An Exclusion Zone for Ca2+ Channels around Docked Vesicles Explains Release Control by Multiple Channels at a CNS Synapse

The spatial arrangement of Ca2+ channels and vesicles remains unknown for most CNS synapses, despite of the crucial importance of this geometrical parameter for the Ca2+ control of transmitter release. At a large model synapse, the calyx of Held, transmitter release is controlled by several Ca2+ channels in a "domain overlap" mode, at least in young animals. To study the geometrical constraints of Ca2+ channel placement in domain overlap control of release, we used stochastic MCell modelling, at active zones for which the position of docked vesicles was derived from electron microscopy (EM). We found that random placement of Ca2+ channels was unable to produce high slope values between release and presynaptic Ca2+ entry, a hallmark of domain overlap, and yielded excessively large release probabilities. The simple assumption that Ca2+ channels can be located anywhere at active zones, except below a critical distance of ~ 30 nm away from docked vesicles ("exclusion zone"), rescued high slope values and low release probabilities. Alternatively, high slope values can also be obtained by placing all Ca2+ channels into a single supercluster, which however results in significantly higher heterogeneity of release probabilities. We also show experimentally that high slope values, and the sensitivity to the slow Ca2+ chelator EGTA-AM, are maintained with developmental maturation of the calyx synapse. Taken together, domain overlap control of release represents a highly organized active zone architecture in which Ca2+ channels must obey a certain distance to docked vesicles. Furthermore, domain overlap can be employed by near-mature, fast-releasing synapses

Characterization of the response of IHEP-IME LGAD with shallow carbon to Gamma Irradiation

Low Gain Avalanche Detectors (LGAD), as part of High-Granularity Timing Detector (HGTD), is crucial to reducing pileup in the upgrading to HL-LHC. Many studies have been done on the bulk damages of the LGAD. However, there's no study about the surface radiation hardness of the LGAD sensors with carbon implanted. The IHEP-IME LGAD version 3 with the shallow carbon and different interpad separations were irradiated up to 2 MGy by gamma irradiation. The performance of the IHEP-IME LGAD version 3 before and after irradiation had been tested, such as the leakage current, break-down voltage, capacitance, V$_{gl}$, and inter-pad resistance. The results showed that apart from minor fluctuations in some samples, no significant changes concerning inter-pad separation were observed before and after irradiation. Leakage current and break-down voltage increase after irradiation, which is considered due to surface passivation; the overall inter-pad resistance are larger than $10^9\ \Omega$before and after irradiation; capacitance is found to be less than 4.5 pF with a slight drop in V$_{gl}$ after irradiation. All parameters meet the requirements of HGTD, and the results indicated that IHEP-IME LGAD v3 has excellent anti-irradiation performance

Leakage current simulations of Low Gain Avalanche Diode with improved Radiation Damage Modeling

We report precise TCAD simulations of IHEP-IME-v1 Low Gain Avalanche Diode (LGAD) calibrated by secondary ion mass spectroscopy (SIMS). Our setup allows us to evaluate the leakage current, capacitance, and breakdown voltage of LGAD, which agree with measurements' results before irradiation. And we propose an improved LGAD Radiation Damage Model (LRDM) which combines local acceptor removal with global deep energy levels. The LRDM is applied to the IHEP-IME-v1 LGAD and able to predict the leakage current well at -30 $^{\circ}$C after an irradiation fluence of $\Phi_{eq}=2.5 \times 10^{15} ~n_{eq}/cm^{2}$. The charge collection efficiency (CCE) is under development

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

RIM Determines Ca²+ Channel Density and Vesicle Docking at the Presynaptic Active Zone

