Statistically unbiased prediction enables accurate denoising of voltage imaging data.

Here we report SUPPORT (statistically unbiased prediction utilizing spatiotemporal information in imaging data), a self-supervised learning method for removing Poisson-Gaussian noise in voltage imaging data. SUPPORT is based on the insight that a pixel value in voltage imaging data is highly dependent on its spatiotemporal neighboring pixels, even when its temporally adjacent frames alone do not provide useful information for statistical prediction. Such dependency is captured and used by a convolutional neural network with a spatiotemporal blind spot to accurately denoise voltage imaging data in which the existence of the action potential in a time frame cannot be inferred by the information in other frames. Through simulations and experiments, we show that SUPPORT enables precise denoising of voltage imaging data and other types of microscopy image while preserving the underlying dynamics within the scene.

L-type Ca2+ channels mediate regulation of glutamate release by subthreshold potential changes.

Subthreshold depolarization enhances neurotransmitter release evoked by action potentials and plays a key role in modulating synaptic transmission by combining analog and digital signals. This process is known to be Ca2+ dependent. However, the underlying mechanism of how small changes in basal Ca2+ caused by subthreshold depolarization can regulate transmitter release triggered by a large increase in local Ca2+ is not well understood. This study aimed to investigate the source and signaling mechanisms of Ca2+ that couple subthreshold depolarization with the enhancement of glutamate release in hippocampal cultures and CA3 pyramidal neurons. Subthreshold depolarization increased presynaptic Ca2+ levels, the frequency of spontaneous release, and the amplitude of evoked release, all of which were abolished by blocking L-type Ca2+ channels. A high concentration of intracellular Ca2+ buffer or blockade of calmodulin abolished depolarization-induced increases in transmitter release. Estimation of the readily releasable pool size using hypertonic sucrose showed depolarization-induced increases in readily releasable pool size, and this increase was abolished by the blockade of calmodulin. Our results provide mechanistic insights into the modulation of transmitter release by subthreshold potential change and highlight the role of L-type Ca2+ channels in coupling subthreshold depolarization to the activation of Ca2+-dependent signaling molecules that regulate transmitter release.

SCAMP5 plays a critical role in axonal trafficking and synaptic localization of NHE6 to adjust quantal size at glutamatergic synapses.

Glutamate uptake into synaptic vesicles (SVs) depends on cation/H+ exchange activity, which converts the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane at the presynaptic terminals. Thus, the proper recruitment of cation/H+ exchanger to SVs is important in determining glutamate quantal size, yet little is known about its localization mechanism. Here, we found that secretory carrier membrane protein 5 (SCAMP5) interacted with the cation/H+ exchanger NHE6, and this interaction regulated NHE6 recruitment to glutamatergic presynaptic terminals. Protein-protein interaction analysis with truncated constructs revealed that the 2/3 loop domain of SCAMP5 is directly associated with the C-terminal region of NHE6. The use of optical imaging and electrophysiological recording showed that small hairpin RNA-mediated knockdown (KD) of SCAMP5 or perturbation of SCAMP5/NHE6 interaction markedly inhibited axonal trafficking and the presynaptic localization of NHE6, leading to hyperacidification of SVs and a reduction in the quantal size of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 KD without causing additional defects. Together, our results reveal that as a key regulator of axonal trafficking and synaptic localization of NHE6, SCAMP5 could adjust presynaptic strength by regulating quantal size at glutamatergic synapses. Since both proteins are autism candidate genes, the reduced quantal size by interrupting their interaction may underscore synaptic dysfunction observed in autism.

DYRK3 phosphorylates SNAPIN to regulate axonal retrograde transport and neurotransmitter release.

