Deep learning-based point-scanning super-resolution imaging.

Point-scanning imaging systems are among the most widely used tools for high-resolution cellular and tissue imaging, benefiting from arbitrarily defined pixel sizes. The resolution, speed, sample preservation and signal-to-noise ratio (SNR) of point-scanning systems are difficult to optimize simultaneously. We show these limitations can be mitigated via the use of deep learning-based supersampling of undersampled images acquired on a point-scanning system, which we term point-scanning super-resolution (PSSR) imaging. We designed a 'crappifier' that computationally degrades high SNR, high-pixel resolution ground truth images to simulate low SNR, low-resolution counterparts for training PSSR models that can restore real-world undersampled images. For high spatiotemporal resolution fluorescence time-lapse data, we developed a 'multi-frame' PSSR approach that uses information in adjacent frames to improve model predictions. PSSR facilitates point-scanning image acquisition with otherwise unattainable resolution, speed and sensitivity. All the training data, models and code for PSSR are publicly available at 3DEM.org.

Water adsorption on lead dioxide from ab initio molecular dynamics simulations.

The electrochemically active lead dioxide (ß-PbO2) contains the hydrogen (H) species inside the bulk and on the surface. The loss of the surface H species is proposed to be one of the factors in lead-acid battery failure. In this study, water adsorption on ß-PbO2 has been investigated using theoretical approaches to reveal the chemical forms of the surface H species and identify a probable cause of H loss mechanisms. For the single water-ß-PbO2, density functional theory (DFT) calculations present intact water molecular adsorption on ß-PbO2 (100) and dissociative water adsorption on ß-PbO2 (110), (101), and (001) surfaces. The geometric distances and the number of hydrogen bonds contribute to the adsorption energy reduction of single water adsorption. For the liquid water-ß-PbO2 slab models, DFT-based molecular dynamics simulations observe that the surface lead sites are fully occupied by a hydroxyl group or intact water molecule, and some of the surface oxygens are protonated at 300 K. On the ß-PbO2 (110) termination, dissociative water adsorption and intact molecular water adsorption occur competitively, leading to about 50% dissociation of adsorbed water molecules. On the ß-PbO2 (100), (101), and (001) terminations, the water molecules adsorb preferably in the dissociative form. The surface dependence of water dissociation is explored in terms of hydrogen bonding interactions relevant to adsorbed aqueous species. It is indicated through the Wulff crystal shape that the increase in the ß-PbO2 crystallite size may be one of the H loss mechanisms associated with the electrochemically inactive ß-PbO2.

The critical balance between dopamine D2 receptor and RGS for the sensitive detection of a transient decay in dopamine signal.

In behavioral learning, reward-related events are encoded into phasic dopamine (DA) signals in the brain. In particular, unexpected reward omission leads to a phasic decrease in DA (DA dip) in the striatum, which triggers long-term potentiation (LTP) in DA D2 receptor (D2R)-expressing spiny-projection neurons (D2 SPNs). While this LTP is required for reward discrimination, it is unclear how such a short DA-dip signal (0.5-2 s) is transferred through intracellular signaling to the coincidence detector, adenylate cyclase (AC). In the present study, we built a computational model of D2 signaling to determine conditions for the DA-dip detection. The DA dip can be detected only if the basal DA signal sufficiently inhibits AC, and the DA-dip signal sufficiently disinhibits AC. We found that those two requirements were simultaneously satisfied only if two key molecules, D2R and regulators of G protein signaling (RGS) were balanced within a certain range; this balance has indeed been observed in experimental studies. We also found that high level of RGS was required for the detection of a 0.5-s short DA dip, and the analytical solutions for these requirements confirmed their universality. The imbalance between D2R and RGS is associated with schizophrenia and DYT1 dystonia, both of which are accompanied by abnormal striatal LTP. Our simulations suggest that D2 SPNs in patients with schizophrenia and DYT1 dystonia cannot detect short DA dips. We finally discussed that such psychiatric and movement disorders can be understood in terms of the imbalance between D2R and RGS.

Control of excitatory hierarchical circuits by parvalbumin-FS basket cells in layer 5 of the frontal cortex: insights for cortical oscillations.

