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Communication within the nervous system relies on the calcium‐triggered release of neurotransmitter molecules by exocytosis of synaptic vesicles (SVs) at defined active zone release sites. While decades of research have provided detailed insight into the molecular machinery for SV fusion, much less is known about the mechanisms that form functional SVs during the development of synapses and that control local SV reformation following exocytosis in the mature nervous system. Here we review the current state of knowledge in the field, focusing on the pathways implicated in the formation and axonal transport of SV precursor organelles and the mechanisms involved in the local reformation of SVs within nerve terminals in mature neurons. We discuss open questions and outline perspectives for future research. [ABSTRACT FROM AUTHOR]

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as "gold standard" fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R's performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts. [ABSTRACT FROM AUTHOR]

Full-length RIM1 and 2 are key components of the presynaptic active zone that ubiquitously control excitatory and inhibitory neurotransmitter release. Here, we report that the function of the small RIM isoform RIM4, consisting of a single C2 domain, is strikingly different from that of the long isoforms. RIM4 is dispensable for neurotransmitter release but plays a postsynaptic, cell type-specific role in cerebellar Purkinje cells that is essential for normal motor function. In the absence of RIM4, Purkinje cell intrinsic firing is reduced and caffeine-sensitive, and dendritic integration of climbing fibre input is disturbed. Mice lacking RIM4, but not mice lacking RIM1/2, selectively in Purkinje cells exhibit a severe, hours-long paroxysmal dystonia. These episodes can also be induced by caffeine, ethanol or stress and closely resemble the deficits seen with mutations of the PNKD (paroxysmal non-kinesigenic dystonia) gene. Our data reveal essential postsynaptic functions of RIM proteins and show non-overlapping specialized functions of a small isoform despite high homology to a single domain in the full-length proteins. [ABSTRACT FROM AUTHOR]

The abundance of CaV2 voltage-gated calcium channels is linked to presynaptic homeostatic plasticity (PHP), a process that recalibrates synaptic strength to maintain the stability of neural circuits. However, the molecular and cellular mechanisms governing PHP and CaV2 channels are not completely understood. Here, we uncover a previously not described form of PHP in Caenorhabditis elegans, revealing an inverse regulatory relationship between the efficiency of neurotransmitter release and the abundance of UNC-2/CaV2 channels. Gain-of-function unc-2SL(S240L) mutants, which carry a mutation analogous to the one causing familial hemiplegic migraine type 1 in humans, showed markedly reduced channel abundance despite increased channel functionality. Reducing synaptic release in these unc-2SL(S240L) mutants restored channel levels to those observed in wild-type animals. Conversely, loss-of-function unc-2DA(D726A) mutants, which harbor the D726A mutation in the channel pore, exhibited a marked increase in channel abundance. Enhancing synaptic release in unc-2DA mutants reversed this increase in channel levels. Importantly, this homeostatic regulation of UNC-2 channel levels is accompanied by the structural remodeling of the active zone (AZ); specifically, unc-2DA mutants, which exhibit increased channel abundance, showed parallel increases in select AZ proteins. Finally, our forward genetic screen revealed that WWP-1, a HECT family E3 ubiquitin ligase, is a key homeostatic mediator that removes UNC-2 from synapses. These findings highlight a self-tuning PHP regulating UNC-2/CaV2 channel abundance along with AZ reorganization, ensuring synaptic strength and stability. [ABSTRACT FROM AUTHOR]

At chemical synapses, voltage-gated Ca2+ channels (VGCCs) translate electrical signals into a trigger for synaptic vesicle (SV) fusion. VGCCs and the Ca2+ microdomains they elicit must be located precisely to primed SVs to evoke rapid transmitter release. Localization is mediated by Rab3-interacting molecule (RIM) and RIM-binding proteins, which interact and bind to the C terminus of the CaV2 VGCC α-subunit. We studied this machinery at the mixed cholinergic/GABAergic neuromuscular junction of Caenorhabditis elegans hermaphrodites. rimb-1 mutants had mild synaptic defects, through loosening the anchoring of UNC-2/CaV2 and delaying the onset of SV fusion. UNC-10/RIM deletion much more severely affected transmission. Although postsynaptic depolarization was reduced, rimb-1 mutants had increased cholinergic (but reduced GABAergic) transmission, to compensate for the delayed release. This did not occur when the excitation–inhibition (E–I) balance was altered by removing GABA transmission. Further analyses of GABA defective mutants and GABAA or GABAB receptor deletions, as well as cholinergic rescue of RIMB-1, emphasized that GABA neurons may be more affected than cholinergic neurons. Thus, RIMB-1 function differentially affects excitation–inhibition balance in the different motor neurons, and RIMB-1 thus may differentially regulate transmission within circuits. Untethering the UNC-2/CaV2 channel by removing its C-terminal PDZ ligand exacerbated the rimb-1 defects, and similar phenotypes resulted from acute degradation of the CaV2 β-subunit CCB-1. Therefore, untethering of the CaV2 complex is as severe as its elimination, yet it does not abolish transmission, likely due to compensation by CaV1. Thus, robustness and flexibility of synaptic transmission emerge from VGCC regulation. [ABSTRACT FROM AUTHOR]