Among the five members of the dual-specificity tyrosine-phosphorylation-regulated kinase (DYRK) family, the cellular functions of DYRK3 have not been fully elucidated. Some studies have indicated limited physiological roles and substrates of DYRK3, including promotion of glioblastoma, requirement in influenza virus replication, and coupling of stress granule condensation with mammalian target of rapamycin complex 1 signaling. Here, we demonstrate that serum deprivation causes a decrease in intracellular DYRK3 levels via the proteolytic autophagy pathway, as well as the suppression of DYRK3 gene expression. To further demonstrate how DYRK3 affects cell viability, especially in neurons, we used a yeast two-hybrid assay and identified multiple DYRK3-binding proteins, including SNAPIN, a SNARE-associated protein implicated in synaptic transmission. We also found that DYRK3 directly phosphorylates SNAPIN at the threonine (Thr) 14 residue, increasing the interaction of SNAPIN with other proteins such as dynein and synaptotagmin-1. In central nervous system neurons, SNAPIN is associated with and mediate the retrograde axonal transport of diverse cellular products from the distal axon terminal to the soma and the synaptic release of neurotransmitters, respectively. Moreover, phosphorylation of SNAPIN at Thr-14 was found to positively modulate mitochondrial retrograde transport in mouse cortical neurons and the recycling pool size of synaptic vesicles, contributing to neuronal viability. In conclusion, the present study demonstrates that DYRK3 phosphorylates SNAPIN, positively regulating the dynein-mediated retrograde transport of mitochondria and SNARE complex-mediated exocytosis of synaptic vesicles within the neurons. This finding further suggests that DYRK3 affects cell viability and provides a novel neuroprotective mechanism.

Potent Analgesic Action of 2-acetoxy-5-(2-4 (trifluoromethyl)-phenethylamino)-benzoic Acid (Flusalazine) in Experimental Mice.

Purpose: Nonsteroidal anti-inflammatory drugs (NSAIDs) and cyclooxygenase (COX)-2 selective inhibitors are the most widely used drugs to treat pain. Conventional NSAIDs and COX-2 selective inhibitors, however, cause several side effects such as gastric damage, kidney damage, and cardiovascular problems. Our previous study showed that 2-acetoxy-5-(2-4-(trifluoromethyl)-phenethylamino)-benzoic acid ie, flusalazine (also known as ND-07), which exerts dual actions by serving both as an anti-inflammatory agent and a free radical scavenger, is an effective and safe treatment for severe inflammatory diseases in mice. The goal of the present study was to examine the potential analgesic action and safety of flusalazine in mice models of pain. Methods and Results: Flusalazine showed a significant analgesic effect in an acetic acid-induced abdominal constriction model. Likewise, total paw licking was reduced significantly in neurogenic (early stage) and inflammatory (late stage) pain induced by formalin in flusalazine-treated mice. In the tail immersion test, flusalazine significantly increased tail withdrawal time at 2 h after its administration. Also, the formation of paw edema in the flusalazine-treated group was significantly inhibited in a carrageenan-induced inflammatory pain model. Gastric damage was not induced by flusalazine even up to 1000 mg/kg, while aspirin and indomethacin caused critical gastric bleeding. Conclusion: These findings suggest that flusalazine's safety profile and analgesic effects have high translational potential for the clinical treatment of patients experiencing pain.

SCAMP5 mediates activity-dependent enhancement of NHE6 recruitment to synaptic vesicles during synaptic plasticity.

Na+(K+)/H+ exchanger 6 (NHE6) on synaptic vesicle (SV) is critical for the presynaptic regulation of quantal size at the glutamatergic synapses by converting the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane. We recently found that NHE6 directly interacts with secretory carrier membrane protein 5 (SCAMP5), and SCAMP5-dependent recruitment of NHE6 to SVs controls the strength of synaptic transmission by modulation of quantal size of glutamate release at rest. It is, however, unknown whether NHE6 recruitment by SCAMP5 plays a role during synaptic plasticity. Here, we found that the number of NHE6-positive presynaptic boutons was significantly increased by the chemical long-term potentiation (cLTP). Since cLTP involves new synapse formation, our results indicated that NHE6 was recruited not only to the existing presynaptic boutons but also to the newly formed presynaptic boutons. Knock down of SCAMP5 completely abrogated the enhancement of NHE6 recruitment by cLTP. Interestingly, despite an increase in the number of NHE6-positive boutons by cLTP, the quantal size of glutamate release at the presynaptic terminals remained unaltered. Together with our recent results, our findings indicate that SCAMP5-dependent recruitment of NHE6 plays a critical role in manifesting presynaptic efficacy not only at rest but also during synaptic plasticity. Since both are autism candidate genes, reduced presynaptic efficacy by interfering with their interaction may underlie the molecular mechanism of synaptic dysfunction observed in autism.