Fast neurotransmitter release is essential for neuron-neuron communication and is initiated by the opening of voltage-gated Ca2+ channels close to docked vesicles at the presynaptic active zone. The high concentration of Ca2+ channels at the active zone is important to secure fast transmitter release. However, the mechanism that enriches Ca2+ channels at active zones is largely unknown, maybe because of the limited accessibility of most model synapses to direct measurements of Ca2+ signaling in the nerve terminal. RIM's (Rab3 interacting molecule) are scaffolding proteins in the active zone which interact with several presynaptic proteins. As revealed by previous studies in C. elegans and cultured neurons, RIM proteins affect synaptic transmission. However, their complex functions have not been clearly dissected, because direct access to the presynaptic nerve terminal has so far not been possible in synapses that are amenable to genetic manipulation. Here, we have established a Cre-lox based conditional KO approach at a presynaptically accessible CNS synapse, the calyx of Held. This has allowed us to use presynaptic recordings and Ca2+ uncaging, as well as electron microscopic analyses of synapses which have developed in vivo, to directly study the presynaptic functions of RIM proteins. We discovered three major functions of RIM proteins: First, RIM's hold Ca2+ channels in the presynaptic terminals to secure the high Ca2+ channel density at active zones. Removal of RIM proteins leads to a decrease of presynaptic Ca2+ current amplitude (by ∼ 50%) as well as to a decreased immunostaining signal using an antibody against a Ca2+ channel α-subunit (by ∼ 40%). Second, RIM proteins dock vesicles to the active zone and therefore control the size of readily releasable pool. In RIM1/2 cDKO synapses, the number of docked vesicles is reduced by ∼ 80% and the functional pool size is decreased by ∼ 75%. The good correlation of morphological and physiological data identified RIM as the first presynaptic protein in a CNS synapse with a clearly corresponding role in vesicle docking, and priming. Third, RIM proteins increase the release probability and speed up the release kinetics. This is carried out by two new functions: increasing the intrinsic Ca2+ sensitivity of release and tightly coupling docked vesicles to Ca2+ channels. Taken together, this study shows that RIM proteins function as the central organizers to coordinate active zone function: holding the Ca2+ channels in the presynaptic terminal, docking vesicles to the active zones, and regulating the release probability of any given readily-releasable vesicle. All the three functions together secure the fast vesicle release when the AP arrives in the presynaptic terminal

The Effect of FDI Agglomeration on Carbon Emission Intensity: Evidence from City-Level Data in China

How to accelerate the reduction of carbon emissions in the context of the “double carbon” target has become a key concern for all sectors of society. This paper firstly analyzes the influence mechanism of foreign direct investment (FDI) agglomeration on carbon emission intensity, from a theoretical perspective. Then, based on a panel data of 270 cities in China from 2006 to 2019, this paper uses ArcGIS software to visually analyze the spatial and temporal characteristics of FDI agglomeration and carbon emission intensity, and constructs traditional fixed effect models and spatial econometric models for empirical analysis. The results show that, first, FDI agglomeration has a significantly positive impact on the carbon emission intensity of local and neighboring cities with crowding effect. Second, the level of technological innovation can mitigate the crowding effect of FDI agglomeration on carbon emission intensity in local and neighboring cities. Third, there is a negative spatial autocorrelation between the local carbon emission intensity and the carbon emission intensity of neighboring cities. Fourth, the crowding effect of FDI agglomeration on carbon emission intensity is mainly concentrated in the central and western regions. Based on the research conclusions, this paper puts forward corresponding countermeasure suggestions

Developmental regulation of the intracellular Ca2+ sensitivity of vesicle fusion and Ca2+–secretion coupling at the rat calyx of Held

Developmental refinement of synaptic transmission can occur via changes in several pre- and postsynaptic factors, but it has been unknown whether the intrinsic Ca2+ sensitivity of vesicle fusion in the nerve terminal can be regulated during development. Using the calyx of Held, a giant synapse in the auditory pathway, we studied the presynaptic mechanisms underlying the developmental regulation of Ca2+–secretion coupling, comparing a time period before, and shortly after the onset of hearing in rats. We found an ∼2-fold leftward shift in the relationship between EPSC amplitude and presynaptic Ca2+ current charge (QCa), indicating that brief presynaptic Ca2+ currents become significantly more efficient in driving release. Using a Ca2+ tail current protocol, we also found that the high cooperativity between EPSC amplitude and QCa was slightly reduced with development. In contrast, in presynaptic Ca2+ uncaging experiments, the intrinsic Ca2+ cooperativity of vesicle fusion was identical, and the intrinsic Ca2+ sensitivity was slightly reduced with development. This indicates that the significantly enhanced release efficiency of brief Ca2+ currents must be caused by a tighter co-localization of Ca2+ channels and readily releasable vesicles, but not by changes in the intrinsic properties of Ca2+-dependent release. Using the parameters of the intrinsic Ca2+ sensitivity measured at each developmental stage, we estimate that during a presynaptic action potential (AP), a given readily releasable vesicle experiences an about 1.3-fold higher ‘local’ intracellular Ca2+ concentration ([Ca2+]i) signal with development. Thus, the data indicate a tightening in the Ca2+ channel–vesicle co-localization during development, without a major change in the intrinsic Ca2+ sensitivity of vesicle fusion