Long-lasting forms of plasticity through patterned ultrasound-induced brainwave entrainment.

Achieving long-lasting neuronal modulation with low-intensity, low-frequency ultrasound is challenging. Here, we devised theta burst ultrasound stimulation (TBUS) with gamma bursts for brain entrainment and modulation of neuronal plasticity in the mouse motor cortex. We demonstrate that two types of TBUS, intermittent and continuous TBUS, induce bidirectional long-term potentiation or depression-like plasticity, respectively, as evidenced by changes in motor-evoked potentials. These effects depended on molecular pathways associated with long-term plasticity, including N-methyl-d-aspartate receptor and brain-derived neurotrophic factor/tropomyosin receptor kinase B activation, as well as de novo protein synthesis. Notably, bestrophin-1 and transient receptor potential ankyrin 1 play important roles in these enduring effects. Moreover, pretraining TBUS enhances the acquisition of previously unidentified motor skills. Our study unveils a promising protocol for ultrasound neuromodulation, enabling noninvasive and sustained modulation of brain function.

Ultrasonocoverslip: In-vitro platform for high-throughput assay of cell type-specific neuromodulation with ultra-low-intensity ultrasound stimulation.

Brain stimulation with ultra-low-intensity ultrasound has rarely been investigated due to the lack of a reliable device to measure small neuronal signal changes made by the ultra-low intensity range. We propose Ultrasonocoverslip, an ultrasound-transducer-integrated-glass-coverslip that determines the minimum intensity for brain cell activation. Brain cells can be cultured directly on Ultrasonocoverslip to simultaneously deliver uniform ultrasonic pressure to hundreds of cells with real-time monitoring of cellular responses using fluorescence microscopy and single-cell electrophysiology. The sensitivity for detecting small responses to ultra-low-intensity ultrasound can be improved by averaging simultaneously obtained responses. Acoustic absorbers can be placed under Ultrasonocoverslip, and stimuli distortions are substantially reduced to precisely deliver user-intended acoustic stimulations. With the proposed device, we discover the lowest acoustic threshold to induce reliable neuronal excitation releasing glutamate. Furthermore, mechanistic studies on the device show that the ultra-low-intensity ultrasound stimulation induces cell type-specific neuromodulation by activating astrocyte-mediated neuronal excitation without direct neuronal involvement. The performance of ultra-low-intensity stimulation is validated by in vivo experiments demonstrating improved safety and specificity in motor modulation of tail movement compared to that with supra-watt-intensity.

Glutamate Permeability of Chicken Best1.

Bestrophin-1 (Best1) is a calcium (Ca2+)-activated chloride (Cl-) channel which has a phylogenetically conserved channel structure with an aperture and neck in the ion-conducting pathway. Mammalian mouse Best1 (mBest1) has been known to have a permeability for large organic anions including gluconate, glutamate, and D-serine, in addition to several small monovalent anions, such as Cl­, bromine (Br-), iodine (I-), and thiocyanate (SCN-). However, it is still unclear whether non-mammalian Best1 has a glutamate permeability through the ion-conducting pathway. Here, we report that chicken Best1 (cBest1) is permeable to glutamate in a Ca2+-dependent manner. The molecular docking and molecular dynamics simulation showed a glutamate binding at the aperture and neck of cBest1 and a glutamate permeation through the ion-conducting pore, respectively. Moreover, through electrophysiological recordings, we calculated the permeability ratio of glutamate to Cl- (PGlutamate/PCl) as 0.28 based on the reversal potential shift by ion substitution from Cl- to glutamate in the internal solution. Finally, we directly detected the Ca2+-dependent glutamate release through cBest1 using the ultrasensitive two-cell sniffer patch technique. Our results propose that Best1 homologs from non-mammalian (cBest1) to mammalian (mBest1) have a conserved permeability for glutamate.

Unaltered Tonic Inhibition in the Arcuate Nucleus of Diet-induced Obese Mice.