Detection of Chilomastix mesnili in Common Marmoset (Callithrix jacchus) and Treatment with Metronidazole.

BACKGROUND: Recently, the use of common marmoset (Callithrix jacchus) has increased in biomedical research as an animal model. This study aimed to test fecal samples to monitor bacterial and parasite infections in common marmoset at the Laboratory Animal Center of Osong Medical Innovation Foundation in Korea. METHODS: To monitor bacteria and parasites in common marmoset, we tested 43 fecal samples of 43 common marmosets by culture and parasitological test in 2014. Infection by Chilomastix mesnili was determined by PCR method. RESULTS: We identified nonpathogenic bacteria such as Proteus mirabilis and Escherichia coli in feces of normal common marmosets. Interestingly, C. mesnili was isolated from a healthy common marmoset by fecal centrifugation concentration and PCR. The monkey infected with C. mesnili was treated with metronidazole. After the treatment, C. mesnili were not found in feces using fecal centrifugation concentration and PCR. CONCLUSION: This is the first case report of C. mesnili infection in common marmoset. Treatment with metronidazole is found to be highly effective in eradicating C. mesnili infection in common marmoset.

A murine colitis model developed using a combination of dextran sulfate sodium and Citrobacter rodentium.

Adult mice were treated with dextran sulfate sodium (DSS) and infected with Citrobacter rodentium for developing a novel murine colitis model. C57BL/6N mice (7-week-old) were divided into four groups. Each group composed of control, dextran sodium sulfate-treated (DSS), C. rodentium-infected (CT), and DSS-treated and C. rodentium-infected (DSS-CT) mice. The DSS group was administered 1% DSS in drinking water for 7 days. The CT group was supplied with normal drinking water for 7 days and subsequently infected with C. rodentium via oral gavage. The DSS-CT group was supplied with 1% DSS in drinking water for 7 days and subsequently infected with C. rodentium via oral gavage. The mice were sacrificed 10 days after the induction of C. rodentium infection. The DSS-CT group displayed significantly shorter colon length, higher spleen to body weight ratio, and higher histopathological score compared to the other three groups. The mRNA expression levels of tumor necrosis factor (TNF)-α and interferon (INF)-γ were significantly upregulated; however, those of interleukin (IL)-6 and IL-10 were significantly downregulated in the DSS-CT group than in the control group. These results demonstrated that a combination of low DSS concentration (1%) and C. rodentium infection could effectively induce inflammatory bowel disease (IBD) in mice. This may potentially be used as a novel IBD model, in which colitis is induced in mice by the combination of a chemical and a pathogen.

Erratum to: A murine colitis model developed using a combination of dextran sulfate sodium and Citrobacter rodentium.

In the article by Park et al. published in Journal of Microbiology 2018; 56, 272-279, the supplementary data Figs S1 and S2 should be corrected as below. The original article can be found online at https://doi.org/10.1007/s12275-018-7504-x .

Anti-obesity effect of robusta fermented with Leuconostoc mesenteroides in high-fat diet-induced obese mice.

Robusta beans cultivated with Monascus ruber (RMR) were successively fermented with Leuconostoc mesenteroides (LM) and the antiobesity effects were examined. To produce an obese mouse model to investigate the hypolipidemic effects, ICR mice were fed the same high-fat diet for 6 weeks. Treatment groups were given 10 or 20% RMR-LM. Body weight changes in the 20% RMR-LM group were lower compared with those in the control group. Visceral adipose tissue weight and adipose size were significantly lower in the 20% RMR-LM group compared with those in the control group. Significant improvement in glucose tolerance was observed in the 10 and 20% RMR-LM groups compared with the control group. The 20% RMR-LM group exhibited a significant reduction in serum glucose concentration. Hepatic mRNA levels of sterol regulatory element-binding protein 1, fas cell surface death receptor, and peroxisome proliferator-activated receptor γ, which are associated with lipid, and fatty acid metabolism, in the 20% RMR-LM group were significantly lower compared with those in the control group. The results of the present study demonstrated that 20% RMR-LM may be used to prevent obesity, and ameliorate diabetes and lipid metabolism imbalances.