Fine-tuning TrailMap: The utility of transfer learning to improve the performance of deep learning in axon segmentation of light-sheet microscopy images.

Light-sheet microscopy has made possible the 3D imaging of both fixed and live biological tissue, with samples as large as the entire mouse brain. However, segmentation and quantification of that data remains a time-consuming manual undertaking. Machine learning methods promise the possibility of automating this process. This study seeks to advance the performance of prior models through optimizing transfer learning. We fine-tuned the existing TrailMap model using expert-labeled data from noradrenergic axonal structures in the mouse brain. By changing the cross-entropy weights and using augmentation, we demonstrate a generally improved adjusted F1-score over using the originally trained TrailMap model within our test datasets.

Fine-tuning TrailMap: The utility of transfer learning to improve the performance of deep learning in axon segmentation of light-sheet microscopy images.

Light-sheet microscopy has made possible the 3D imaging of both fixed and live biological tissue, with samples as large as the entire mouse brain. However, segmentation and quantification of that data remains a time-consuming manual undertaking. Machine learning methods promise the possibility of automating this process. This study seeks to advance the performance of prior models through optimizing transfer learning. We fine-tuned the existing TrailMap model using expert-labeled data from noradrenergic axonal structures in the mouse brain. By fine-tuning the final two layers of the neural network at a lower learning rate of the TrailMap model, we demonstrate an improved recall and an occasionally improved adjusted F1-score within our test dataset over using the originally trained TrailMap model.

Treatment of paradoxical eczematous eruption in psoriasis treated with secukinumab: A case report.

RATIONALE: Eczematous eruption is an increasingly recognized form of drug-related eruption, typically reported in association with interleukin 17 (IL-17)A inhibitors. However, severe paradoxical eczematous eruption due to IL-17A inhibitors has been rarely reported. Herein, we reported a case of a man with severe psoriasis with erythematous scaly plaques on the scalp, trunk, and arms and legs after the administration of secukinumab was initiated. PATIENT CONCERNS: We reported a case of a 20-year-old man with severe psoriasis with erythematous scaly plaques on the scalp, trunk, and arms and legs after the administration of secukinumab was initiated. A skin biopsy was performed. It revealed spongiotic dermatitis consistent with eczematous reaction. Direct and indirect immunofluorescence assays were negative. DIAGNOSES: He was diagnosed with eczematous eruption. INTERVENTIONS: Discontinuation of secukinumab and administration of cyclosporine and prednisone were considered. OUTCOMES: Significant improvement was observed, with no adverse events. CONCLUSION: Our case shows that eczematous eruption can paradoxically occur in patients on IL-17A inhibitors and this report is expected to increase awareness of the rising number of cutaneous eruptions related to biological agents.

Valence-arousal classification of emotion evoked by Chinese ancient-style music using 1D-CNN-BiLSTM model on EEG signals for college students.

During the COVID-19 pandemic, young people are using multimedia content more frequently to communicate with each other on Internet platforms. Among them, music, as psychological support for a lonely life in this special period, is a powerful tool for emotional self-regulation and getting rid of loneliness. More and more attention has been paid to the music recommender system based on emotion. In recent years, Chinese music has tended to be considered an independent genre. Chinese ancient-style music is one of the new folk music styles in Chinese music and is becoming more and more popular among young people. The complexity of Chinese-style music brings significant challenges to the quantitative calculation of music. To effectively solve the problem of emotion classification in music information search, emotion is often characterized by valence and arousal. This paper focuses on the valence and arousal classification of Chinese ancient-style music-evoked emotion. It proposes a hybrid one-dimensional convolutional neural network and bidirectional and unidirectional long short-term memory model (1D-CNN-BiLSTM). And a self-acquisition EEG dataset for Chinese college students was designed to classify music-induced emotion by valence-arousal based on EEG. In addition to that, the proposed 1D-CNN-BILSTM model verified the performance of public datasets DEAP and DREAMER, as well as the self-acquisition dataset DESC. The experimental results show that, compared with traditional LSTM and 1D-CNN-LSTM models, the proposed method has the highest accuracy in the valence classification task of music-induced emotion, reaching 94.85%, 98.41%, and 99.27%, respectively. The accuracy of the arousal classification task also gained 93.40%, 98.23%, and 99.20%, respectively. In addition, compared with the positive valence classification results of emotion, this method has obvious advantages in negative valence classification. This study provides a computational classification model for a music recommender system with emotion. It also provides some theoretical support for the brain-computer interactive (BCI) application products of Chinese ancient-style music which is popular among young people.

