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Multiscale neurophysiology reveals that simple motor actions are associated with changes in neuronal firing in virtually every brain region studied. Accordingly, the assessment of focal pathology such as stroke or progressive neurodegenerative diseases must also extend widely across brain areas. To derive mechanistic information through imaging, multiple resolution scales and multimodal factors must be included, such as the structure and function of specific neurons and glial cells and the dynamics of specific neurotransmitters. Emerging multiscale methods in preclinical animal studies that span micro- to macroscale examinations fill this gap, allowing a circuit-based understanding of pathophysiological mechanisms. Combined with high-performance computation and open-source data repositories, these emerging multiscale and large field-of-view techniques include live functional ultrasound, multi- and single-photon wide-scale light microscopy, video-based miniscopes, and tissue-penetrating fiber photometry, as well as variants of post-mortem expansion microscopy. We present these technologies and outline use cases and data pipelines to uncover new knowledge within animal models of stroke, Alzheimer's disease, and movement disorders.

In this research, the deep-learning optimizers Adagrad, AdaDelta, Adaptive Moment Estimation (Adam), and Stochastic Gradient Descent (SGD) were applied to the deep convolutional neural networks AlexNet, GoogLeNet, VGGNet, and ResNet that were trained to recognize weeds among alfalfa using photographic images taken at 200×200, 400×400, 600×600, and 800×800 pixels. An increase in the image sizes reduced the classification accuracy of all neural networks. The neural networks that were trained with images of 200×200 pixels resulted in better classification accuracy than the other image sizes investigated here. The optimizers AlexNet and GoogLeNet trained with AdaDelta and SGD outperformed the Adagrad and Adam optimizers; VGGNet trained with AdaDelta outperformed Adagrad, Adam, and SGD; and ResNet trained with AdaDelta and Adagrad outperformed the Adam and SGD optimizers. When the neural networks were trained with the best-performing input image size (200×200 pixels) and the best-performing deep learning optimizer, VGGNet was the most effective neural network, with high precision and recall values (≥0.99) when validation and testing datasets were used. Alternatively, ResNet was the least effective neural network in its ability to classify images containing weeds. However, there was no difference among the different neural networks in their ability to differentiate between broadleaf and grass weeds. The neural networks discussed herein may be used for scouting weed infestations in alfalfa and further integrated into the machine vision subsystem of smart sprayers for site-specific weed control.

Huntington Disease (HD), caused by dominantly inherited expansions of a CAG repeat results in characteristic motor dysfunction. Although gross motor and balance defects have been extensively characterized in multiple HD mouse models using tasks such as rotarod, beam walking and gait analysis, little is known about forelimb deficits. Here we use a high-throughput alternating reward/non-reward water-reaching task conducted daily over ∼2 months to simultaneously monitor forelimb impairment and mesoscale cortical changes in GCaMP activity, comparing female zQ175 (HD) and wildtype (WT) littermate mice, starting at ∼5.5 months of age. Behavioral analysis of the water-reaching task reveals that HD mice, despite learning the water-reaching task as proficiently as WT mice, take longer to learn the alternating event sequence. Although WT mice displayed no significant changes in cortical activity and reaching trajectory throughout the testing period, HD mice exhibited an increase in cortical activity – especially in the secondary motor and retrosplenial cortices – over time, as well as longer and more variable reaching trajectories by ∼7 months of age. HD mice also experienced a progressive reduction in successful performance rates. Tapered beam and rotarod tests before and/or after water-reaching assessment confirmed these early and manifest stages of HD characterized by the absence and presence of failed water-reaching trials, respectively. Reduced DARPP-32 (marker for striatal medium spiny neurons) expression in HD mice further confirmed disease pathology. The water-reaching task can be used to inform HD and potentially other movement disorder onset, therapeutic intervention windows and test drug efficacy.Significance statementThe movement disorder, Huntington Disease (HD), has been extensively studied in preclinical settings using mouse models of disease examining gross motor and balance defects. Little however, is known regarding forelimb deficits and underlying cortical circuit changes. Using a high-throughput alternating reward/non-reward water-reaching task, we characterized early event sequence learning defects in HD mice aged ∼5.5 months. Progressive forelimb movement defects first become apparent at ∼6.5 months of age with corresponding increases in cortical activity associated with reaching observed over time. These forelimb defects revealed in the water-reaching task are coincident with gross motor defects characterized using the tapered beam and rotarod tasks, demonstrating the suitability of the water-reaching task in phenotyping HD motor deficits.