The cortex contains multiple neuron types with specific connectivity and functions. Recent progress has provided a better understanding of the interactions of these neuron types as well as their output organization particularly for the frontal cortex, with implications for the circuit mechanisms underlying cortical oscillations that have cognitive functions. Layer 5 pyramidal cells (PCs) in the frontal cortex comprise two major subtypes: crossed-corticostriatal (CCS) and corticopontine (CPn) cells. Functionally, CCS and CPn cells exhibit similar phase-dependent firing during gamma waves but participate in two distinct subnetworks that are linked unidirectionally from CCS to CPn cells. GABAergic parvalbumin-expressing fast-spiking (PV-FS) cells, necessary for gamma oscillation, innervate PCs, with stronger and global inhibition to somata and weaker and localized inhibitions to dendritic shafts/spines. While PV-FS cells form reciprocal connections with both CCS and CPn cells, the excitation from CPn to PV-FS cells exhibits short-term synaptic dynamics conducive for oscillation induction. The electrical coupling between PV-FS cells facilitates spike synchronization among PV-FS cells receiving common excitatory inputs from local PCs and inhibits other PV-FS cells via electrically communicated spike afterhyperpolarizations. These connectivity characteristics can promote synchronous firing in the local networks of CPn cells and firing of some CCS cells by anode-break excitation. Thus subsets of L5 CCS and CPn cells within different levels of connection hierarchy exhibit coordinated activity via their common connections with PV-FS cells, and the resulting PC output drives diverse neuronal targets in cortical layer 1 and the striatum with specific temporal precision, expanding the computational power of the cortical network.

New insights into the classification and nomenclature of cortical GABAergic interneurons.

A systematic classification and accepted nomenclature of neuron types is much needed but is currently lacking. This article describes a possible taxonomical solution for classifying GABAergic interneurons of the cerebral cortex based on a novel, web-based interactive system that allows experts to classify neurons with pre-determined criteria. Using Bayesian analysis and clustering algorithms on the resulting data, we investigated the suitability of several anatomical terms and neuron names for cortical GABAergic interneurons. Moreover, we show that supervised classification models could automatically categorize interneurons in agreement with experts' assignments. These results demonstrate a practical and objective approach to the naming, characterization and classification of neurons based on community consensus.

Carbon dioxide capture in 2,2'-iminodiethanol aqueous solution from ab initio molecular dynamics simulations.

The reaction of carbon dioxide (CO2) with aqueous 2,2'-iminodiethanol (trivial name is diethanolamine: DEA) has been investigated using both blue moon ensemble and metadynamics approaches combined with ab initio molecular dynamics (AIMD) simulations. A spontaneous direct proton transfer from DEA zwitterion (DEAZW) to DEA but not to H2O has been observed in straightforward AIMD simulation in the time scale of ps. The ab initio free-energy calculations reproduced the overall free-energy difference, predicting the ionic products DEA carbamate ion (DEAC) and the protonated DEA (DEAH). The computed free-energy barrier for the first reaction step, which is the CO2 binding (48 kJ mol-1), is found to agree reasonably well with the available experimental data (52-56 kJ mol-1). By contrast, the barriers for the next step, the deprotonation of zwitterion realized either via reaction with DEA or H2O, are underestimated by 25-35 kJ mol-1 compared to the experimental reference. A part of this error is attributed to the neglected reversible work needed to bring two reactants together, which might significantly contribute to the free-energy of activation of bimolecular reactions in a dilute solution. The computed free-energy profile is compared with our results [Y. Kubota et al., J. Chem. Phys. 146, 094303 (2017)] for the same reaction in 2-aminoethanol (trivial name is monoethanolamine: MEA).

Selective Thalamic Innervation of Rat Frontal Cortical Neurons.

