Physiologic and Nanoscale Distinctions Define Glutamatergic Synapses in Tonic vs Phasic Neurons.

Neurons exhibit a striking degree of functional diversity, each one tuned to the needs of the circuitry in which it is embedded. A fundamental functional dichotomy occurs in activity patterns, with some neurons firing at a relatively constant "tonic" rate, while others fire in bursts, a "phasic" pattern. Synapses formed by tonic versus phasic neurons are also functionally differentiated, yet the bases of their distinctive properties remain enigmatic. A major challenge toward illuminating the synaptic differences between tonic and phasic neurons is the difficulty in isolating their physiological properties. At the Drosophila neuromuscular junction, most muscle fibers are coinnervated by two motor neurons: the tonic "MN-Ib" and phasic "MN-Is." Here, we used selective expression of a newly developed botulinum neurotoxin transgene to silence tonic or phasic motor neurons in Drosophila larvae of either sex. This approach highlighted major differences in their neurotransmitter release properties, including probability, short-term plasticity, and vesicle pools. Furthermore, Ca2+ imaging demonstrated ∼2-fold greater Ca2+ influx at phasic neuron release sites relative to tonic, along with an enhanced synaptic vesicle coupling. Finally, confocal and super-resolution imaging revealed that phasic neuron release sites are organized in a more compact arrangement, with enhanced stoichiometry of voltage-gated Ca2+ channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca2+ influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes.SIGNIFICANCE STATEMENT "Tonic" and "phasic" neuronal subtypes, based on differential firing properties, are common across many nervous systems. Using a recently developed approach to selectively silence transmission from one of these two neurons, we reveal specialized synaptic functional and structural properties that distinguish these specialized neurons. This study provides important insights into how input-specific synaptic diversity is achieved, which could have implications for neurologic disorders that involve changes in synaptic function.

Ergodic speckle contrast optical coherence tomography velocimetry of rapid blood flow.

Visualizing a 3D blood flow velocity field through noninvasive imaging is crucial for analyzing hemodynamic mechanisms in areas prone to disorders. However, traditional correlation-based optical coherence tomography (OCT) velocimetry techniques have a maximum measurable flow velocity depending on the A-line rate. We presented the ergodic speckle contrast OCT (ESCOCT) to break the bottleneck in measuring the rapid blood flow velocity. It achieved a measurement of blood flow velocity ranging from 9.5 to 280 mm/s using a 100 kHz swept-source (SS) OCT based on 100 A-repeats scanning mode. Addressing the non-ergodic problem of temporal OCT signals by integrating more consecutive A-scans, ESCOCT can enable the estimation for lower velocity flows by increasing A-repeats. ESCOCT provided a wide dynamic range with no upper limit on measuring blood flow velocity with an adequate signal-to-noise ratio and improved the sensitivity and accuracy of the hemodynamic assessment.

Ethylene Signaling Modulates Cortical Microtubule Reassembly in Response to Salt Stress.

Regulation of cortical microtubule reorganization is essential for plant cell survival under high salinity conditions. In response to salt stress, microtubules undergo rapid depolymerization followed by reassembly to form a new microtubule network that promotes cell survival; however, the upstream regulatory mechanisms for this recovery response are largely unknown. In this study, we demonstrate that ethylene signaling facilitates salt stress-induced reassembly of cortical microtubules in Arabidopsis (Arabidopsis thaliana). Microtubule depolymerization was not affected under salt stress following the suppression of ethylene signaling with Ag+ or in ethylene-insensitive mutants, whereas microtubule reassembly was significantly inhibited. ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, was shown to play a central role in microtubule reassembly under salt stress. In addition, we performed functional characterization of the microtubule-stabilizing protein WAVE-DAMPENED2-LIKE5 (WDL5), which was found to promote ethylene-associated microtubule reassembly and plant salt stress tolerance. These findings indicate that ethylene signaling regulates microtubule reassembly by up-regulating WDL5 expression in response to salt stress, thereby implicating ethylene signaling in salt-stress tolerance in plants.