Huntington disease (HD), caused by dominantly inherited expansions of a CAG repeat results in characteristic motor dysfunction. Although gross motor defects have been extensively characterized in multiple HD mouse models using tasks such as rotarod and beam walking, less is known about forelimb deficits. We develop a high-throughput alternating reward/nonreward water-reaching task and training protocol conducted daily over approximately two months to simultaneously monitor forelimb impairment and mesoscale cortical changes in GCaMP activity, comparing female zQ175 (HD) and wild-type (WT) littermate mice, starting at ∼5.5 months. Behavioral analysis of the water-reaching task reveals that HD mice, despite learning the water-reaching task as proficiently as wild-type mice, take longer to learn the alternating event sequence as evident by impulsive (noncued) reaches and initially display reduced cortical activity associated with successful reaches. At this age gross motor defects determined by tapered beam assessment were not apparent. Although wild-type mice displayed no significant changes in cortical activity and reaching trajectory throughout the testing period, HD mice exhibited an increase in cortical activity, especially in the secondary motor and retrosplenial cortices, over time, as well as longer and more variable reaching trajectories by approximately seven months. HD mice also experienced a progressive reduction in successful performance. Tapered beam and rotarod tests as well as reduced DARPP-32 expression (striatal medium spiny neuron marker) after water-reaching assessment confirmed HD pathology. The water-reaching task can be used to inform on a daily basis, HD and other movement disorder onset and manifestation, therapeutic intervention windows, and test drug efficacy.

KCNQ1 is a pore-forming K+ channel subunit critically important to cardiac repolarization at high heart rates. (2R)-N-[4-(4-methoxyphenyl)-2-thiazolyl]-1-[(4-methylphenyl)sulfonyl]-2 piperidinecarboxamide, or ML277, is an activator of this channel that rescues function of pathophysiologically important mutant channel complexes in human induced pluripotent stem cell–derived cardiomyocytes, and that therefore may have therapeutic potential. Here we extend our understanding of ML277 actions through cell-attached single-channel recordings of wild-type and mutant KCNQ1 channels with voltage sensor domains fixed in resting, intermediate, and activated states. ML277 has profound effects on KCNQ1 single-channel kinetics, eliminating the flickering nature of the openings, converting them to discrete opening bursts, and increasing their amplitudes approximately threefold. KCNQ1 single-channel behavior after ML277 treatment most resembles IO state-locked channels (E160R/R231E) rather than AO state channels (E160R/R237E), suggesting that at least during ML277 treatment, KCNQ1 does not frequently visit the AO state. Introduction of KCNE1 subunits reduces the effectiveness of ML277, but some enhancement of single-channel openings is still observed.

In-field weed detection in wheat (Triticum aestivum L.) is challenging due to the occurrence of weeds in close proximity with the crop. The objective of this research was to evaluate the feasibility of using deep convolutional neural networks for detecting broadleaf weed seedlings growing in wheat.The object detection neural networks, including CenterNet, Faster R-CNN, TridenNet, VFNet, and You Only Look Once Version 3 (YOLOv3) were insufficient for weed detection in wheat because the recall never exceeded 0.58 in the testing dataset. The image classification neural networks including AlexNet, DenseNet, ResNet, and VGGNet were trained with small (5500 negative and 5500 positive images) or large training datasets (11 000 negative and 11 000 positive images) and three training image sizes (200 × 200, 300 × 300, and 400 × 400 pixels). For the small training dataset, increasing image sizes decreased the F1 scores of AlexNet and VGGNet but generally increased the F1 scores of DenseNet and ResNet. For the large training dataset, no obvious difference was detected between the training image sizes since all neural networks exhibited remarkable classification accuracies with high F1 scores (≥0.96). All image classification neural networks exhibited high F1 scores (≥0.99) when trained with the large training dataset and the training images of 200 × 200 pixels.CenterNet, Faster R-CNN, TridentNet, VFNet, and YOLOv3 were insufficient, while AlexNet, DenseNet, ResNet, and VGGNet trained with a large training dataset were highly effective for detection of broadleaf weed seedlings in wheat. © 2021 Society of Chemical Industry.

