FosGFP expression does not capture a sensory learning-related engram in superficial layers of mouse barrel cortex.

Immediate-early gene (IEG) expression has been used to identify small neural ensembles linked to a particular experience, based on the principle that a selective subset of activated neurons will encode specific memories or behavioral responses. The majority of these studies have focused on "engrams" in higher-order brain areas where more abstract or convergent sensory information is represented, such as the hippocampus, prefrontal cortex, or amygdala. In primary sensory cortex, IEG expression can label neurons that are responsive to specific sensory stimuli, but experience-dependent shaping of neural ensembles marked by IEG expression has not been demonstrated. Here, we use a fosGFP transgenic mouse to longitudinally monitor in vivo expression of the activity-dependent gene c-fos in superficial layers (L2/3) of primary somatosensory cortex (S1) during a whisker-dependent learning task. We find that sensory association training does not detectably alter fosGFP expression in L2/3 neurons. Although training broadly enhances thalamocortical synaptic strength in pyramidal neurons, we find that synapses onto fosGFP+ neurons are not selectively increased by training; rather, synaptic strengthening is concentrated in fosGFP- neurons. Taken together, these data indicate that expression of the IEG reporter fosGFP does not facilitate identification of a learning-specific engram in L2/3 in barrel cortex during whisker-dependent sensory association learning.

Dynamic multimodal holograms of conjugated organogels via dithering mask lithography.

Polymeric materials have been used to realize optical systems that, through periodic variations of their structural or optical properties, interact with light-generating holographic signals. Complex holographic systems can also be dynamically controlled through exposure to external stimuli, yet they usually contain only a single type of holographic mode. Here, we report a conjugated organogel that reversibly displays three modes of holograms in a single architecture. Using dithering mask lithography, we realized two-dimensional patterns with varying cross-linking densities on a conjugated polydiacetylene. In protic solvents, the organogel contracts anisotropically to develop optical and structural heterogeneities along the third dimension, displaying holograms in the form of three-dimensional full parallax signals, both in fluorescence and bright-field microscopy imaging. In aprotic solvents, these heterogeneities diminish as organogels expand, recovering the two-dimensional periodicity to display a third hologram mode based on iridescent structural colours. Our study presents a next-generation hologram manufacturing method for multilevel encryption technologies.

Hydrogel-Based Colorimetric Assay for Multiplexed MicroRNA Detection in a Microfluidic Device.

Although microRNA (miRNA) expression levels provide important information regarding disease states owing to their unique dysregulation patterns in tissues, translation of miRNA diagnostics into point-of-care (POC) settings has been limited by practical challenges. Here, we developed a hydrogel-based microfluidic platform for colorimetric profiling of miRNAs, without the use of complex external equipment for fluidics and imaging. For sensitive and reliable measurement without the risk of sequence bias, we employed a gold deposition-based signal amplification scheme and dark-field imaging, and seamlessly integrated a previously developed miRNA assay scheme into this platform. The assay demonstrated a limit of detection of 260 fM, along with multiplexing of small panels of miRNAs in healthy and cancer samples. We anticipate this versatile platform to facilitate a broad range of POC profiling of miRNAs in cancer-associated dysregulation with high-confidence by exploiting the unique features of hydrogel substrate in an on-chip format and colorimetric analysis.

Autistic-like social behaviour in Shank2-mutant mice improved by restoring NMDA receptor function.