Most glutamatergic inputs in the neocortex originate from the thalamus or neocortical pyramidal cells. To test whether thalamocortical afferents selectively innervate specific cortical cell subtypes and surface domains, we investigated the distribution patterns of thalamocortical and corticocortical excitatory synaptic inputs in identified postsynaptic cortical cell subtypes using intracellular and immunohistochemical staining combined with confocal laser scanning and electron microscopic observations in 2 thalamorecipient sublayers, lower layer 2/3 (L2/3b) and lower layer 5 (L5b) of rat frontal cortex. The dendrites of GABAergic parvalbumin (PV) cells preferentially received corticocortical inputs in both sublayers. The somata of L2/3b PV cells received thalamic inputs in similar proportions to the basal dendritic spines of L2/3b pyramidal cells, whereas L5b PV somata were mostly innervated by cortical inputs. The basal dendrites of L2/3b pyramidal and L5b corticopontine pyramidal cells received cortical and thalamic glutamatergic inputs in proportion to their local abundance, whereas crossed-corticostriatal pyramidal cells in L5b exhibited a preference for thalamic inputs, particularly in their distal dendrites. Our data demonstrate an exquisite selectivity among thalamocortical afferents in which synaptic connectivity is dependent on the postsynaptic neuron subtype, cortical sublayer, and cell surface domain.

Correlative light and electron microscopic observation of calcium phosphate particles in a mouse kidney formed under a high-phosphate diet.

Calcium phosphate forms particles under excessive urinary excretion of phosphate in the kidney. While the formation of calcium phosphate particles (CaPs) has been implicated in the damage to renal tubular cells and renal dysfunction, clarifying the ultrastructural information and the elemental composition of the small CaPs in the wide areas of kidney tissue has been technically difficult. This study introduces correlative and sequential light as well as electron microscopic CaP observation in the kidney tissue by combining fluorescent staining for CaPs and energy-dispersive X-ray spectroscopy (EDS) in scanning electron microscopy (SEM) on resin sections prepared using high-pressure freezing and freeze substitution. CaPs formed in mouse kidneys under long-term feeding of a high-phosphate diet were clearly visualized on resin sections by fluorescence-conjugated alendronate derivatives and toluidine blue metachromasia. These CaPs were verified by correlative observation with EDS. Furthermore, small CaPs formed in the kidney under short-term feeding were detected using fluorescent probes. The elemental composition of the particles, including calcium and magnesium, was identified following EDS analyses. These results suggest that the correlative microscopy approach is helpful for observing in situ distribution and elemental composition of CaPs in the kidney and contributing to studies regarding CaP formation-associated pathophysiology.

Highly differentiated projection-specific cortical subnetworks.

Pyramidal cells in the neocortex are differentiated into several subgroups based on their extracortical projection targets. However, little is known regarding the relative intracortical connectivity of pyramidal neurons specialized for these specific output channels. We used paired recordings and quantitative morphological analysis to reveal distinct synaptic transmission properties, connection patterns, and morphological differentiation correlated with heterogeneous thalamic input to two different groups of pyramidal cells residing in layer 5 (L5) of rat frontal cortex. Retrograde tracers were used to label two projection subtypes in L5: crossed-corticostriatal (CCS) cells projecting to both sides of the striatum, and corticopontine (CPn) cells projecting to the ipsilateral pons. Although CPn/CPn and CCS/CCS pairs had similar connection probabilities, CPn/CPn pairs exhibited greater reciprocal connectivity, stronger unitary synaptic transmission, and more facilitation of paired-pulse responses. These synaptic characteristics were strongly correlated to the projection subtype of the presynaptic neuron. CPn and CCS cells were further differentiated according to their somatic position (L5a and L5b, the latter denser thalamic afferent fibers) and their dendritic/axonal arborizations. Together, our data demonstrate that the pyramidal projection system is segregated into different output channels according to subcortical target and thalamic input, and that information flow within and between these channels is selectively organized.

Selective coexpression of multiple chemical markers defines discrete populations of neocortical GABAergic neurons.