Ca2+ imaging of synaptic compartments using subcellularly targeted GCaMP8f in Drosophila.

GCaMP8f is a sensitive genetically encoded Ca2+ indicator that enables imaging of neuronal activity. Here, we present a protocol to perform Ca2+ imaging of the Drosophila neuromuscular junction using GCaMP8f targeted to pre- or postsynaptic compartments. We describe ratiometric Ca2+ imaging using GCaMP8f fused to mScarlet and synaptotagmin that reveals Ca2+ dynamics at presynaptic terminals. We then detail "quantal" imaging of miniature transmission events using GCaMP8f targeted to postsynaptic compartments by fusion to a PDZ-binding motif. For complete details on the use and execution of this protocol, please refer to Li et al.,1 Han et al.,2 Perry et al.,3 and Han et al.4.

Electrostatically fabricated heterostructure of interfacial-polarization-enhanced Fe3O4/C/MXene for ultra-wideband electromagnetic wave absorption.

Electromagnetic (EM) pollution can disrupt the functioning of advanced electronic devices, hence it's necessary to design EM wave absorbers with high-level absorption capabilities. The Ti3C2Tx (MXene) is classified as a potential EM absorbing material; nevertheless, the lack of magnetic loss mechanism leads to its inadequate EM absorbing performance. On this basis, a novel composite design with promising EM absorption properties is hypothesized to be the integration of few-layer MXene and heterogeneous magnetic MOF derivatives (Fe3O4/C) with complementary advantages. Herein, we synthesized two-dimensional (2D) interfacial-polarization-enhanced MXene hybrid (Fe3O4/C/MXene) by electrostatic assembly. It is notable that the interfacial polarization is realized by adding a small amount of magnetic Fe3O4/C. Furthermore, the Fe3O4/C/ MXene demonstrates an astonishing effective absorption bandwidth (EAB) of 10.7 GHz and an excellent EM wave absorption performance (RLmin) of -66.9 dB. Moreover, the radar cross section (RCS) of Fe3O4/C/MXene is lower than -15.1 dB m2 from -90° to 90° with a minimum RCS value of -52.6 dB m2 at 32°. In addition, the significant attenuation of the EM wave is due to the synergistic effect of improved impedance matching, dielectric loss, and magnetic loss. Thus, the magnetized Fe3O4/C/MXene hybrid is expected to emerge as a strong contender for high-performance EM wave absorbers.

Distinct input-specific mechanisms enable presynaptic homeostatic plasticity.

Synapses are endowed with the flexibility to change through experience, but must be sufficiently stable to last a lifetime. This tension is illustrated at the Drosophila neuromuscular junction (NMJ), where two motor inputs that differ in structural and functional properties co-innervate most muscles to coordinate locomotion. To stabilize NMJ activity, motor neurons augment neurotransmitter release following diminished postsynaptic glutamate receptor functionality, termed presynaptic homeostatic potentiation (PHP). How these distinct inputs contribute to PHP plasticity remains enigmatic. We have used a botulinum neurotoxin to selectively silence each input and resolve their roles in PHP, demonstrating that PHP is input-specific: Chronic (genetic) PHP selectively targets the tonic MN-Ib, where active zone remodeling enhances Ca2+ influx to promote increased glutamate release. In contrast, acute (pharmacological) PHP selectively increases vesicle pools to potentiate phasic MN-Is. Thus, distinct homeostatic modulations in active zone nanoarchitecture, vesicle pools, and Ca2+ influx collaborate to enable input-specific PHP expression.

Geochemical impact of dissolved organic matter on antimony mobilization in shallow groundwater of the Xikuangshan antimony mine, Hunan Province, China.