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (Pr). Surprisingly, VGCC levels are highly predictive of heterogeneous Pr among individual synapses of either low- or high-Pr inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-Pr synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ–3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-Pr inputs, yet positively correlate with Pr among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses.

Pronounced differences in neurotransmitter release from a given presynaptic neuron, depending on the synaptic target, are among the most intriguing features of cortical networks. Hippocampal pyramidal cells (PCs) release glutamate with low probability to somatostatin expressing oriens- lacunosum- moleculare (O- LM) interneurons (INs), and the postsynaptic responses show robust short- term facilitation, whereas the release from the same presynaptic axons onto fast- spiking INs (FSINs) is -10- fold higher and the excitatory postsynaptic currents (EPSCs) display depression. The mechanisms underlying these vastly different synaptic behaviors have not been conclusively identified. Here, we applied a combined functional, pharmacological, and modeling approach to address whether the main difference lies in the action potential- evoked fusion or else in upstream priming processes of synaptic vesicles (SVs). A sequential two- step SV priming model was fitted to the peak amplitudes of unitary EPSCs recorded in response to complex trains of presynaptic stimuli in acute hippocampal slices of adult mice. At PC-FSIN connections, the fusion probability (Pfusion) of well- primed SVs is 0.6, and 44% of docked SVs are in a fusion- competent state. At PC-O- LM synapses, Pfusion is only 40% lower (0.36), whereas the fraction of well- primed SVs is 6.5- fold smaller. Pharmacological enhancement of fusion by 4- AP and priming by PDBU was recaptured by the model with a selective increase of Pfusion and the fraction of well- primed SVs, respectively. Our results demonstrate that the low fidelity of transmission at PC-O- LM synapses can be explained by a low occupancy of the release sites by well- primed SVs. [ABSTRACT FROM AUTHOR]

Super-resolution single-molecule localization microscopy (SMLM) of presynaptic active zones (AZs) and postsynaptic densities contributed to the observation of protein nanoclusters that are involved in defining functional characteristics and in plasticity of synaptic connections. Among SMLM techniques, direct stochastic optical reconstruction microscopy (dSTORM) depends on organic fluorophores that exert high brightness and reliable photoswitching. While multicolor imaging is highly desirable, the requirements necessary for high-quality dSTORM make it challenging to identify combinations of equally performing, spectrally separated dyes. Red-excited carbocyanine dyes, e.g., Alexa Fluor 647 (AF647) or Cy5, are currently regarded as “gold standard” fluorophores for dSTORM imaging. However, a recent study introduced a set of chemically modified rhodamine dyes, including CF583R, that promise to display similar performance in dSTORM. In this study, we defined CF583R’s performance compared to AF647 and CF568 based on a nanoscopic analysis of Bruchpilot (Brp), a nanotopologically well-characterized scaffold protein at Drosophila melanogaster AZs. We demonstrate equal suitability of AF647, CF568 and CF583R for basal AZ morphometry, while in Brp subcluster analysis CF583R outperforms CF568 and is on par with AF647. Thus, the AF647/CF583R combination will be useful in future dSTORM-based analyses of AZs and other subcellularly located marker molecules and their role in physiological and pathophysiological contexts.

The processes of creating an electromagnetic force acting on the molten metal in the active zone of a magnetodynamic pump are studied depending on the spectral composition of the higher harmonic components of the current in the channel and the induction of the magnetic flux induced by an electromagnet. The peculiarities of the regulation of the magnitude and direction of the resultant vector of this force, the favorable conditions of the vibrational action on the molten metal in the active zone are defined. Ref. 8, fig. 4, tables 3.