Whether neocortical γ-aminobutyric acid (GABA) cells are composed of a limited number of distinct classes of neuron, or whether they are continuously differentiated with much higher diversity, remains a contentious issue for the field. Most GABA cells of rat frontal cortex have at least 1 of 6 chemical markers (parvalbumin, calretinin, alpha-actinin-2, somatostatin, vasoactive intestinal polypeptide, and cholecystokinin), with each chemical class comprising several distinct neuronal subtypes having specific physiological and morphological characteristics. To better clarify GABAergic neuron diversity, we assessed the colocalization of these 6 chemical markers with corticotropin-releasing factor (CRF), neuropeptide Y (NPY), the substance P receptor (SPR), and nitric oxide synthase (NOS); these 4 additional chemical markers suggested to be expressed diversely or specifically among cortical GABA cells. We further correlated morphological and physiological characteristics of identified some chemical subclasses of inhibitory neurons. Our results reveal expression specificity of CRF, NPY, SPR, and NOS in morphologically and physiologically distinct interneuron classes. These observations support the existence of a limited number of functionally distinct subtypes of GABA cells in the neocortex.

Anatomical and Functional Connectivity at the Dendrodendritic Reciprocal Mitral Cell-Granule Cell Synapse: Impact on Recurrent and Lateral Inhibition.

In the vertebrate olfactory bulb, reciprocal dendrodendritic interactions between its principal neurons, the mitral and tufted cells, and inhibitory interneurons in the external plexiform layer mediate both recurrent and lateral inhibition, with the most numerous of these interneurons being granule cells. Here, we used recently established anatomical parameters and functional data on unitary synaptic transmission to simulate the strength of recurrent inhibition of mitral cells specifically from the reciprocal spines of rat olfactory bulb granule cells in a quantitative manner. Our functional data allowed us to derive a unitary synaptic conductance on the order of 0.2 nS. The simulations predicted that somatic voltage deflections by even proximal individual granule cell inputs are below the detection threshold and that attenuation with distance is roughly linear, with a passive length constant of 650 µm. However, since recurrent inhibition in the wake of a mitral cell action potential will originate from hundreds of reciprocal spines, the summated recurrent IPSP will be much larger, even though there will be substantial mutual shunting across the many inputs. Next, we updated and refined a preexisting model of connectivity within the entire rat olfactory bulb, first between pairs of mitral and granule cells, to estimate the likelihood and impact of recurrent inhibition depending on the distance between cells. Moreover, to characterize the substrate of lateral inhibition, we estimated the connectivity via granule cells between any two mitral cells or all the mitral cells that belong to a functional glomerular ensemble (i.e., which receive their input from the same glomerulus), again as a function of the distance between mitral cells and/or entire glomerular mitral cell ensembles. Our results predict the extent of the three regimes of anatomical connectivity between glomerular ensembles: high connectivity within a glomerular ensemble and across the first four rings of adjacent glomeruli, substantial connectivity to up to eleven glomeruli away, and negligible connectivity beyond. Finally, in a first attempt to estimate the functional strength of granule-cell mediated lateral inhibition, we combined this anatomical estimate with our above simulation results on attenuation with distance, resulting in slightly narrowed regimes of a functional impact compared to the anatomical connectivity.

Presynaptic supervision of cortical spine dynamics in motor learning.

In mammalian neocortex, learning triggers the formation and turnover of new postsynaptic spines on pyramidal cell dendrites. However, the biological principles of spine reorganization during learning remain elusive because the identity of their presynaptic neuronal partners is unknown. Here, we show that two presynaptic neural circuits supervise distinct programs of spine dynamics to execute learning. We imaged spine dynamics in motor cortex during learning and performed post hoc identification of their afferent presynaptic neurons. New spines that appeared during learning formed small transient contacts with corticocortical neurons that were eliminated on skill acquisition. In contrast, persistent spines with axons from thalamic neurons were formed and enlarged. These results suggest that pyramidal cell dendrites in motor cortex use a neural circuit division of labor during skill learning, with dynamic teaching contacts from top-down intracortical axons followed by synaptic memory formation driven by thalamic axons. Dual spine supervision may govern diverse skill learning in the neocortex.

UNI-EM: An Environment for Deep Neural Network-Based Automated Segmentation of Neuronal Electron Microscopic Images.