Dissolved organic matter (DOM) is widely used in aquatic systems to control the environmental fate of As. However, similar to the behavior of As, Sb mobilization driven by DOM is poorly understood. A total of 25 samples were collected from shallow groundwater in the Xikuangshan mine to compare the spectroscopic characteristics and chemical properties of DOM between high- and low-Sb groundwater and to determine the roles of DOM in Sb mobility. The concentrations of Sb and DOM varied from 0.003 to 18.402 mg/L (mean: 3.407 mg/L) and 0.38 to 9.90 mg/L (mean: 2.49 mg/L), respectively. The DOM of the D3x4 water was primarily dominated by terrestrial and microbial humic-like and fulvic acid substances, with a relatively small contribution of tryptophan-like components. Complexing agents, competitive adsorption, and photopromoted oxidation under sunlight were considered as the formation mechanisms for DOM-controlled Sb(V)-dominated Sb species in D3x4 water. The weakly alkaline and oxidizing conditions, and the presence of Fe hydroxides facilitated the promotion of Sb(V) concentration. The findings of this study further enhance our understanding of the Sb migration mechanism in oxic groundwater.

2-Arachidonoylglycerol mobilization following brief synaptic stimulation in the dorsal lateral striatum requires glutamatergic and cholinergic neurotransmission.

Several forms of endocannabinoid (eCB) signaling have been described in the dorsal lateral striatum (DLS), however most experimental protocols used to generate eCBs do not recapitulate the firing patterns of striatal-projecting pyramidal neurons in the cortex or firing patterns of striatal medium spiny neurons. Therefore, it is unclear if current models of eCB signaling in the DLS provide a reliable description of mechanisms engaged under physiological conditions. To address this uncertainty, we investigated mechanisms of eCB mobilization following brief synaptic stimulation that mimics in vivo patterns of neural activity in the DLS. To monitor eCB mobilization, the novel genetically encoded fluorescent eCB biosensor, GRABeCB2.0, was expressed presynaptically in corticostriatal afferents of C57BL6J mice and evoked eCB transients were measured in the DLS using a brain slice photometry technique. We found that brief bouts of synaptic stimulation induce long lasting eCB transients that were generated predominantly by 2-arachidonoylglycerol (2-AG) mobilization. Efficient 2-AG mobilization required coactivation of AMPA and NMDA ionotropic glutamate receptors and muscarinic M1 receptors. Dopamine D2 receptors expressed on cholinergic interneurons inhibited 2-AG mobilization by inhibiting acetylcholine release. Collectively, these data uncover unrecognized mechanisms underlying 2-AG mobilization in the DLS.

Botulinum neurotoxin accurately separates tonic vs. phasic transmission and reveals heterosynaptic plasticity rules in Drosophila.

In developing and mature nervous systems, diverse neuronal subtypes innervate common targets to establish, maintain, and modify neural circuit function. A major challenge towards understanding the structural and functional architecture of neural circuits is to separate these inputs and determine their intrinsic and heterosynaptic relationships. The Drosophila larval neuromuscular junction is a powerful model system to study these questions, where two glutamatergic motor neurons, the strong phasic-like Is and weak tonic-like Ib, co-innervate individual muscle targets to coordinate locomotor behavior. However, complete neurotransmission from each input has never been electrophysiologically separated. We have employed a botulinum neurotoxin, BoNT-C, that eliminates both spontaneous and evoked neurotransmission without perturbing synaptic growth or structure, enabling the first approach that accurately isolates input-specific neurotransmission. Selective expression of BoNT-C in Is or Ib motor neurons disambiguates the functional properties of each input. Importantly, the blended values of Is+Ib neurotransmission can be fully recapitulated by isolated physiology from each input. Finally, selective silencing by BoNT-C does not induce heterosynaptic structural or functional plasticity at the convergent input. Thus, BoNT-C establishes the first approach to accurately separate neurotransmission between tonic vs. phasic neurons and defines heterosynaptic plasticity rules in a powerful model glutamatergic circuit.

A sensitive GRAB sensor for detecting extracellular ATP in vitro and in vivo.