Autism spectrum disorder (ASD) is a group of conditions characterized by impaired social interaction and communication, and restricted and repetitive behaviours. ASD is a highly heritable disorder involving various genetic determinants. Shank2 (also known as ProSAP1) is a multi-domain scaffolding protein and signalling adaptor enriched at excitatory neuronal synapses, and mutations in the human SHANK2 gene have recently been associated with ASD and intellectual disability. Although ASD-associated genes are being increasingly identified and studied using various approaches, including mouse genetics, further efforts are required to delineate important causal mechanisms with the potential for therapeutic application. Here we show that Shank2-mutant (Shank2(-/-)) mice carrying a mutation identical to the ASD-associated microdeletion in the human SHANK2 gene exhibit ASD-like behaviours including reduced social interaction, reduced social communication by ultrasonic vocalizations, and repetitive jumping. These mice show a marked decrease in NMDA (N-methyl-D-aspartate) glutamate receptor (NMDAR) function. Direct stimulation of NMDARs with D-cycloserine, a partial agonist of NMDARs, normalizes NMDAR function and improves social interaction in Shank2(-/-) mice. Furthermore, treatment of Shank2(-/-) mice with a positive allosteric modulator of metabotropic glutamate receptor 5 (mGluR5), which enhances NMDAR function via mGluR5 activation, also normalizes NMDAR function and markedly enhances social interaction. These results suggest that reduced NMDAR function may contribute to the development of ASD-like phenotypes in Shank2(-/-) mice, and mGluR modulation of NMDARs offers a potential strategy to treat ASD.

Cerebellar Shank2 Regulates Excitatory Synapse Density, Motor Coordination, and Specific Repetitive and Anxiety-Like Behaviors.

Shank2 is a multidomain scaffolding protein implicated in the structural and functional coordination of multiprotein complexes at excitatory postsynaptic sites as well as in psychiatric disorders, including autism spectrum disorders. While Shank2 is strongly expressed in the cerebellum, whether Shank2 regulates cerebellar excitatory synapses, or contributes to the behavioral abnormalities observed in Shank2-/- mice, remains unexplored. Here we show that Shank2-/- mice show reduced excitatory synapse density in cerebellar Purkinje cells in association with reduced levels of excitatory postsynaptic proteins, including GluD2 and PSD-93, and impaired motor coordination in the Erasmus test. Shank2 deletion restricted to Purkinje cells (Pcp2-Cre;Shank2fl/fl mice) leads to similar reductions in excitatory synapse density, synaptic protein levels, and motor coordination. Pcp2-Cre;Shank2fl/fl mice do not recapitulate autistic-like behaviors observed in Shank2-/- mice, such as social interaction deficits, altered ultrasonic vocalizations, repetitive behaviors, and hyperactivity. However, Pcp2-Cre;Shank2fl/fl mice display enhanced repetitive behavior in the hole-board test and anxiety-like behavior in the light-dark test, which are not observed in Shank2-/- mice. These results implicate Shank2 in the regulation of cerebellar excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors. SIGNIFICANCE STATEMENT: The postsynaptic side of excitatory synapses contains multiprotein complexes, termed the postsynaptic density, which contains receptors, scaffolding/adaptor proteins, and signaling molecules. Shank2 is an excitatory postsynaptic scaffolding protein implicated in the formation and functional coordination of the postsynaptic density and has been linked to autism spectrum disorders. Using Shank2-null mice and Shank2-conditional knock-out mice with a gene deletion restricted to cerebellar Purkinje cells, we explored functions of Shank2 in the cerebellum. We found that Shank2 regulates excitatory synapse density, motor coordination, and specific repetitive and anxiety-like behaviors, but is not associated with autistic-like social deficits or repetitive behaviors.

Universal process-inert encoding architecture for polymer microparticles.

Polymer microparticles with unique, decodable identities are versatile information carriers with a small footprint. Widespread incorporation into industrial processes, however, is limited by a trade-off between encoding density, scalability and decoding robustness in diverse physicochemical environments. Here, we report an encoding strategy that combines spatial patterning with rare-earth upconversion nanocrystals, single-wavelength near-infrared excitation and portable CCD (charge-coupled device)-based decoding to distinguish particles synthesized by means of flow lithography. This architecture exhibits large, exponentially scalable encoding capacities (>10(6) particles), an ultralow decoding false-alarm rate (<10(-9)), the ability to manipulate particles by applying magnetic fields, and pronounced insensitivity to both particle chemistry and harsh processing conditions. We demonstrate quantitative agreement between observed and predicted decoding for a range of practical applications with orthogonal requirements, including covert multiparticle barcoding of pharmaceutical packaging (refractive-index matching), multiplexed microRNA detection (biocompatibility) and embedded labelling of high-temperature-cast objects (temperature resistance).

Polydiacetylene liposome microarray toward influenza a virus detection: effect of target size on turn-on signaling.