Recently, there has been rapid expansion in the field of micro-connectomics, which targets the three-dimensional (3D) reconstruction of neuronal networks from stacks of two-dimensional (2D) electron microscopy (EM) images. The spatial scale of the 3D reconstruction increases rapidly owing to deep convolutional neural networks (CNNs) that enable automated image segmentation. Several research teams have developed their own software pipelines for CNN-based segmentation. However, the complexity of such pipelines makes their use difficult even for computer experts and impossible for non-experts. In this study, we developed a new software program, called UNI-EM, for 2D and 3D CNN-based segmentation. UNI-EM is a software collection for CNN-based EM image segmentation, including ground truth generation, training, inference, postprocessing, proofreading, and visualization. UNI-EM incorporates a set of 2D CNNs, i.e., U-Net, ResNet, HighwayNet, and DenseNet. We further wrapped flood-filling networks (FFNs) as a representative 3D CNN-based neuron segmentation algorithm. The 2D- and 3D-CNNs are known to demonstrate state-of-the-art level segmentation performance. We then provided two example workflows: mitochondria segmentation using a 2D CNN and neuron segmentation using FFNs. By following these example workflows, users can benefit from CNN-based segmentation without possessing knowledge of Python programming or CNN frameworks.

Neocortical inhibitory terminals innervate dendritic spines targeted by thalamocortical afferents.

Fast inhibition in the cortex is gated primarily at GABAergic synapses formed by local interneurons onto postsynaptic targets. Although GABAergic inputs to the somata and axon initial segments of neocortical pyramidal neurons are associated with direct inhibition of action potential generation, the role of GABAergic inputs to distal dendritic segments, including spines, is less well characterized. Because a significant proportion of inhibitory input occurs on distal dendrites and spines, it will be important to determine whether these GABAergic synapses are formed selectively by certain classes of presynaptic cells onto specific postsynaptic elements. By electron microscopic observations of synapses formed by different subtypes of nonpyramidal cells, we found that a surprisingly large fraction (33.4 +/- 9.3%) of terminals formed symmetrical synaptic junctions onto a subset of cortical spines that were mostly coinnervated by an asymmetrical terminal. Using VGLUT1 and VGLUT2 isoform of the glutamate vesicular transporter immunohistochemistry, we found that the double-innervated spines selectively received thalamocortical afferents expressing the VGLUT2 but almost never intracortical inputs expressing the VGLUT1. When comparing the volumes of differentially innervated spines and their synaptic junction areas, we found that spines innervated by VGLUT2-positive terminal were significantly larger than spines innervated by VGLUT1-positive terminal and that these spines had larger, and more often perforated, synapses than those of spines innervated by VGLUT1-positive afferent. These results demonstrate that inhibitory inputs to pyramidal cell spines may preferentially reduce thalamocortical rather than intracortical synaptic transmission and are therefore positioned to selectively gate extracortical information.

Large Volume Electron Microscopy and Neural Microcircuit Analysis.

One recent technical innovation in neuroscience is microcircuit analysis using three-dimensional reconstructions of neural elements with a large volume Electron microscopy (EM) data set. Large-scale data sets are acquired with newly-developed electron microscope systems such as automated tape-collecting ultramicrotomy (ATUM) with scanning EM (SEM), serial block-face EM (SBEM) and focused ion beam-SEM (FIB-SEM). Currently, projects are also underway to develop computer applications for the registration and segmentation of the serially-captured electron micrographs that are suitable for analyzing large volume EM data sets thoroughly and efficiently. The analysis of large volume data sets can bring innovative research results. These recently available techniques promote our understanding of the functional architecture of the brain.

A carbon nanotube tape for serial-section electron microscopy of brain ultrastructure.

Automated tape-collecting ultramicrotomy in conjunction with scanning electron microscopy (SEM) is a powerful approach for volume electron microscopy and three-dimensional neuronal circuit analysis. Current tapes are limited by section wrinkle formation, surface scratches and sample charging during imaging. Here we show that a plasma-hydrophilized carbon nanotube (CNT)-coated polyethylene terephthalate (PET) tape effectively resolves these issues and produces SEM images of comparable quality to those from transmission electron microscopy. CNT tape can withstand multiple rounds of imaging, offer low surface resistance across the entire tape length and generate no wrinkles during the collection of ultrathin sections. When combined with an enhanced en bloc staining protocol, CNT tape-processed brain sections reveal detailed synaptic ultrastructure. In addition, CNT tape is compatible with post-embedding immunostaining for light and electron microscopy. We conclude that CNT tape can enable high-resolution volume electron microscopy for brain ultrastructure analysis.