The principal inhibitory transmitter, γ-aminobutyric acid (GABA), is critical for maintaining hypothalamic homeostasis and released from neurons phasically, as well as from astrocytes tonically. Although astrocytes in the arcuate nucleus (ARC) of the hypothalamus are shown to transform into reactive astrocytes, the tonic inhibition by astrocytic GABA has not been adequately investigated in diet-induced obesity (DIO). Here, we investigated the expression of monoamine oxidase-B (MAOB), a GABA-synthesizing enzyme, in reactive astrocytes in obese mice. We observed that a chronic high-fat diet (HFD) significantly increased astrocytic MAOB and cellular GABA content, along with enhanced hypertrophy of astrocytes in the ARC. Unexpectedly, we found that the level of tonic GABA was unaltered in chronic HFD mice using whole-cell patch-clamp recordings in the ARC. Furthermore, the GABA-induced current was increased with elevated GABAA receptor α5 (GABRA5) expression. Surprisingly, we found that a nonselective GABA transporter (GAT) inhibitor, nipecotic acid (NPA)-induced current was significantly increased in chronic HFD mice. We observed that GAT1 inhibitor, NO711-induced current was significantly increased, whereas GAT3 inhibitor, SNAP5114-induced current was not altered. The unexpected unaltered tonic inhibition was due to an increase of GABA clearance in the ARC by neuronal GAT1 rather than astrocytic GAT3. These results imply that increased astrocytic GABA synthesis and neuronal GABAA receptor were compensated by GABA clearance, resulting in unaltered tonic GABA inhibition in the ARC of the hypothalamus in obese mice. Taken together, GABA-related molecular pathways in the ARC dynamically regulate the tonic inhibition to maintain hypothalamic homeostasis against the HFD challenge.

Generation of Astrocyte-Specific MAOB Conditional Knockout Mouse with Minimal Tonic GABA Inhibition.

Monoamine oxidase B (MAOB) is a key enzyme for GABA production in astrocytes in several brain regions. To date, the role of astrocytic MAOB has been studied in MAOB null knockout (KO) mice, although MAOB is expressed throughout the body. Therefore, there has been a need for genetically engineered mice in which only astrocytic MAOB is targeted. Here, we generated an astrocyte-specific MAOB conditional KO (cKO) mouse line and characterized it in the cerebellar and striatal regions of the brain. Using the CRISPR-Cas9 gene-editing technique, we generated Maob floxed mice (B6-Maobem1Cjl/Ibs) which have floxed exons 2 and 3 of Maob with two loxP sites. By crossing these mice with hGFAP-CreERT2, we obtained Maob floxed::hGFAP-CreERT2 mice which have a property of tamoxifen-inducible ablation of Maob under the human GFAP (hGFAP) promoter. When we treated Maob floxed::hGFAP-CreERT2 mice with tamoxifen for 5 consecutive days, MAOB and GABA immunoreactivity were significantly reduced in striatal astrocytes as well as in Bergmann glia and lamellar astrocytes in the cerebellum, compared to sunflower oil-injected control mice. Moreover, astrocyte-specific MAOB cKO led to a 74.6% reduction in tonic GABA currents from granule cells and a 76.8% reduction from medium spiny neurons. Our results validate that astrocytic MAOB is a critical enzyme for the synthesis of GABA in astrocytes. We propose that this new mouse line could be widely used in studies of various brain diseases to elucidate the pathological role of astrocytic MAOB in the future.

Common bacterial metabolite indole directly activates nociceptive neuron through transient receptor potential ankyrin 1 channel.

ABSTRACT: Nociceptors are known to directly recognize bacterial cell wall components or secreted toxins, thereby leading to pain induced by bacterial infection. However, direct activation of nociceptors by bacterial metabolites remains unclear although bacteria produce numerous metabolites related to health and disease. In this study, we investigated whether and how a common bacterial metabolite, indole, which is produced by normal microflora of the gastrointestinal tract and oral cavity, can directly activate nociceptive sensory neurons. We found that indole elicits calcium response and evokes inward currents in subsets of dorsal root ganglia (DRG) neurons. Intraplantar (i.pl.) injection of indole produced nocifensive behaviors in adult mice, which were enhanced in complete Freund's adjuvant-induced chronic inflammatory condition. Indole increased calcitonin gene-related peptide release in DRG neurons, and i.pl. injection of indole increased hind paw thickness, suggesting its role in generation of neurogenic inflammation. These in vitro and in vivo indole-induced responses were pharmacologically blocked by transient receptor potential ankyrin 1 (TRPA1) antagonist, HC-030031, and significantly abolished in TRPA1 knockout (KO) mice, indicating that indole targets TRPA1 for its action in DRG neurons. Nocifensive licking behavior induced by the injection of live Escherichia coli was significantly decreased in tryptophanase mutant (TnaA KO) E. coli- injected mice that lack indole production, further supporting the idea that bacteria-derived indole can induce pain during infection. Identifying the mechanism of action of indole through TRPA1 provides insights into bacteria-neuron interactions and the role of bacterial metabolites in pain signaling, especially in inflammation-accompanied bacterial infection.