Target size effect on the sensory signaling intensity of polydiacetylene (PDA) liposome microarrays was systematically investigated. Influenza A virus M1 peptide and M1 antibody were selected as a probe-target pair. While red fluorescence from the PDA liposome microarrays was observed when the larger M1 antibody was used as a target, when the same M1 antibody was used as a probe to detect the smaller M1 peptide sensory signal did not appear. The results reveal that the intensity of the PDA sensory signal is mainly related to the steric repulsion between probe-target complexes not the strength of the probe-target binding force. Based on this finding, we devised a PDA sensory system that directly detects influenza A whole virus as a larger target, and confirmed the target size effect on the signaling efficiency of PDA.

Nanoscale Vertical Resolution in Optical Printing of Inorganic Nanoparticles.

Direct optical printing of functional inorganics shows tremendous potential as it enables the creation of intricate two-dimensional (2D) patterns and affordable design and production of various devices. Although there have been recent advancements in printing processes using short-wavelength light or pulsed lasers, the precise control of the vertical thickness in printed 3D structures has received little attention. This control is vital to the diverse functionalities of inorganic thin films and their devices, as they rely heavily on their thicknesses. This lack of research is attributed to the technical intricacy and complexity involved in the lithographic processes. Herein, we present a generalized optical 3D printing process for inorganic nanoparticles using maskless digital light processing. We develop a range of photocurable inorganic nanoparticle inks encompassing metals, semiconductors, and oxides, combined with photolinkable ligands and photoacid generators, enabling the direct solidification of nanoparticles in the ink medium. Our process creates complex and large-area patterns with a vertical resolution of ∼50 nm, producing 50-nm-thick 2D films and several micrometer-thick 3D architectures with no layer height difference via layer-by-layer deposition. Through fabrication and operation of multilayered switching devices with Au electrodes and Ag-organic resistive layers, the feasibility of our process for cost-effective manufacturing of multilayered devices is demonstrated.

Systematic investigation of allelic regulatory activity of schizophrenia-associated common variants.

Genome-wide association studies (GWASs) have successfully identified 145 genomic regions that contribute to schizophrenia risk, but linkage disequilibrium makes it challenging to discern causal variants. We performed a massively parallel reporter assay (MPRA) on 5,173 fine-mapped schizophrenia GWAS variants in primary human neural progenitors and identified 439 variants with allelic regulatory effects (MPRA-positive variants). Transcription factor binding had modest predictive power, while fine-map posterior probability, enhancer overlap, and evolutionary conservation failed to predict MPRA-positive variants. Furthermore, 64% of MPRA-positive variants did not exhibit expressive quantitative trait loci signature, suggesting that MPRA could identify yet unexplored variants with regulatory potentials. To predict the combinatorial effect of MPRA-positive variants on gene regulation, we propose an accessibility-by-contact model that combines MPRA-measured allelic activity with neuronal chromatin architecture.

Design of polydiacetylene-phospholipid supramolecules for enhanced stability and sensitivity.

We present polydiacetylene (PDA) liposome assemblies with various phospholipids that have different headgroup charges and phase transition temperatures (T(m)). 10,12-Pentacosadiynoic acid (PCDA)-epoxy was used as a base PDA monomer and the insertion of highly charged phospholipids resulted in notable changes in the size of liposome and reduction of the aggregation of PDA liposome. Among the various phospholipids, the phospholipid with a moderate T(m) demonstrated enhanced stability and sensitivity, as measured by the size and zeta potential over storage time, thermochoromic response, and transmission electron microscopy images. By combining these results, we were able to detect immunologically an antibody of bovine viral diarrhea virus over a wide dynamic range of 0.001 to 100 µg/mL.

Detection method for transparent window cleaning device, image processing approach.