Cochlear inner hair cells (IHCs) form specialized ribbon synapses with spiral ganglion neurons that tirelessly transmit sound information at high rates over long time periods with extreme temporal precision. This functional specialization is essential for sound encoding and is attributed to a distinct molecular machinery with unique players or splice variants compared to conventional neuronal synapses. Among these is the active zone (AZ) scaffold protein piccolo/aczonin, which is represented by its short splice variant piccolino at cochlear and retinal ribbon synapses. While the function of piccolo at synapses of the central nervous system has been intensively investigated, the role of piccolino at IHC synapses remains unclear. In this study, we characterize the structure and function of IHC synapses in piccolo gene‐trap mutant rats (Pclogt/gt). We find a mild hearing deficit with elevated thresholds and reduced amplitudes of auditory brainstem responses. Ca2+ channel distribution and ribbon morphology are altered in apical IHCs, while their presynaptic function seems to be unchanged. We conclude that piccolino contributes to the AZ organization in IHCs and is essential for normal hearing. Synopsis: Piccolino regulates ribbon morphology as well as the arrangement of Ca2+ channels at cochlear ribbon synapses. Lack of piccolino results in mild hearing impairment. Rats lacking piccolino show elevated thresholds and reduced amplitudes of auditory brainstem responses for middle and high sound frequencies, indicating an impairment in synchronous synaptic transmission of sound information.Disruption of piccolino results in two distinct categories of active zones with different ribbon synapse morphologies in mutant inner hair cells, while tethering of synaptic vesicles is normal.Ca2+ channel clustering is impaired in piccolino‐deficient hair cells, but Ca2+ currents and exocytosis are unaffected. [ABSTRACT FROM AUTHOR]

This paper is dedicated to the modeling, design, and consequent construction of the practical setup of the small-size solid-state laser with Nd:YAG gain medium and the diode pumping. Solid-state lasers have been in existence for around 50 years and have experienced both positive and negative aspects during their evolution. Nevertheless, they have always held an essential position in numerous industries, scientific endeavors, and everyday activities. Nowadays, such devices are widely used and developed in location, manufacturing, medicine, and of course in military. Hence, the development and optimization of their structure remain a pertinent and important issue. The research reveals the basic component parameters for a compact Nd:YAG laser with diode pump, which can be implemented in the construction of range-finder. The main results of the work. Nd:YAG laser with overall dimensions of 350 x 25 x 25 mm was developed and modeled; the dimensions of the laser active head are 110 x25 x 25 mm. Diode pumping is a matrix of six diodes with a power of 90 W each, which operate in a pulse mode with a duration of 250 ns. The resonator consists of two mirrors, with an output reflectance from 0.66 to 0.74. The active element is 72 mm long. With these laser parameters, the first giant pulse after the start of pumping was achieved at 59 μs, an output energy of 25 mJ, and a laser system efficiency of 22 %. Additionally, the following radiation pulses were generated with a time interval of 4 μs. The results obtained in this work make it possible to create a diode-pumped solid-state laser with an output radiation energy of up to 25 mJ and have compact cavity dimensions of about 80 x 25 x 25 mm. [ABSTRACT FROM AUTHOR]

We still face fundamental gaps in understanding how molecular plastic changes of synapses intersect with circuit operation to define behavioral states. Here, we show that an antagonism between two conserved regulatory proteins, Spinophilin (Spn) and Syd-1, controls presynaptic long-term plasticity and the maintenance of olfactory memories in Drosophila. While Spn mutants could not trigger nanoscopic active zone remodeling under homeostatic challenge and failed to stably potentiate neurotransmitter release, concomitant reduction of Syd-1 rescued all these deficits. The Spn/Syd-1 antagonism converged on active zone close F-actin, and genetic or acute pharmacological depolymerization of F-actin rescued the Spn deficits by allowing access to synaptic vesicle release sites. Within the intrinsic mushroom body neurons, the Spn/Syd-1 antagonism specifically controlled olfactory memory stabilization but not initial learning. Thus, this evolutionarily conserved protein complex controls behaviorally relevant presynaptic long-term plasticity, also observed in the mammalian brain but still enigmatic concerning its molecular mechanisms and behavioral relevance.