The purinergic transmitter ATP (adenosine 5'-triphosphate) plays an essential role in both the central and peripheral nervous systems, and the ability to directly measure extracellular ATP in real time will increase our understanding of its physiological functions. Here, we developed a sensitive GPCR activation-based ATP sensor called GRABATP1.0, with a robust fluorescence response to extracellular ATP when expressed in several cell types. This sensor has sub-second kinetics, has ATP affinity in the range of tens of nanomolar, and can be used to localize ATP release with subcellular resolution. Using this sensor, we monitored ATP release under a variety of in vitro and in vivo conditions, including stimuli-induced and spontaneous ATP release in primary hippocampal cultures, injury-induced ATP release in a zebrafish model, and lipopolysaccharides-induced ATP-release events in individual astrocytes in the mouse cortex. Thus, the GRABATP1.0 sensor is a sensitive, versatile tool for monitoring ATP release and dynamics under both physiological and pathophysiological conditions.

A fluorescent sensor for spatiotemporally resolved imaging of endocannabinoid dynamics in vivo.

Endocannabinoids (eCBs) are retrograde neuromodulators with important functions in a wide range of physiological processes, but their in vivo dynamics remain largely uncharacterized. Here we developed a genetically encoded eCB sensor called GRABeCB2.0. GRABeCB2.0 consists of a circular-permutated EGFP and the human CB1 cannabinoid receptor, providing cell membrane trafficking, second-resolution kinetics with high specificity for eCBs, and shows a robust fluorescence response at physiological eCB concentrations. Using GRABeCB2.0, we monitored evoked and spontaneous changes in eCB dynamics in cultured neurons and acute brain slices. We observed spontaneous compartmentalized eCB transients in cultured neurons and eCB transients from single axonal boutons in acute brain slices, suggesting constrained, localized eCB signaling. When GRABeCB2.0 was expressed in the mouse brain, we observed foot shock-elicited and running-triggered eCB signaling in the basolateral amygdala and hippocampus, respectively. In a mouse model of epilepsy, we observed a spreading wave of eCB release that followed a Ca2+ wave through the hippocampus. GRABeCB2.0 is a robust probe for eCB release in vivo.

Deficiency in endocannabinoid synthase DAGLB contributes to early onset Parkinsonism and murine nigral dopaminergic neuron dysfunction.

Endocannabinoid (eCB), 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain, regulates diverse neural functions. Here we linked multiple homozygous loss-of-function mutations in 2-AG synthase diacylglycerol lipase ß (DAGLB) to an early onset autosomal recessive Parkinsonism. DAGLB is the main 2-AG synthase in human and mouse substantia nigra (SN) dopaminergic neurons (DANs). In mice, the SN 2-AG levels were markedly correlated with motor performance during locomotor skill acquisition. Genetic knockdown of Daglb in nigral DANs substantially reduced SN 2-AG levels and impaired locomotor skill learning, particularly the across-session learning. Conversely, pharmacological inhibition of 2-AG degradation increased nigral 2-AG levels, DAN activity and dopamine release and rescued the locomotor skill learning deficits. Together, we demonstrate that DAGLB-deficiency contributes to the pathogenesis of Parkinsonism, reveal the importance of DAGLB-mediated 2-AG biosynthesis in nigral DANs in regulating neuronal activity and dopamine release, and suggest potential benefits of 2-AG augmentation in alleviating Parkinsonism.

The E3 ligase MREL57 modulates microtubule stability and stomatal closure in response to ABA.