Astrocytes Control Sensory Acuity via Tonic Inhibition in the Thalamus.

Sensory discrimination is essential for survival. However, how sensory information is finely controlled in the brain is not well defined. Here, we show that astrocytes control tactile acuity via tonic inhibition in the thalamus. Mechanistically, diamine oxidase (DAO) and the subsequent aldehyde dehydrogenase 1a1 (Aldh1a1) convert putrescine into GABA, which is released via Best1. The GABA from astrocytes inhibits synaptically evoked firing at the lemniscal synapses to fine-tune the dynamic range of the stimulation-response relationship, the precision of spike timing, and tactile discrimination. Our findings reveal a novel role of astrocytes in the control of sensory acuity through tonic GABA release.

Excessive Astrocytic GABA Causes Cortical Hypometabolism and Impedes Functional Recovery after Subcortical Stroke.

Glucose hypometabolism in cortical structures after functional disconnection is frequently reported in patients with white matter diseases such as subcortical stroke. However, the molecular and cellular mechanisms have been poorly elucidated. Here we show, in an animal model of internal capsular infarct, that GABA-synthesizing reactive astrocytes in distant cortical areas cause glucose hypometabolism via tonic inhibition of neighboring neurons. We find that reversal of aberrant astrocytic GABA synthesis, by pharmacological inhibition and astrocyte-specific gene silencing of MAO-B, reverses the reduction in cortical glucose metabolism. Moreover, induction of aberrant astrocytic GABA synthesis by cortical injection of putrescine or adenovirus recapitulates cortical hypometabolism. Furthermore, MAO-B inhibition causes a remarkable recovery from post-stroke motor deficits when combined with a rehabilitation regimen. Collectively, our data indicate that cortical glucose hypometabolism in subcortical stroke is caused by aberrant astrocytic GABA and MAO-B inhibition and that attenuating cortical hypometabolism can be a therapeutic approach in subcortical stroke.

Quantitative approach to realization of ultrasonic grain refinement of Al-7Si-2Cu-1Mg alloy.

Ultrasonic melt treatment (UST) was applied to Al-7Si-2Cu-1Mg melt at various temperatures of 620, 650, 700 and 785 °C. MgAl2O4 particles which were often found to be densely populated along oxide films, became effectively dispersed and well-wetted by UST. Transmission electron microscopy work combined with crystallography analysis clearly indicates that MgAl2O4 particles can act as α-Al nucleation site with the aid of UST. However, with UST, grain refinement occurred only at temperature of 620 °C and the grain size increased from 97 to 351 µm with increase of melt temperature to 785 °C for UST. In quantitative analysis of grain size and MgAl2O4 particle diameter, it was found that ultrasonic de-agglomeration decreased mean particle size of the MgAl2O4 particles, significantly reducing size from 1.2 to 0.4 µm when temperature increased from 620 to 785 °C. Such a size reduction with increased number of MgAl2O4 particles does not always guarantee grain refinement. Thus, in this work, detailed condition for achieving grain refinement by UST is discussed based on quantitative measurement. Furthermore, we tried to suggest the most valid grain refinement mechanism among the known mechanisms by investigation of the relationship between grain size and particle size with variation of melt temperature.

Ultrasonic Neuromodulation via Astrocytic TRPA1.

Low-intensity, low-frequency ultrasound (LILFU) is the next-generation, non-invasive brain stimulation technology for treating various neurological and psychiatric disorders. However, the underlying cellular and molecular mechanism of LILFU-induced neuromodulation has remained unknown. Here, we report that LILFU-induced neuromodulation is initiated by opening of TRPA1 channels in astrocytes. The Ca2+ entry through TRPA1 causes a release of gliotransmitters including glutamate through Best1 channels in astrocytes. The released glutamate activates NMDA receptors in neighboring neurons to elicit action potential firing. Our results reveal an unprecedented mechanism of LILFU-induced neuromodulation, involving TRPA1 as a unique sensor for LILFU and glutamate-releasing Best1 as a mediator of glia-neuron interaction. These discoveries should prove to be useful for optimization of human brain stimulation and ultrasonogenetic manipulations of TRPA1.