In the process of designing the new NEPTUN pulsed reactor, the phenomenon of dynamic bending was discovered; it is a feature of the dynamics of pulsed reactors that does not appear in reactors of other types; however, it plays a significant role in evaluating the stability of reactor operation. Movements of fuel elements (fuel rods) under the influence of periodically changing temperature can lead to significant changes in the reactivity and fluctuations of the reactor power. To accurately simulate the operation of the reactor, it is necessary to know the shape of the fuel element at the time of the next power pulse. Since numerical methods for solving this dynamic problem of thermoelasticity require a computation time that is too large, an analytical method was applied. In this study, the first results of the numerical–analytical solution of the equation of induced oscillations of a fuel rod, as well as the prospects for using the method in studying the dynamics of the NEPTUN reactor, are presented. [ABSTRACT FROM AUTHOR]

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, Ca21 imaging demonstrated ;2-fold greater Ca21 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 Ca21 channels relative to other active zone scaffolds. These data suggest that distinctions in active zone nano-architecture and Ca21 influx collaborate to differentially tune glutamate release at tonic versus phasic synaptic subtypes. [ABSTRACT FROM AUTHOR]

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, "spontaneous" transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the nonspecific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses. [ABSTRACT FROM AUTHOR]

A recent characterization of the role of autophagy in two different neuron types during brain development in Drosophila revealed two different mechanisms to regulate synapse formation. In photoreceptor neurons, autophagosome formation in synaptogenic filopodia destabilizes presumptive synaptic contacts and thereby restricts incorrect synaptic partnerships. In dorsal cluster neurons, autophagy is actively suppressed to keep mature synapses stable during axonal branching. These findings indicate that different neuron types can require activation or suppression of synaptic autophagy during the same developmental period to ensure proper synapse formation and brain connectivity. [ABSTRACT FROM AUTHOR]

Presynaptic active zones (AZs) contain many molecules essential for neurotransmitter release and are assembled in a highly organized manner. A network of adaptor proteins known as cytomatrix at the AZ (CAZ) is important for shaping the structural characteristics of AZ. Rab3-interacting molecule (RIM)-binding protein (RBP) family are binding partners of the CAZ protein RIM and also bind the voltage-gated calcium channels (VGCCs) in mice and flies. Here, we investigated the physiological roles of RIMB-1, the homolog of RBPs in the nematode Caenorhabditis elegans RIMB-1 is expressed broadly in neurons and predominantly localized at presynaptic sites. Loss-of-function animals of rimb-1 displayed slight defects in motility and response to pharmacological inhibition of synaptic transmission, suggesting a modest involvement of rimb-1 in synapse function. We analyzed genetic interactions of rimb-1 by testing candidate genes and by an unbiased forward genetic screen for rimb-1 enhancer. Both analyses identified the RIM homolog UNC-10 that acts together with RIMB-1 to regulate presynaptic localization of the P/Q-type VGCC UNC-2/Cav2. We also find that the precise localization of RIMB-1 to presynaptic sites requires presynaptic UNC-2/Cav2. RIMB-1 has multiple FN3 and SH3 domains. Our transgenic rescue analysis with RIMB-1 deletion constructs revealed a functional requirement of a C-terminal SH3 in regulating UNC-2/Cav2 localization. Together, these findings suggest a redundant role of RIMB-1/RBP and UNC-10/RIM to regulate the abundance of UNC-2/Cav2 at the presynaptic AZ in C. elegans, depending on the bidirectional interplay between CAZ adaptor and channel proteins.SIGNIFICANCE STATEMENT Presynaptic active zones (AZs) are highly organized structures for synaptic transmission with characteristic networks of adaptor proteins called cytomatrix at the AZ (CAZ). In this study, we characterized a CAZ protein RIMB-1, named for RIM-binding protein (RBP), in the nematode Caenorhabditis elegans Through systematic analyses of genetic interactions and an unbiased genetic enhancer screen of rimb-1, we revealed a redundant role of two CAZ proteins RIMB-1/RBP and UNC-10/RIM in regulating presynaptic localization of UNC-2/Cav2, a voltage-gated calcium channel (VGCC) critical for proper neurotransmitter release. Additionally, the precise localization of RIMB-1/RBP requires presynaptic UNC-2/Cav2. These findings provide new mechanistic insight about how the interplay among multiple CAZ adaptor proteins and VGCC contributes to the organization of presynaptic AZ.