Recent years, there has been an increase in the number of high-rise buildings, and subsequently, the interest in external wall cleaning methods has similarly increased. While a number of exterior wall cleaning robots are being developed, a method to detect contaminants on the exterior walls is still required. The exteriors of most high-rise buildings today take the form of a window curtain-wall made of translucent glass. Detecting dust on translucent glass is a significant challenge. Here, we have attempted to overcome this challenge using image processing, inspired by the fact that people typically use just the 'naked eye' to recognize dust on windows. In this paper, we propose a method that detects dust through simple image processing techniques and estimates its density. This method only uses processing techniques that are not significantly restricted by global brightness and background, making it easily applicable in outdoor conditions. Dust separation was performed using a median filter, and dust density was estimated through a mean shift analysis technique. This dust detection method can perform dust separation and density estimation using only an image of the dust on a translucent window with blurry background.

Generalised optical printing of photocurable metal chalcogenides.

Optical three-dimensional (3D) printing techniques have attracted tremendous attention owing to their applicability to mask-less additive manufacturing, which enables the cost-effective and straightforward creation of patterned architectures. However, despite their potential use as alternatives to traditional lithography, the printable materials obtained from these methods are strictly limited to photocurable resins, thereby restricting the functionality of the printed objects and their application areas. Herein, we report a generalised direct optical printing technique to obtain functional metal chalcogenides via digital light processing. We developed universally applicable photocurable chalcogenidometallate inks that could be directly used to create 2D patterns or micrometre-thick 2.5D architectures of various sizes and shapes. Our process is applicable to a diverse range of functional metal chalcogenides for compound semiconductors and 2D transition-metal dichalcogenides. We then demonstrated the feasibility of our technique by fabricating and evaluating a micro-scale thermoelectric generator bearing tens of patterned semiconductors. Our approach shows potential for simple and cost-effective architecturing of functional inorganic materials.

Nonpolymeric thermosensitive benzenetricarboxamides.

A new class of nonpolymeric thermosensitive materials based on the benzene-1,3,5-tricarboxamide (BTC) structural platform are described. We observed that the benzocrown ether-substituted BTC derivatives undergo an unusual temperature-dependent reversible solubility change in aqueous solution. Thus, a clear nonfluorescent solution of BTC derivatives becomes turbid and generates fluorescent aggregates above the LCST temperature. The aggregates disappear, and a clear solution is reformed when the solution is cooled to 20 °C. It is believed that the LCST behavior of BTC derivatives results from the removal of water molecules from crown ether moieties at elevated temperature. Thus, BTC derivatives exist in fully hydrated forms below the LCST. Heating the BTC derivatives solutions causes the expulsion of water and induces the formation of aggregates. At room temperature, the removal of water from BTC derivatives occurs slowly and leads to the formation of long nanofibers.

Programmable multilayer printing of a mechanically-tunable 3D hydrogel co-culture system for high-throughput investigation of complex cellular behavior.

Hydrogels are widely used as a 3D cell culture platform, as they can be tailored to provide suitable microenvironments to induce cellular phenotypes with physiological significance. Hydrogels are especially deemed attractive as a co-culture platform, in which two or more different types of cells are cultured together in close proximity, since the spatial distribution of different cell types can be rendered possible by advanced microfabrication schemes. Herein, programmable multilayer photolithography is employed to develop a 3D hydrogel-based co-culture system in an efficient and scalable manner, which consists of an inner microgel array containing one cell type covered by an outer hydrogel overlay containing another cell type. In particular, the mechanical properties of microgel array and hydrogel overlay are independently controlled in a wide range, with elastic moduli ranging from 1.7 to 31.6 kPa, allowing the high-throughput investigation of both individual hydrogel mechanics and mechanical gradients generated at their interface. Utilizing this system, phenotypical changes (i.e. proliferation, spheroid formation and Mφ polarization) of macrophages encapsulated in microgel array, in response to complex mechanical microenvironment and co-cultured fibroblasts, are comprehensively explored.

An automated homecage system for multiwhisker detection and discrimination learning in mice.