Repositioning of the antipsychotic trifluoperazine: Synthesis, biological evaluation and in silico study of trifluoperazine analogs as anti-glioblastoma agents.

Repositioning of the antipsychotic drug trifluoperazine for treatment of glioblastoma, an aggressive brain tumor, has been previously suggested. However, trifluoperazine did not increase the survival time in mice models of glioblastoma. In attempt to identify an effective trifluoperazine analog, fourteen compounds have been synthesized and biologically in vitro and in vivo assessed. Using MTT assay, compounds 3dc and 3dd elicited 4-5 times more potent inhibitory activity than trifluoperazine with IC50 = 2.3 and 2.2 µM against U87MG glioblastoma cells, as well as, IC50 = 2.2 and 2.1 µM against GBL28 human glioblastoma patient derived primary cells, respectively. Furthermore, they have shown a reasonable selectivity for glioblastoma cells over NSC normal neural cell. In vivo evaluation of analog 3dc confirmed its advantageous effect on reduction of tumor size and increasing the survival time in brain xenograft mouse model of glioblastoma. Molecular modeling simulation provided a reasonable explanation for the observed variation in the capability of the synthesized analogs to increase the intracellular Ca2+ levels. In summary, this study presents compound 3dc as a proposed new tool for the adjuvant chemotherapy of glioblastoma.

Trifluoperazine, a Well-Known Antipsychotic, Inhibits Glioblastoma Invasion by Binding to Calmodulin and Disinhibiting Calcium Release Channel IP3R.

Calcium (Ca2+) signaling is an important signaling process, implicated in cancer cell proliferation and motility of the deadly glioblastomas that aggressively invade neighboring brain tissue. We have previously demonstrated that caffeine blocks glioblastoma invasion and extends survival by inhibiting Ca2+ release channel inositol 1,4,5-trisphosphate receptor (IP3R) subtype 3. Trifluoperazine (TFP) is an FDA-approved antipsychotic drug for schizophrenia. Interestingly, TFP has been recently reported to show a strong anticancer effect on lung cancer, hepatocellular carcinoma, and T-cell lymphoma. However, the possible anticancer effect of TFP on glioblastoma has not been tested. Here, we report that TFP potently suppresses proliferation, motility, and invasion of glioblastoma cells in vitro, and tumor growth in in vivo xenograft mouse model. Unlike caffeine, TFP triggers massive and irreversible release of Ca2+ from intracellular stores by IP3R subtype 1 and 2 by directly interacting at the TFP-binding site of a Ca2+-binding protein, calmodulin subtype 2 (CaM2). TFP binding to CaM2 causes a dissociation of CaM2 from IP3R and subsequent opening of IP3R. Compared with the control neural stem cells, various glioblastoma cell lines showed enhanced expression of CaM2 and thus enhanced sensitivity to TFP. On the basis of these findings, we propose TFP as a potential therapeutic drug for glioblastoma by aberrantly and irreversibly increasing Ca2+ in glioblastoma cells. Mol Cancer Ther; 16(1); 217-27. ©2016 AACR.

The extent and historical trend of metal pollution recorded in core sediments from the artificial Lake Shihwa, Korea.

The vertical distribution of trace metals in sediment cores was investigated to evaluate the extent and the historical record of metal pollution over 30 years in the artificial Lake Shihwa in Korea. A marked increase of trace metals after 1980 was observed due to the operation of two large industrial complexes and dike construction for a reclamation project. There was a decreasing trend of metal concentrations with the distance from the pollution source. The enrichment factor and pollution load index of the metals indicated that the metal pollution was mainly derived from Cu, Zn and Cd loads due to anthropogenic activities. The concentrations of Cr, Ni, Cu, Zn, As and Pb in the upper part of all core sediments exceeded the ERL criteria of NOAA. Our results indicate that inadequate planning and management of industrialization and a large reclamation project accomplished by dike construction have continued to strongly accelerate metal pollution in Lake Shihwa.

Enhancement of CO2 sorption uptake on hydrotalcite by impregnation with K2CO3.

