential expression of genes related to apicobasal polarization, cell migration, and endocytosis. Accordingly, different kinematics and mechanical dynamics were found between center and edge cells, and perturbed actin dynamics disrupted the position-dependent apical polarity. In addition, our dye-labeling approach could be utilized to sort out a certain cell population in differentiated micropatterned colonies. In summary, hPSC colonies have position-dependent differences in apical structures and properties, and actin dynamics appear to play an important role in the establishment of this position-dependent cell polarity.

Interpreting the Entire Connectivity of Individual Neurons in Micropatterned Neural Culture With an Integrated Connectome Analyzer of a Neuronal Network (iCANN).

The function of a neural circuit can be determined by the following: (1) characteristics of individual neurons composing the circuit, (2) their distinct connection structure, and (3) their neural circuit activity. However, prior research on correlations between these three factors revealed many limitations. In particular, profiling and modeling of the connectivity of complex neural circuits at the cellular level are highly challenging. To reduce the burden of the analysis, we suggest a new approach with simplification of the neural connection in an array of honeycomb patterns on 2D, using a microcontact printing technique. Through a series of guided neuronal growths in defined honeycomb patterns, a simplified neuronal circuit was achieved. Our approach allowed us to obtain the whole network connectivity at cellular resolution using a combination of stochastic multicolor labeling via viral transfection. Therefore, we were able to identify several types of hub neurons with distinct connectivity features. We also compared the structural differences between different circuits using three-node motif analysis. This new model system, iCANN, is the first experimental model of neural computation at the cellular level, providing neuronal circuit structures for the study of the relationship between anatomical structure and function of the neuronal network.

Sensitive label-free imaging of brain samples using FxClear-based tissue clearing technique.

Optical clearing has emerged as a powerful tool for volume imaging. Although volume imaging with immunostaining have been successful in many protocols, yet obtaining homogeneously stained thick samples remains challenging. Here, we propose a method for label-free imaging of brain slices by enhancing the regional heterogeneity of the optical properties using the tissue clearing principle. We used FxClear, a method for delipidation of brain tissue, to retain a larger proportion of lipids at the white matter (WM). Furthermore, the embedding media affected the contrasts for the lipid-rich or extracellular matrix-rich areas, depending on their chemical properties. Thus, we tailored clearing conditions for the enhancement of the refractive indices (RIs) differences between gray and WM, or several pathological features. RI differences can be imaged using conventional light microscopy or optical coherence tomography. We propose that our protocol is simple, reliable, and flexible for label-free imaging, easily implementable to routine histology laboratory.

Label-free microendoscopy using a micro-needle imaging probe for in vivo deep tissue imaging.

We report a label-free imaging method for microendoscopy that uses a needle-type imaging probe. We inserted a thin GRIN lens that had been attached to a fiber bundle into a medical-grade needle that was used as an imaging probe. The introduction of the needle probe into biological tissue allows for direct access to deep regions that we otherwise could not achieve because of the multiple light scattering. To minimize invasiveness, we introduced the illuminating probe on the tissue surface, using an oblique back-illumination configuration. We achieved three-dimensional depth imaging by changing the depth of penetration. Since only the imaging probe goes deep into the tissue while leaving the illumination channels outside, the achievable signal depends on the location of the illumination channels. We explored this point and investigated the optimal condition for the illumination distance in a systematic way. We also applied this method to ex vivo, as well as in vivo, imaging of a mouse brain, and confirmed that we had visualized the microvasculature embedded deep within the brain.

FxClear, A Free-hydrogel Electrophoretic Tissue Clearing Method for Rapid De-lipidation of Tissues with High Preservation of Immunoreactivity.

Over the last two decades, several tissue clearing methodologies have been established that render tissues optically transparent and allow imaging of unsectioned tissues of significant volumes, thus improving the capacity to study the relationships between cell and 3D tissue architecture. Despite these technical advances, the important unsolved challenges that these methods face include complexity, time, consistency of tissue size before and after clearing, and ability to immunolabel various antibodies in cleared tissue. Here, we established very simple and fast tissue clearing protocol, FxClear, which involves acrylamide-free electrophoretic tissue clearing (ETC). By removal of the acrylamide infusion step, we were able to achieve fast reaction time, smaller tissue expansion, and higher immunoreactivity. Especially, immunoreactivity and fluorescence intensity were increased in FxClear-processed tissues compared to un-cleared tissues. Our protocol may be suitable for small-sized biopsy samples for 3D pathological examinations.

Farnesylation-defective Rheb Increases Axonal Length Independently of mTORC1 Activity in Embryonic Primary Neurons.