Automated, homecage behavioral training for rodents has many advantages: it is low stress, requires little interaction with the experimenter, and can be easily manipulated to adapt to different experimental conditions. We have developed an inexpensive, Arduino-based, homecage training apparatus for sensory association training in freely-moving mice using multiwhisker air current stimulation coupled to a water reward. Animals learn this task readily, within 1-2 days of training, and performance progressively improves with training. We examined the parameters that regulate task acquisition using different stimulus intensities, directions, and reward valence. Learning was assessed by comparing anticipatory licking for the stimulus compared to the no-stimulus (blank) trials. At high stimulus intensities (>9 psi), animals showed markedly less participation in the task. Conversely, very weak air current intensities (1-2 psi) were not sufficient to generate rapid learning behavior. At intermediate stimulus intensities (5-6 psi), a majority of mice learned that the multiwhisker stimulus predicted the water reward after 24-48 hrs of training. Both exposure to isoflurane and lack of whiskers decreased animals' ability to learn the task. Following training at an intermediate stimulus intensity, mice were able to transfer learning behavior when exposed to a lower stimulus intensity, an indicator of perceptual learning. Mice learned to discriminate between two directions of stimulation rapidly and accurately, even when the angular distance between the stimuli was <15 degrees. Switching the reward to a more desirable reward, aspartame, had little effect on learning trajectory. Our results show that a tactile association task in an automated homecage environment can be monitored by anticipatory licking to reveal rapid and progressive behavioral change. These Arduino-based, automated mouse cages enable high-throughput training that facilitate analysis of large numbers of genetically modified mice with targeted manipulations of neural activity.

Highly Stable Upconverting Nanocrystal-Polydiacetylenes Nanoplates for Orthogonal Dual Signaling-Based Detection of Cyanide.

Although the unique optical signaling properties of polydiacetylene (PDA) have been exploited in diverse bio-chemosensors, the practical application of most PDA sensor systems is limited by their instability in harsh environments and fluorescence signal weakness. Herein, a universal design principle for a highly stable PDA sensor system with a practical dual signaling capability is developed to detect cyanide (CN) ions, which are commonly found in drinking water. Effective metal intercalation and enhanced hydrophobic intermolecular interactions between PDA-metal supramolecules are used to construct highly stacked PDA-metal nanoplates that feature unusual optical stability upon exposure to strong acids, bases, organic solvents, and thermal/mechanical stresses, and can selectively detect CN anions, concomitantly undergoing a specific supramolecular structure change. To realize the practical dual signaling capability of the PDA sensor system, upconverting nanocrystals (UCNs) are incorporated into highly stacked PDA-metal nanoplates, and practical dual signaling (orthogonal changes in luminescence and visible color) is demonstrated using a portable detection system. The presented universal design principle is expected to be suitable for the development of other highly stable and selective PDA sensor systems with practical dual signaling capability.

Multi-Color Luminescence Transition of Upconversion Nanocrystals via Crystal Phase Control with SiO2 for High Temperature Thermal Labels.

Upconversion nanocrystals (UCNs)-embedded microarchitectures with luminescence color transition capability and enhanced luminescence intensity under extreme conditions are suitable for developing a robust labeling system in a high-temperature thermal industrial process. However, most UCNs based labeling systems are limited by the loss of luminescence owing to the destruction of the crystalline phase or by a predetermined luminescence color without color transition capability. Herein, an unusual crystal phase transition of UCNs to a hexagonal apatite phase in the presence of SiO2 nanoparticles is reported with the enhancements of 130-fold green luminescence and 52-fold luminance as compared to that of the SiO2-free counterpart. By rationally combining this strategy with an additive color mixing method using a mask-less flow lithography technique, single to multiple luminescence color transition, scalable labeling systems with hidden letters-, and multi-luminescence colored microparticles are demonstrated for a UCNs luminescence color change-based high temperature labeling system.

Recyclable High-Performance Polymer Electrolyte Based on a Modified Methyl Cellulose-Lithium Trifluoromethanesulfonate Salt Composite for Sustainable Energy Systems.