The Diversity of Cortical Inhibitory Synapses.

The most typical and well known inhibitory action in the cortical microcircuit is a strong inhibition on the target neuron by axo-somatic synapses. However, it has become clear that synaptic inhibition in the cortex is much more diverse and complicated. Firstly, at least ten or more inhibitory non-pyramidal cell subtypes engage in diverse inhibitory functions to produce the elaborate activity characteristic of the different cortical states. Each distinct non-pyramidal cell subtype has its own independent inhibitory function. Secondly, the inhibitory synapses innervate different neuronal domains, such as axons, spines, dendrites and soma, and their inhibitory postsynaptic potential (IPSP) size is not uniform. Thus, cortical inhibition is highly complex, with a wide variety of anatomical and physiological modes. Moreover, the functional significance of the various inhibitory synapse innervation styles and their unique structural dynamic behaviors differ from those of excitatory synapses. In this review, we summarize our current understanding of the inhibitory mechanisms of the cortical microcircuit.

Inhibitory Synapses Are Repeatedly Assembled and Removed at Persistent Sites In Vivo.

Older concepts of a hard-wired adult brain have been overturned in recent years by in vivo imaging studies revealing synaptic remodeling, now thought to mediate rearrangements in microcircuit connectivity. Using three-color labeling and spectrally resolved two-photon microscopy, we monitor in parallel the daily structural dynamics (assembly or removal) of excitatory and inhibitory postsynaptic sites on the same neurons in mouse visual cortex in vivo. We find that dynamic inhibitory synapses often disappear and reappear again in the same location. The starkest contrast between excitatory and inhibitory synapse dynamics is on dually innervated spines, where inhibitory synapses frequently recur while excitatory synapses are stable. Monocular deprivation, a model of sensory input-dependent plasticity, shortens inhibitory synapse lifetimes and lengthens intervals to recurrence, resulting in a new dynamic state with reduced inhibitory synaptic presence. Reversible structural dynamics indicate a fundamentally new role for inhibitory synaptic remodeling--flexible, input-specific modulation of stable excitatory connections.

Comment on "Principles of connectivity among morphologically defined cell types in adult neocortex".

Jiang et al (Research Article, 27 November 2015, aac9462) describe detailed experiments that substantially add to the knowledge of cortical microcircuitry and are unique in the number of connections reported and the quality of interneuron reconstruction. The work appeals to experts and laypersons because of the notion that it unveils new principles and provides a complete description of cortical circuits. We provide a counterbalance to the authors' claims to give those less familiar with the minutiae of cortical circuits a better sense of the contributions and the limitations of this study.

Axon branching and synaptic bouton phenotypes in GABAergic nonpyramidal cell subtypes.

GABAergic nonpyramidal cells, cortical interneurons, consist of heterogeneous subtypes differing in their axonal field and target selectivity. It remains to be investigated how the diverse innervation patterns are generated and how these spatially complicated, but synaptically specific wirings are achieved. Here, we asked whether a particular cell type obeys a specific branching and bouton arrangement principle or differs from others only in average morphometric values of the morphological template common to nonpyramidal cells. For this purpose, we subclassified nonpyramidal cells within each physiological class by quantitative parameters of somata, dendrites, and axons and characterized axon branching and bouton distribution patterns quantitatively. Each subtype showed a characteristic set of vertical and horizontal bouton spreads around the somata. Each parameter, such as branching angles, internode or interbouton intervals, followed its own characteristic distribution pattern irrespective of subtypes, suggesting that nonpyramidal cells have the common mechanism for formation of the axon branching pattern and bouton arrangement. Fitting of internode and interbouton interval distributions to the exponential indicated their apparent random occurrence. Decay constants of the fitted exponentials varied among nonpyramidal cells, but each subtype expressed a particular set of interbouton and internode interval averages. The distinctive combination of innervation field shape and local axon phenotypes suggests a marked functional difference in the laminar and columnar integration properties of different GABAergic subtypes, as well as the subtype-specific density of inhibited targets.