Genetically encoded fluorescent sensors for imaging neuronal dynamics in vivo.

The brain relies on many forms of dynamic activities in individual neurons, from synaptic transmission to electrical activity and intracellular signaling events. Monitoring these neuronal activities with high spatiotemporal resolution in the context of animal behavior is a necessary step to achieve a mechanistic understanding of brain function. With the rapid development and dissemination of highly optimized genetically encoded fluorescent sensors, a growing number of brain activities can now be visualized in vivo. To date, cellular calcium imaging, which has been largely used as a proxy for electrical activity, has become a mainstay in systems neuroscience. While challenges remain, voltage imaging of neural populations is now possible. In addition, it is becoming increasingly practical to image over half a dozen neurotransmitters, as well as certain intracellular signaling and metabolic activities. These new capabilities enable neuroscientists to test previously unattainable hypotheses and questions. This review summarizes recent progress in the development and delivery of genetically encoded fluorescent sensors, and highlights example applications in the context of in vivo imaging.

An unusual case report of porokeratosis ptychotropica on the buttocks.

RATIONALE: Porokeratosis ptychotropica represents an unusual form of porokeratosis characterized by symmetrical dyskeratotic skin lesions on the gluteal clefts. Herein, we report a case of porokeratosis ptychotropica. PATIENT CONCERNS: A 33-year-old man, who complained of itching papules and plaques in the gluteal cleft and the buttocks for the last 7 years. Clinical examination showed a large well-defined reddish brown verrucous plaque located on both buttocks along with satellite papules on the inner thigh. Dermoscopy and histopathological findings were consistent with porokeratosis. DIAGNOSIS: He was diagnosed with porokeratosis ptychotropica. OUTCOMES: No significant improvement was observed following treatment with oral acitretin and a topical retinoid. LESSONS: The case report highlights the need for awareness amongst dermatologists for porokeratosis ptychotropica as a differential diagnosis for pruritic papules in the gluteal fold.

Locomotion activates PKA through dopamine and adenosine in striatal neurons.

The canonical model of striatal function predicts that animal locomotion is associated with the opposing regulation of protein kinase A (PKA) in direct and indirect pathway striatal spiny projection neurons (SPNs) by dopamine1-7. However, the precise dynamics of PKA in dorsolateral SPNs during locomotion remain to be determined. It is also unclear whether other neuromodulators are involved. Here we show that PKA activity in both types of SPNs is essential for normal locomotion. Using two-photon fluorescence lifetime imaging8-10 of a PKA sensor10 through gradient index lenses, we measured PKA activity within individual SPNs of the mouse dorsolateral striatum during locomotion. Consistent with the canonical view, dopamine activated PKA activity in direct pathway SPNs during locomotion through the dopamine D1 receptor. However, indirect pathway SPNs exhibited a greater increase in PKA activity, which was largely abolished through the blockade of adenosine A2A receptors. In agreement with these results, fibre photometry measurements of an adenosine sensor11 revealed an acute increase in extracellular adenosine during locomotion. Functionally, antagonism of dopamine or adenosine receptors resulted in distinct changes in SPN PKA activity, neuronal activity and locomotion. Together, our results suggest that acute adenosine accumulation interplays with dopamine release to orchestrate PKA activity in SPNs and proper striatal function during animal locomotion.

Sensitive genetically encoded sensors for population and subcellular imaging of cAMP in vivo.