The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (IKs), which is partly responsible for cardiomyocyte repolarization and physiological shortening of the cardiac action potential. Mefenamic acid, an NSAID, has been identified as an IKs activator. Here, we provide a biophysical and pharmacological characterization of mefenamic acid9s effect on IKs. Using whole-cell patch-clamp, we show that mefenamic acid enhances IKs activity in both a dose- and stoichiometry-dependent fashion by changing the slowly activating and deactivating IKs current into an almost linear current with instantaneous onset and slowed tail current decay, all of which are sensitive to the IKs blocker, HMR1556. Both single channels, which reveal no change in the maximum conductance, and whole-cell studies which reveal a dramatically altered G-V relationship despite increasingly longer interpulse intervals, suggest mefenamic acid decreases the voltage sensitivity of the IKs channel, and shifts channel gating kinetics towards more negative potentials. Modeling studies revealed that changes in voltage sensor activation kinetics are sufficient to reproduce the dose- and frequency-dependence of mefenamic acid action on IKs channels. Mutational analysis showed that mefenamic acid9s effect on IKs required residue K41 and potentially other surrounding residues on the extracellular surface of KCNE1, and explains why the KCNQ1 channel alone is insensitive to up to 1 mM mefenamic acid. Given that mefenamic acid can enhance all IKs channel complexes containing different ratios of KCNQ1 to KCNE1, it may provide a promising therapeutic approach to treating life-threatening cardiac arrhythmia syndromes. SIGNIFICANCE STATEMENT The channels which generate the IKs current are composed of KCNQ1 and KCNE1 subunits. Due to the critical role played by IKs in heartbeat regulation, enhancing IKs current has been identified as a promising therapeutic strategy to treat various heart rhythm diseases. Most IKs activators unfortunately, only work on KCNQ1 alone and not the physiologically relevant IKs channel. We have demonstrated that mefenamic acid can enhance IKs in a dose- and stoichiometry-dependent fashion, regulated by its interactions with KCNE1.

The stream processing model continuously processes online data in an on-pass fashion that can be more vulnerable to failures than other offline-data processing schemes. Checkpoint-based fault-tolerant methods have been widely used to enhance the reliability of stream processing systems. To ensure exact data recoveries upon failures, full-backup mechanisms are used to store a complete copy of data, which introduces substantial runtime overhead and increases output latency. In the meantime, a wide range of online processing applications prefer quick-and-dirty results with a slight degradation inaccuracy to delayed exact results. This paper introduces a novel approximate fault-tolerant problem (OAFP) with the objective of reducing the failure-free fault-tolerant overhead and ensuring user-defiled output accuracy requirement upon failure at the same time. We present an approximate fault-tolerant scheme based on sampling backup mechanism and study the trade-off between fault-tolerant overhead and output accuracy in stream processing systems. We proposed two algorithms to compute backup plans for both single-node failure and correlated failure scenarios. Extensive experiments with different types of stream topologies are conducted on our simulator to verify the correctness and effectiveness of our approach. We prove our solution guarantees the output accuracy requirement with minimum FT latency for directed acyclic graph (DAG) stream topologies with single-node failures.

The IKs channel complex is formed by the co-assembly of Kv7.1 (KCNQ1), a voltage-gated potassium channel, with its β-subunit, KCNE1 and the association of numerous accessory regulatory molecules such as PIP2, calmodulin, and yotiao. As a result, the IKs potassium current shows kinetic and regulatory flexibility, which not only allows IKs to fulfill physiological roles as disparate as cardiac repolarization and the maintenance of endolymph K+ homeostasis, but also to cause significant disease when it malfunctions. Here, we review new areas of understanding in the assembly, kinetics of activation and inactivation, voltage-sensor pore coupling, unitary events and regulation of this important ion channel complex, all of which have been given further impetus by the recent solution of cryo-EM structural representations of KCNQ1 alone and KCNQ1+KCNE3. Recently, the stoichiometric ratio of KCNE1 to KCNQ1 subunits has been confirmed to be variable up to a ratio of 4:4, rather than fixed at 2:4, and we will review the results and new methodologies that support this conclusion. Significant advances have been made in understanding differences between KCNQ1 and IKs gating using voltage clamp fluorimetry and mutational analysis to illuminate voltage sensor activation and inactivation, and the relationship between voltage sensor translation and pore domain opening. We now understand that the KCNQ1 pore can open with different permeabilities and conductance when the voltage sensor is in partially or fully activated positions, and the ability to make robust single channel recordings from IKs channels has also revealed the complicated pore subconductance architecture during these opening steps, during inactivation, and regulation by 1−4 associated KCNE1 subunits. Experiments placing mutations into individual voltage sensors to drastically change voltage dependence or prevent their movement altogether have demonstrated that the activation of KCNQ1 alone and IKs can best be explained using allosteric models of channel gating. Finally, we discuss how the intrinsic gating properties of KCNQ1 and IKs are highly modulated through the impact of intracellular signaling molecules and co-factors such as PIP2, protein kinase A, calmodulin and ATP, all of which modulate IKs current kinetics and contribute to diverse IKs channel complex function.