Rheb (Ras homolog enriched in the brain) is a small GTPase protein that plays an important role in cell signaling for development of the neocortex through modulation of mTORC1 (mammalian-target-of-rapamycin-complex-1) activity. mTORC1 is known to control various biological processes including axonal growth in forming complexes at the lysosomal membrane compartment. As such, anchoring of Rheb on the lysosomal membrane via the farnesylation of Rheb at its cysteine residue (C180) is required for its promotion of mTOR activity. To test the significance of Rheb farnesylation, we overexpressed a farnesylation mutant form of Rheb, Rheb C180S, in primary rat hippocampal neurons and also in mouse embryonic neurons using in utero electroporation. Interestingly, we found that Rheb C180S maintained promotional effect of axonal elongation similar to the wild-type Rheb in both test systems. On the other hand, Rheb C180S failed to exhibit the multiple axon-promoting effect which is found in wild-type Rheb. The levels of phospho-4EBP1, a downstream target of mTORC1, were surprisingly increased in Rheb C180S transfected neurons, despite the levels of phosphorylated mTOR being significantly decreased compared to control vector transfectants. A specific mTORC1 inhibitor, rapamycin, also could not completely abolish axon elongation characteristics of Rheb C180S in transfected cells. Our data suggests that Rheb in a non-membrane compartment can promote the axonal elongation via phosphorylation of 4EBP1 and through an mTORC1-independent pathway.

Encoding information into autonomously bursting neural network with pairs of time-delayed pulses.

Biological neural networks with many plastic synaptic connections can store external input information in the map of synaptic weights as a form of unsupervised learning. However, the same neural network often produces dramatic reverberating events in which many neurons fire almost simultaneously - a phenomenon coined as 'population burst.' The autonomous bursting activity is a consequence of the delicate balance between recurrent excitation and self-inhibition; as such, any periodic sequences of burst-generating stimuli delivered even at a low frequency (~1 Hz) can easily suppress the entire network connectivity. Here we demonstrate that 'Δt paired-pulse stimulation', can be a novel way for encoding spatially-distributed high-frequency (~10 Hz) information into such a system without causing a complete suppression. The encoded memory can be probed simply by delivering multiple probing pulses and then estimating the precision of the arrival times of the subsequent evoked recurrent bursts.

A monitoring system for axonal growth dynamics using micropatterns of permissive and Semaphorin 3F chemorepulsive signals.

Neurons reach their correct targets by directional outgrowth of axons, which is mediated by attractive or repulsive cues. Growing axons occasionally cross a field of repulsive cues and stop at intermediate targets on the journey to their final destination. However, it is not well-understood how individual growth cones make decisions, and pass through repulsive territory to reach their permissive target regions. We developed a microcontact printing culture system that could trap individual axonal tips in a permissive dot area surrounded by the repulsive signal, semaphorin 3F (Sema3F). Axons of rat hippocampal neurons on the Sema3F/PLL dot array extended in the checkboard pattern with a significantly slow growth rate. The detailed analysis of the behaviors of axonal growth cones revealed the saccadic dynamics in the dot array system. The trapped axonal tips in the permissive area underwent growth cone enlargement with remarkably spiky filopodia, promoting their escape from the Sema3F constraints with straight extension of axons. This structured axonal growth on the dot pattern was disrupted by increased inter-dot distance, or perturbing intracellular signaling machineries. These data indicate that axons grow against repulsive signals by jumping over the repulsive cues, depending on contact signals and intracellular milieu. Our study suggests that our dot array culture system can be used as a screening system to easily and efficiently evaluate ECM or small molecule inhibitors interfering growth cone dynamics leading to controlling axonal growth.

Drp1-Zip1 Interaction Regulates Mitochondrial Quality Surveillance System.

Mitophagy, a mitochondrial quality control process for eliminating dysfunctional mitochondria, can be induced by a response of dynamin-related protein 1 (Drp1) to a reduction in mitochondrial membrane potential (MMP) and mitochondrial division. However, the coordination between MMP and mitochondrial division for selecting the damaged portion of the mitochondrial network is less understood. Here, we found that MMP is reduced focally at a fission site by the Drp1 recruitment, which is initiated by the interaction of Drp1 with mitochondrial zinc transporter Zip1 and Zn2+ entry through the Zip1-MCU complex. After division, healthy mitochondria restore MMP levels and participate in the fusion-fission cycle again, but mitochondria that fail to restore MMP undergo mitophagy. Thus, interfering with the interaction between Drp1 and Zip1 blocks the reduction of MMP and the subsequent mitophagic selection of damaged mitochondria. These results suggest that Drp1-dependent fission provides selective pressure for eliminating "bad sectors" in the mitochondrial network, serving as a mitochondrial quality surveillance system.

Optimizing tissue-clearing conditions based on analysis of the critical factors affecting tissue-clearing procedures.

Tissue-clearing techniques have received great attention for volume imaging and for the potential to be applied in optical diagnosis. In principle, tissue clearing is achieved by reducing light scattering through a combination of lipid removal, size change, and matching of the refractive index (RI) between the imaging solution and the tissue. However, the contributions of these major factors in tissue clearing have not been systematically evaluated yet. In this study, we experimentally measured and mathematically calculated the contribution of these factors to the clearing of four organs (brain, liver, kidney, and lung). We found that these factors differentially influence the maximal clearing efficacy of tissues and the diffusivity of materials inside the tissue. We propose that these physical properties of organs can be utilized for the quality control (Q/C) process during tissue clearing, as well as for the monitoring of the pathological changes of tissues.