Cyclic adenosine monophosphate (cAMP) signaling integrates information from diverse G-protein-coupled receptors, such as neuromodulator receptors, to regulate pivotal biological processes in a cellular-specific and subcellular-specific manner. However, in vivo cellular-resolution imaging of cAMP dynamics remains challenging. Here, we screen existing genetically encoded cAMP sensors and further develop the best performer to derive three improved variants, called cAMPFIREs. Compared with their parental sensor, these sensors exhibit up to 10-fold increased sensitivity to cAMP and a cytosolic distribution. cAMPFIREs are compatible with both ratiometric and fluorescence lifetime imaging and can detect cAMP dynamics elicited by norepinephrine at physiologically relevant, nanomolar concentrations. Imaging of cAMPFIREs in awake mice reveals tonic levels of cAMP in cortical neurons that are associated with wakefulness, modulated by opioids, and differentially regulated across subcellular compartments. Furthermore, enforced locomotion elicits neuron-specific, bidirectional cAMP dynamics. cAMPFIREs also function in Drosophila. Overall, cAMPFIREs may have broad applicability for studying intracellular signaling in vivo.

Labeling Endogenous Proteins Using CRISPR-mediated Insertion of Exon (CRISPIE).

The CRISPR/Cas9 technology has transformed our ability to edit eukaryotic genomes. Despite this breakthrough, it remains challenging to precisely knock-in large DNA sequences, such as those encoding a fluorescent protein, for labeling or modifying a target protein in post-mitotic cells. Previous efforts focusing on sequence insertion to the protein coding sequence often suffer from insertions/deletions (INDELs) resulting from the efficient non-homologous end joining pathway (NHEJ). To overcome this limitation, we have developed CRISPR-mediated insertion of exon (CRISPIE). CRISPIE circumvents INDELs and other editing errors by inserting a designer exon flanked by adjacent intron sequences into an appropriate intronic location of the targeted gene. Because INDELs at the insertion junction can be spliced out, "CRISPIEd" genes produce precisely edited mRNA transcripts that are virtually error-free. In part due to the elimination of INDELs, high-efficiency labeling can be achieved in vivo. CRISPIE is compatible with both N- and C-terminal labels, and with all common transfection methods. Importantly, CRISPIE allows for later removal of the protein modification by including exogenous single-guide RNA (sgRNA) sites in the intronic region of the donor module. This protocol provides the detailed CRISPIE methodology, using endogenous labeling of ß-actin in human U-2 OS cells with enhanced green fluorescent protein (EGFP) as an example. When combined with the appropriate gene delivery methods, the same methodology can be applied to label post-mitotic neurons in culture and in vivo. This methodology can also be readily adapted for use in other gene editing contexts.

Static coded aperture in robotic X-ray tomography systems.

Coded aperture X-ray computed tomography is a computational imaging technique capable of reconstructing inner structures of an object from a reduced set of X-ray projection measurements. Coded apertures are placed in front of the X-ray sources from different views and thus significantly reduce the radiation dose. This paper introduces coded aperture X-ray computed tomography for robotic X-ray systems which offer positioning flexibility. While single coded-aperture 3D tomography was recently introduced for standard trajectory CT scanning, it is shown that significant gains in imaging performance can be attained by simple modifications in the CT scanning trajectories enabled by emerging dual robotic CT systems. In particular, the subject is fixed on a plane and the CT system uniformly rotates around the r -axis which is misaligned with the coordinate axes. A single stationary coded aperture is placed on front of the robotic X-ray source above the plane and the corresponding X-ray projections are measured by a two-dimensional detector on the second arm of the robotic system. The compressive measurements with misalignment enable the reconstruction of high-resolution three-dimensional volumetric images from the low-resolution coded projections on the detector at a sub-sampling rate. An efficient algorithm is proposed to generate the rotation matrix with two basic sub-matrices and thus the forward model is formulated. The stationary coded aperture is designed based on the Pearson product-moment correlation coefficient analysis and the direct binary search algorithm is used to obtain the optimized coded aperture. Simulations using simulated datasets show significant gains in reconstruction performance compared to conventional coded aperture CT systems.

Photon-efficient 3D reconstruction employing a edge enhancement method.