The pairing of KCNQ1 and KCNE1 subunits together mediates the cardiac slow delayed rectifier current (

The cardiac Ca2+ release channel [ryanodine receptor type 2 (RyR2)] is modulated by thiol reactive agents, but the molecular basis of RyR2 modulation by thiol reagents is poorly understood. Cys3635 in the skeletal muscle RyR1 is one of the most hyper-reactive thiols and is important for the redox and calmodulin (CaM) regulation of the RyR1 channel. However, little is known about the role of the corresponding cysteine residue in RyR2 (Cys3602) in the function and regulation of the RyR2 channel. In the present study, we assessed the impact of mutating Cys3602 (C3602A) on store overload-induced Ca2+ release (SOICR) and the regulation of RyR2 by thiol reagents and CaM. We found that the C3602A mutation suppressed SOICR by raising the activation threshold and delayed the termination of Ca2+ release by reducing the termination threshold. As a result, C3602A markedly increased the fractional Ca2+ release. Furthermore, the C3602A mutation diminished the inhibitory effect of N-ethylmaleimide on Ca2+ release, but it had no effect on the stimulatory action of 4,4′-dithiodipyridine (DTDP) on Ca2+ release. In addition, Cys3602 mutations (C3602A or C3602R) did not abolish the effect of CaM on Ca2+-release termination. Therefore, RyR2–Cys3602 is a major site mediating the action of thiol alkylating agent N-ethylmaleimide, but not the action of the oxidant DTDP. Our data also indicate that residue Cys3602 plays an important role in the activation and termination of Ca2+ release, but it is not essential for CaM regulation of RyR2.

Recent three-dimensional structural studies reveal that the central domain of ryanodine receptor (RyR) serves as a transducer that converts long-range conformational changes into the gating of the channel pore. Interestingly, the central domain encompasses one of the mutation hotspots (corresponding to amino acid residues 3778–4201) that contains a number of cardiac RyR (RyR2) mutations associated with catecholaminergic polymorphic ventricular tachycardia (CPVT) and atrial fibrillation (AF). However, the functional consequences of these central domain RyR2 mutations are not well understood. To gain insights into the impact of the mutation and the role of the central domain in channel function, we generated and characterized eight disease-associated RyR2 mutations in the central domain. We found that all eight central domain RyR2 mutations enhanced the Ca2+-dependent activation of [3H]ryanodine binding, increased cytosolic Ca2+-induced fractional Ca2+ release, and reduced the activation and termination thresholds for spontaneous Ca2+ release in HEK293 cells. We also showed that racemic carvedilol and the non-beta-blocking carvedilol enantiomer, (R)-carvedilol, suppressed spontaneous Ca2+ oscillations in HEK293 cells expressing the central domain RyR2 mutations associated with CPVT and AF. These data indicate that the central domain is an important determinant of cytosolic Ca2+ activation of RyR2. These results also suggest that altered cytosolic Ca2+ activation of RyR2 represents a common defect of RyR2 mutations associated with CPVT and AF, which could potentially be suppressed by carvedilol or (R)-carvedilol.

Activation of the cardiac ryanodine receptor (RyR2) by elevating cytosolic Ca(2+) is a central step in the process of Ca(2+)-induced Ca(2+) release, but the molecular basis of RyR2 activation by cytosolic Ca(2+) is poorly defined. It has been proposed recently that the putative Ca(2+) binding domain encompassing a pair of EF-hand motifs (EF1 and EF2) in the skeletal muscle ryanodine receptor (RyR1) functions as a Ca(2+) sensor that regulates the gating of RyR1. Although the role of the EF-hand domain in RyR1 function has been studied extensively, little is known about the functional significance of the corresponding EF-hand domain in RyR2. Here we investigate the effect of mutations in the EF-hand motifs on the Ca(2+) activation of RyR2. We found that mutations in the EF-hand motifs or deletion of the entire EF-hand domain did not affect the Ca(2+)-dependent activation of [(3)H]ryanodine binding or the cytosolic Ca(2+) activation of RyR2. On the other hand, deletion of the EF-hand domain markedly suppressed the luminal Ca(2+) activation of RyR2 and spontaneous Ca(2+) release in HEK293 cells during store Ca(2+) overload or store overload-induced Ca(2+) release (SOICR). Furthermore, mutations in the EF2 motif, but not EF1 motif, of RyR2 raised the threshold for SOICR termination, whereas deletion of the EF-hand domain of RyR2 increased both the activation and termination thresholds for SOICR. These results indicate that, although the EF-hand domain is not required for RyR2 activation by cytosolic Ca(2+), it plays an important role in luminal Ca(2+) activation and SOICR.

The NH2-terminal region (residues 1-543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH2-terminal region is involved in the activation and termination of Ca(2+) release. The three-dimensional structure of the NH2-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca(2+) release activation and termination are largely unknown. To understand the functional significance of each of these NH2-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca(2+)-induced Ca(2+) release and store overload-induced Ca(2+) release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH2-terminal region is not essential for channel gating. Ca(2+) release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca(2+) activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca(2+) activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca(2+) activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH2-terminal domains of RyR2 and the action of NH2-terminal disease mutations.