Regulation of stomatal movement is critical for plant adaptation to environmental stresses. The microtubule cytoskeleton undergoes disassembly, which is critical for stomatal closure in response to abscisic acid (ABA). However, the mechanism underlying this regulation largely remains unclear. Here we show that a ubiquitin-26S proteasome (UPS)-dependent pathway mediates microtubule disassembly and is required for ABA-induced stomatal closure. Moreover, we identify and characterize the ubiquitin E3 ligase MREL57 (MICROTUBULE-RELATED E3 LIGASE57) and the microtubule-stabilizing protein WDL7 (WAVE-DAMPENED2-LIKE7) in Arabidopsis and show that the MREL57-WDL7 module regulates microtubule disassembly to mediate stomatal closure in response to drought stress and ABA treatment. MREL57 interacts with, ubiquitinates and degrades WDL7, and this effect is clearly enhanced by ABA. ABA-induced stomatal closure and microtubule disassembly are significantly suppressed in mrel57 mutants, and these phenotypes can be restored when WDL7 expression is decreased. Our results unravel UPS-dependent mechanisms and the role of an MREL57-WDL7 module in microtubule disassembly and stomatal closure in response to drought stress and ABA.

Control Mechanism of the Effective Stress on Nano-Micro Pores and the Permeability of High-Rank Coals.

To study the change and main control factors of the high-rank coal reservoir permeability in deep coal seams, permeability tests under different stresses and gas pressures were carried out in the laboratory. The development and distribution of nano-micro pores and fractures in the coal matrix were analyzed and observed by mercury intrusion porosimetry, gas adsorption, scanning electron microscope and computed tomography to reveal the permeability variation mechanism. The results showed that the initial permeability of the coal samples ranged from 0.0114 mD to 0.2349 mD when the effective stress was 0 MPa, and it clearly varied among different samples. The permeability of all the coal samples was very sensitive to the effective stress and decreased exponentially with the increase of the effective stress. The increase of the pore pressure also led to a decrease of the permeability, whereas the impact of the pore pressure on permeability was less obvious compared with the effective stress. Sub-nanopores, nanopores, micro-fractures and larger fractures are all developed in the coal samples. Connected larger fractures were the main gas migration channels in permeability determination, and the narrowing, disconnection, and closure of the fractures caused by the increase of the effective stress were the most important reasons for significant reduction of permeability.

In vivo endocannabinoid dynamics at the timescale of physiological and pathological neural activity.

The brain's endocannabinoid system is a powerful controller of neurotransmitter release, shaping synaptic communication under physiological and pathological conditions. However, our understanding of endocannabinoid signaling in vivo is limited by the inability to measure their changes at timescales commensurate with the high lability of lipid signals, leaving fundamental questions of whether, how, and which endocannabinoids fluctuate with neural activity unresolved. Using novel imaging approaches in awake behaving mice, we now demonstrate that the endocannabinoid 2-arachidonoylglycerol, not anandamide, is dynamically coupled to hippocampal neural activity with high spatiotemporal specificity. Furthermore, we show that seizures amplify the physiological endocannabinoid increase by orders of magnitude and drive the downstream synthesis of vasoactive prostaglandins that culminate in a prolonged stroke-like event. These results shed new light on normal and pathological endocannabinoid signaling in vivo.

A highly sensitive label-free electrochemical immunosensor based on poly(indole-5-carboxylicacid) with ultra-high redox stability.

The high stability of redox signal is one of the most crucial factors in construction of electrochemical immunosensors. However, the redox-active species usually show low stability and poor conductivity, which inhibits their application in electrochemical immunosensors. In this work, we report that the conductive polymer poly(indole-5-carboxylic acid) (PIn-5-COOH) possesses ultra-high redox stability. The redox signal of PIn-5-COOH could remain 96.03% after 500 cyclic voltammery (CV) cycles in buffer solution with pH of 6.2, while the redox signals in most of the previous reports only remained less than 90% after 50 CV cycles. Our mechanism investigation indicated that the ultra-high redox stability of PIn-5-COOH should be attributed to its stable structure. The electrochemical immunosensors fabricated with PIn-5-COOH/MWCNTs-COOH nanocomposite showed a wide linear range from 0.001 ng mL-1 to 100 ng mL-1 and a low detection limit of 0.33 pg mL-1 for the detection of alpha fetoprotein. This study opens up a new avenue for the construction of electrochemical immunosensors with ultra-stable redox signal.