The awareness of symptoms of global warming and its seriousness urges the development of technologies to reduce greenhouse gas emissions. Carbon dioxide (CO(2)) is a representative greenhouse gas, and numerous methods to capture and storage CO(2) have been considered. Recently, the technology to remove high-temperature CO(2) by sorption has received lots of attention. In this study, hydrotalcite, which has been known to have CO(2) sorption capability at high temperature, was impregnated with K(2)CO(3) to enhance CO(2) sorption uptake, and the mechanism of CO(2) sorption enhancement on K(2)CO(3)-promoted hydrotalcite was investigated. Thermogravimetric analysis was used to measure equilibrium CO(2) sorption uptake and to estimate CO(2) sorption kinetics. The analyses based on N(2) gas physisorption, X-ray diffractometry, Fourier transform infrared spectrometry, Raman spectrometry, transmission electron microscopy, scanning electron microscopy, and energy dispersive X-ray spectroscopy were carried out to elucidate the characteristics of sorbents and the mechanism of enhanced CO(2) sorption. The equilibrium CO(2) sorption uptake on hydrotalcite could be increased up to 10 times by impregnation with K(2)CO(3), and there was an optimal amount of K(2)CO(3) for a maximum equilibrium CO(2) sorption uptake. In the K(2)CO(3)-promoted hydrotalcite, K(2)CO(3) was incorporated without changing the structure of hydrotalcite and it was thermally stabilized, resulting in the enhanced equilibrium CO(2) sorption uptake and fast CO(2) sorption kinetics.

Calcium and neostigmine antagonize gentamicin, but augment clindamycin-induced tetanic fade in rat phrenic nerve-hemidiaphragm preparations.

PURPOSE: A reduction in acetylcholine release induced by gentamicin may limit neostigmine-induced increases in acetylcholine concentration in the neuromuscular junction. An increase in acetylcholine concentration caused by neostigmine and calcium may enhance the use-dependent ion channel block of the nicotinic acetylcholine receptor caused by clindamycin. The purpose of this study was to determine whether calcium and neostigmine antagonize the neuromuscular blockade caused by gentamicin and augment the blockade caused by clindamycin during both single-twitch (0.1 Hz) and tetanic stimulation (50 Hz for 1.9 s). METHODS: Left phrenic nerve-hemidiaphragm preparations (Male Sprague-Dawley rats, 150-250 g) were mounted in Krebs solution. The concentration-response curves of gentamicin and clindamycin were obtained. The reversal effects of treatment with 5 mM calcium or 250 nM neostigmine on the effects of 1.5 mM gentamicin, which caused 72% reduction of single twitch, were studied. The effects of calcium or neostigmine on the effects of clindamycin were studied by examining the shift of the concentration-response curve of clindamycin with pretreatments with these agents. The effective concentrations were determined by a probit model. RESULTS: Calcium antagonized the single-twitch depression and tetanic fade caused by gentamicin more effectively than neostigmine. The effective concentration of 50% maximal effect (EC(50)) values of clindamycin for tetanic fade in the presence of 5 mM calcium or 250 nM neostigmine were reduced by approximately 52%. CONCLUSION: Clindamycin and gentamicin interfere with neuromuscular transmission and cause tetanic fade. Neostigmine and calcium antagonized the neuromuscular blockade caused by gentamicin, but augmented that caused by clindamycin.

Microstructures and shape memory characteristics of a nanostructured Ti-50.0Ni(at%) alloy.

Nanostructured Ti-Ni alloys were prepared by cold working followed by annealing, and then their shape memory characteristics and superelasticity were investigated by means of differential scanning calorimetry (DSC), transmission electron microscopy (TEM), thermal cycling tests under constant load and tensile tests. Morphology of amorphous phases induced by cold working depended largely on the amount of cold working. They had domain like shape in the 40% cold rolled alloy, while had mainly wide band shape in the 70% cold rolled alloy. In 40% cold rolled alloy, the average grain size increased from 27 nm to 80 nm with increasing annealing temperature from 573 K to 673 K. Transformation elongation increases with raising annealing temperature, which was ascribed to the increase in grain size reducing the constraints of grain boundaries. Transformation hysteresis increased rapidly with raising annealing temperature up to 623 K, above which they almost keep constant, which was ascribed to the small grain size and large constraints of grain boundaries.