Photon-efficient 3D reconstruction under sparse photon conditions remains challenges. Especially for scene edge locations, the light scattering results in a weaker echo signal than non-edge locations. Depth images can be viewed as smooth regions stitched together by edge segmentation, yet none of the existing methods focus on how to improve the accuracy of edge reconstruction when performing 3D reconstruction. Moreover, the impact of edge reconstruction to overall depth reconstruction hasn't been investigated. In this paper, we explore how to improve the edge reconstruction accuracy from various aspects such as improving the network structure, employing hybrid loss functions and taking advantages of the non-local correlation of SPAD measurements. Meanwhile, we investigate the correlation between the edge reconstruction accuracy and the reconstruction accuracy of overall depth based on quantitative metrics. The experimental results show that the proposed method achieves superior performance in both edge reconstruction and overall depth reconstruction compared with other state-of-the-art methods. Besides, it proves that the improvement of edge reconstruction accuracy promotes the reconstruction accuracy of depth map.

Distinct in vivo dynamics of excitatory synapses onto cortical pyramidal neurons and parvalbumin-positive interneurons.

Cortical function relies on the balanced activation of excitatory and inhibitory neurons. However, little is known about the organization and dynamics of shaft excitatory synapses onto cortical inhibitory interneurons. Here, we use the excitatory postsynaptic marker PSD-95, fluorescently labeled at endogenous levels, as a proxy for excitatory synapses onto layer 2/3 pyramidal neurons and parvalbumin-positive (PV+) interneurons in the barrel cortex of adult mice. Longitudinal in vivo imaging under baseline conditions reveals that, although synaptic weights in both neuronal types are log-normally distributed, synapses onto PV+ neurons are less heterogeneous and more stable. Markov model analyses suggest that the synaptic weight distribution is set intrinsically by ongoing cell-type-specific dynamics, and substantial changes are due to accumulated gradual changes. Synaptic weight dynamics are multiplicative, i.e., changes scale with weights, although PV+ synapses also exhibit an additive component. These results reveal that cell-type-specific processes govern cortical synaptic strengths and dynamics.

Frequency upconversion imaging based on Hadamard coding.

A frequency upconversion imaging based on Hadamard coding is presented to remove the distorting effect on condition that the pump beam is tightly focused to optimize the conversion efficiency. The distortion caused by the convolution between the object field and the pump field is ascribed to the point spread function effect. In order to remove the blurring in an upconversion imaging system optimized by tight focused pump, the object is encoded by measurement matrices and the corresponding intensity of the converted field is measured. Thus the intensity distribution of the object can be calculated accurately by the measurements and the measurement matrix. The signal-to-noise ratio (SNR) is improved by employing the Hadamard matrix since the intensity of measured converted signal is far larger than the intensity of each pixel. The experimental results show the proposed method removes the distorting effect caused by the convolution. The converted image still has sharp edges on condition that the conversion efficiency is optimized by tight focusing the pump beam.

Genetically encoded sensors towards imaging cAMP and PKA activity in vivo.

Cyclic adenosine monophosphate (cAMP) is a universal second messenger that plays a crucial role in diverse biological functions, ranging from transcription to neuronal plasticity, and from development to learning and memory. In the nervous system, cAMP integrates inputs from many neuromodulators across a wide range of timescales - from seconds to hours - to modulate neuronal excitability and plasticity in brain circuits during different animal behavioral states. cAMP signaling events are both cell-specific and subcellularly compartmentalized. The same stimulus may result in different, sometimes opposite, cAMP dynamics in different cells or subcellular compartments. Additionally, the activity of protein kinase A (PKA), a major cAMP effector, is also spatiotemporally regulated. For these reasons, many laboratories have made great strides toward visualizing the intracellular dynamics of cAMP and PKA. To date, more than 80 genetically encoded sensors, including original and improved variants, have been published. It is starting to become possible to visualize cAMP and PKA signaling events in vivo, which is required to study behaviorally relevant cAMP/PKA signaling mechanisms. Despite significant progress, further developments are needed to enhance the signal-to-noise ratio and practical utility of these sensors. This review summarizes the recent advances and challenges in genetically encoded cAMP and PKA sensors with an emphasis on in vivo imaging in the brain during behavior.

High-fidelity, efficient, and reversible labeling of endogenous proteins using CRISPR-based designer exon insertion.