Senescent tumor cells building three-dimensional tumor clusters.

Cellular senescence, a permanent cell-cycle arrest, is a common yet intriguing phenomenon, in which its beneficial significance for biological organisms has only begun to be explored. Among others, senescent cells are able to transform tissue structures around them. Tumor cells, whose hallmark is their ability to proliferate indefinitely, are not free from the phenomenon. Here, we report a remarkable observation where senescent cells in a dense mono-layer of breast cancer colony act as aggregating centers for non-senescent cells in their vicinity. Consequently, the senescent cells actively form localized 3D cell-clusters in a confluent 2D tumor layer. The biophysical mechanism underpinning the surprising phenomenon primarily involves mitotic cell-rounding, dynamic and differential cell attachments, and cellular chemotaxis. By incorporating these few biophysical factors, we were able to recapitulate the experimental observation via a cellular Potts Model.

BrainFilm, a novel technique for physical compression of 3D brain slices for efficient image acquisition and post-processing.

Tissue clearing enables us to observe thick tissue at a single cell resolution by reducing light scattering and refractive index matching. However, imaging of a large volume of tissue for 3D reconstruction requires a great deal of time, cost, and efforts. Few methods have been developed to transcend these limitations by mechanical compression or isotropic tissue shrinkage. Tissue shrinkage significantly lessens the imaging burden; however, there is an inevitable trade-off with image resolution. Here, we have developed the "BrainFilm" technique to compress cleared tissue at Z-axis by dehydration, without alteration of the XY-axis. The Z-axis compression was approximately 90%, and resulted in substantial reduction in image acquisition time and data size. The BrainFilm technique was successfully used to trace and characterize the morphology of thick biocytin-labelled neurons following electrophysiological recording and trace the GFP-labelled long nerve projections in irregular tissues such as the limb of mouse embryo. Thus, BrainFilm is a versatile tool that can be applied in diverse studies of 3D tissues in which spatial information of the Z-axis is dispensable.

Coherence resonance in bursting neural networks.

Synchronized neural bursts are one of the most noticeable dynamic features of neural networks, being essential for various phenomena in neuroscience, yet their complex dynamics are not well understood. With extrinsic electrical and optical manipulations on cultured neural networks, we demonstrate that the regularity (or randomness) of burst sequences is in many cases determined by a (few) low-dimensional attractor(s) working under strong neural noise. Moreover, there is an optimal level of noise strength at which the regularity of the interburst interval sequence becomes maximal-a phenomenon of coherence resonance. The experimental observations are successfully reproduced through computer simulations on a well-established neural network model, suggesting that the same phenomena may occur in many in vivo as well as in vitro neural networks.

Modulating the precision of recurrent bursts in cultured neural networks.

Synchronized bursts are a very common feature in biological neural networks, and they play an important role in various brain functions and neurological diseases. This Letter investigates "recurrent synchronized bursts" induced by a single pulse stimulation in cultured networks of rat cortical neurons. We look at how the precision in their arrival times can be modified by a noble time-delayed stimulation protocol, which we term as "Δt training." The emergence of recurrent bursts and the change of the precision in their arrival times can be explained by the stochastic resonance of a damped, subthreshold, neural oscillation.

Effect of beam-flow angle on velocity measurements in modern Doppler ultrasound systems.

OBJECTIVE: The purpose of this article is to examine the effect of different beam-flow angles on the accuracy of Doppler ultrasound velocity measurements in modern ultrasound systems. MATERIALS AND METHODS: A flow phantom was used to create a steady flow of water in a 4.3-mm-diameter tube. Using three different modern university-grade ultrasound systems, flow was measured at 30°, 40°, 50°, 60°, 70°, 80°, and 88° beam-flow angles twice by two radiologists in consensus using a convex and linear probe. Measured flow ratio, defined as measured velocity divided by estimated actual velocity, was calculated. Intraprobe, interprobe, and intermachine mean variation of measured flow ratio were calculated. RESULTS: Measured flow ratio increased as beam-flow angles increased. Measured flow ratios for the angles 30°, 40°, 50°, 60°, 70°, 80°, and 88° were 0.90, 0.97, 1.10, 1.22, 1.62, 2.34, and 10.29, respectively. Intraprobe, interprobe, and intermachine variation did not show marked differences. For angles grouped as 30-40°, 50-60°, 70°, and 80-88°, intraprobe variation was 12%, 15%, 15%, and 26%; interprobe variation was 20%, 16%, 13%, and 26%; and intermachine variation was 16%, 16%, 17%, and 54%, respectively. As beam-flow angle increased, an increase in spectral broadening was also noted. CONCLUSION: There is no simple cutoff beam-flow value, such as the well-quoted less than 60°, at which velocity measurements can be considered accurate. For follow-up imaging, beam-flow angle differences should be considered, and the same beam-flow angles should be used when possible. Follow-up imaging by different sonography machines is feasible.