Although energy-storage devices based on Li ions are considered as the most prominent candidates for immediate application in the near future, concerns with regard to their stability, safety, and environmental impact still remain. As a solution, the development of all-solid-state energy-storage devices with enhanced stability is proposed. A new eco-friendly polymer electrolyte has been synthesized by incorporating lithium trifluoromethanesulfonate into chemically modified methyl cellulose (LiTFS-LiSMC). The transparent and flexible electrolyte exhibits a good conductivity of near 1 mS cm-1 . An all-solid-state supercapacitor fabricated from 20 wt % LiTFS-LiSMC shows comparable specific capacitances to a standard liquid-electrolyte supercapacitor and an excellent stability even after 20 000 charge-discharge cycles. The electrolyte is also compatible with patterned carbon, which enables the simple fabrication of micro-supercapacitors. In addition, the LiTFS-LiSMC electrolyte can be recycled and reused more than 20 times with negligible change in its performance. Thus, it is a promising material for sustainable energy-storage devices.

Shank3 Exons 14-16 Deletion in Glutamatergic Neurons Leads to Social and Repetitive Behavioral Deficits Associated With Increased Cortical Layer 2/3 Neuronal Excitability.

Shank3, an abundant excitatory postsynaptic scaffolding protein, has been associated with multiple brain disorders, including autism spectrum disorders (ASD) and Phelan-McDermid syndrome (PMS). However, how cell type-specific Shank3 deletion affects disease-related neuronal and brain functions remains largely unclear. Here, we investigated the impacts of Shank3 deletion in glutamatergic neurons on synaptic and behavioral phenotypes in mice and compared results with those previously obtained from mice with global Shank3 mutation and GABAergic neuron-specific Shank3 mutation. Neuronal excitability was abnormally increased in layer 2/3 pyramidal neurons in the medial prefrontal cortex (mPFC) in mice with a glutamatergic Shank3 deletion, similar to results obtained in mice with a global Shank3 deletion. In addition, excitatory synaptic transmission was abnormally increased in layer 2/3 neurons in mice with a global, but not a glutamatergic, Shank3 deletion, suggesting that Shank3 in glutamatergic neurons are important for the increased neuronal excitability, but not for the increased excitatory synaptic transmission. Neither excitatory nor inhibitory synaptic transmission was altered in the dorsal striatum of Shank3-deficient glutamatergic neurons, a finding that contrasts with the decreased excitatory synaptic transmission in global and Shank3-deficient GABAergic neurons. Behaviorally, glutamatergic Shank3-deficient mice displayed abnormally increased direct social interaction and repetitive self-grooming, similar to global and GABAergic Shank3-deficient mice. These results suggest that glutamatergic and GABAergic Shank3 deletions lead to distinct synaptic and neuronal changes in cortical layer 2/3 and dorsal striatal neurons, but cause similar social and repetitive behavioral abnormalities likely through distinct mechanisms.

Fluorescence-Based Quantitative Synapse Analysis for Cell Type-Specific Connectomics.

Anatomical methods for determining cell type-specific connectivity are essential to inspire and constrain our understanding of neural circuit function. We developed genetically-encoded reagents for fluorescence-synapse labeling and connectivity analysis in brain tissue, using a fluorogen-activating protein (FAP)-coupled or YFP-coupled, postsynaptically-localized neuroligin-1 (NL-1) targeting sequence (FAP/YFPpost). FAPpost expression did not alter mEPSC or mIPSC properties. Sparse AAV-mediated expression of FAP/YFPpost with the cell-filling, red fluorophore dTomato (dTom) enabled high-throughput, compartment-specific detection of putative synapses across diverse neuron types in mouse somatosensory cortex. We took advantage of the bright, far-red emission of FAPpost puncta for multichannel fluorescence alignment of dendrites, FAPpost puncta, and presynaptic neurites in transgenic mice with saturated labeling of parvalbumin (PV), somatostatin (SST), or vasoactive intestinal peptide (VIP)-expressing neurons using Cre-reporter driven expression of YFP. Subtype-specific inhibitory connectivity onto layer 2/3 (L2/3) neocortical pyramidal (Pyr) neurons was assessed using automated puncta detection and neurite apposition. Quantitative and compartment-specific comparisons show that PV inputs are the predominant source of inhibition at both the soma and the dendrites and were particularly concentrated at the primary apical dendrite. SST inputs were interleaved with PV inputs at all secondary-order and higher-order dendritic branches. These fluorescence-based synapse labeling reagents can facilitate large-scale and cell-type specific quantitation of changes in synaptic connectivity across development, learning, and disease states.