Precise and efficient insertion of large DNA fragments into somatic cells using gene editing technologies to label or modify endogenous proteins remains challenging. Non-specific insertions/deletions (INDELs) resulting from the non-homologous end joining pathway make the process error-prone. Further, the insert is not readily removable. Here, we describe a method called CRISPR-mediated insertion of exon (CRISPIE) that can precisely and reversibly label endogenous proteins using CRISPR/Cas9-based editing. CRISPIE inserts a designer donor module, which consists of an exon encoding the protein sequence flanked by intron sequences, into an intronic location in the target gene. INDELs at the insertion junction will be spliced out, leaving mRNAs nearly error-free. We used CRISPIE to fluorescently label endogenous proteins in mammalian neurons in vivo with previously unachieved efficiency. We demonstrate that this method is broadly applicable, and that the insert can be readily removed later. CRISPIE permits protein sequence insertion with high fidelity, efficiency, and flexibility.

Single-snapshot X-ray imaging for nonlinear compressive tomosynthesis.

Traditional compressive X-ray tomosynthesis uses sequential illumination to interrogate the object, leading to long scanning time and image distortion due to the object variation. This paper proposes a single-snapshot compressive tomosynthesis imaging approach, where the object is simultaneously illuminated by multiple X-ray emitters equipped with coded apertures. Based on rank, intensity and sparsity prior models, a nonlinear image reconstruction framework is established. The coded aperture patterns are optimized based on uniform sensing criteria. Then, a modified split Bregman algorithm is developed to reconstruct the object from the set of nonlinear compressive measurements. It is shown that the proposed method can be used to reduce the inspection time and achieve robust reconstruction with respect to shape variation or motion of objects.

Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy.

Neuromodulation exerts powerful control over brain function. Dysfunction of neuromodulatory systems results in neurological and psychiatric disorders. Despite their importance, technologies for tracking neuromodulatory events with cellular resolution are just beginning to emerge. Neuromodulators, such as dopamine, norepinephrine, acetylcholine, and serotonin, trigger intracellular signaling events via their respective G protein-coupled receptors to modulate neuronal excitability, synaptic communications, and other neuronal functions, thereby regulating information processing in the neuronal network. The above mentioned neuromodulators converge onto the cAMP/protein kinase A (PKA) pathway. Therefore, in vivo PKA imaging with single-cell resolution was developed as a readout for neuromodulatory events in a manner analogous to calcium imaging for neuronal electrical activities. Herein, a method is presented to visualize PKA activity at the level of individual neurons in the cortex of head-fixed behaving mice. To do so, an improved A-kinase activity reporter (AKAR), called tAKARα, is used, which is based on Förster resonance energy transfer (FRET). This genetically-encoded PKA sensor is introduced into the motor cortex via in utero electroporation (IUE) of DNA plasmids, or stereotaxic injection of adeno-associated virus (AAV). FRET changes are imaged using two-photon fluorescence lifetime imaging microscopy (2pFLIM), which offers advantages over ratiometric FRET measurements for quantifying FRET signal in light-scattering brain tissue. To study PKA activities during enforced locomotion, tAKARα is imaged through a chronic cranial window above the cortex of awake, head-fixed mice, which run or rest on a speed-controlled motorized treadmill. This imaging approach will be applicable to many other brain regions to study corresponding behavior-induced PKA activities and to other FLIM-based sensors for in vivo imaging.

Synapse-specific opioid modulation of thalamo-cortico-striatal circuits.

The medial thalamus (MThal), anterior cingulate cortex (ACC) and striatum play important roles in affective-motivational pain processing and reward learning. Opioids affect both pain and reward through uncharacterized modulation of this circuitry. This study examined opioid actions on glutamate transmission between these brain regions in mouse. Mu-opioid receptor (MOR) agonists potently inhibited MThal inputs without affecting ACC inputs to individual striatal medium spiny neurons (MSNs). MOR activation also inhibited MThal inputs to the pyramidal neurons in the ACC. In contrast, delta-opioid receptor (DOR) agonists disinhibited ACC pyramidal neuron responses to MThal inputs by suppressing local feed-forward GABA signaling from parvalbumin-positive interneurons. As a result, DOR activation in the ACC facilitated poly-synaptic (thalamo-cortico-striatal) excitation of MSNs by MThal inputs. These results suggest that opioid effects on pain and reward may be shaped by the relative selectivity of opioid drugs to the specific circuit components.