Asynchronous release is a ubiquitous form of neurotransmitter release that persists for tens to hundreds of milliseconds after an action potential. How asynchronous release is organized and regulated at the synaptic active zone (AZ) remains debatable. Using nanoscale-precision imaging of individual release events in rat hippocampal synapses, we observed two spatially distinct subpopulations of asynchronous events, ~75% of which occurred inside the AZ and with a bias towards the AZ center, while ~25% occurred outside of the functionally defined AZ, that is, ectopically. The two asynchronous event subpopulations also differed from each other in temporal properties, with ectopic events occurring at significantly longer time intervals from synchronous events than the asynchronous events inside the AZ. Both forms of asynchronous release did not, to a large extent, utilize the same release sites as synchronous events. The two asynchronous event subpopulations also differ from synchronous events in some aspects of exo-endocytosis coupling, particularly in the contribution from the fast calcium-dependent endocytosis. These results identify two subpopulations of asynchronous release events with distinctive organization and spatiotemporal dynamics.

Synaptic transmission relies on presynaptic neurotransmitter (NT) release from synaptic vesicles (SVs) and on NT detection by postsynaptic receptors. Transmission exists in two principal modes: action-potential (AP) evoked and AP-independent, “spontaneous” transmission. AP-evoked neurotransmission is considered the primary mode of inter-neuronal communication, whereas spontaneous transmission is required for neuronal development, homeostasis, and plasticity. While some synapses appear dedicated to spontaneous transmission only, all AP-responsive synapses also engage spontaneously, but whether this encodes functional information regarding their excitability is unknown. Here we report on functional interdependence of both transmission modes at individual synaptic contacts of Drosophila larval neuromuscular junctions (NMJs) which were identified by the presynaptic scaffolding protein Bruchpilot (BRP) and whose activities were quantified using the genetically encoded Ca2+ indicator GCaMP. Consistent with the role of BRP in organizing the AP-dependent release machinery (voltage-dependent Ca2+ channels and SV fusion machinery), most active BRP-positive synapses (>85%) responded to APs. At these synapses, the level of spontaneous activity was a predictor for their responsiveness to AP-stimulation. AP-stimulation resulted in cross-depletion of spontaneous activity and both transmission modes were affected by the non-specific Ca2+ channel blocker cadmium and engaged overlapping postsynaptic receptors. Thus, by using overlapping machinery, spontaneous transmission is a continuous, stimulus independent predictor for the AP-responsiveness of individual synapses.

Single-molecule localization microscopy (SMLM) greatly advances structural studies of diverse biological tissues. For example, presynaptic active zone (AZ) nanotopology is resolved in increasing detail. Immunofluorescence imaging of AZ proteins usually relies on epitope preservation using aldehyde-based immunocompetent fixation. Cryofixation techniques, such as high-pressure freezing (HPF) and freeze substitution (FS), are widely used for ultrastructural studies of presynaptic architecture in electron microscopy (EM). HPF/FS demonstrated nearer-to-native preservation of AZ ultrastructure, e.g., by facilitating single filamentous structures. Here, we present a protocol combining the advantages of HPF/FS and direct stochastic optical reconstruction microscopy (dSTORM) to quantify nanotopology of the AZ scaffold protein Bruchpilot (Brp) at neuromuscular junctions (NMJs) of Drosophila melanogaster. Using this standardized model, we tested for preservation of Brp clusters in different FS protocols compared to classical aldehyde fixation. In HPF/FS samples, presynaptic boutons were structurally well preserved with ~22% smaller Brp clusters that allowed quantification of subcluster topology. In summary, we established a standardized near-to-native preparation and immunohistochemistry protocol for SMLM analyses of AZ protein clusters in a defined model synapse. Our protocol could be adapted to study protein arrangements at single-molecule resolution in other intact tissue preparations. [ABSTRACT FROM AUTHOR]

Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Given this functional significance, defining the processes that cooperate to establish AZ VGCC abundance is critical for understanding how these mechanisms set synaptic strength and how they might be regulated to control presynaptic plasticity. VGCC abundance at AZs involves multiple steps, including channel biosynthesis (transcription, translation, and trafficking through the endomembrane system), forward axonal trafficking and delivery to synaptic terminals, incorporation and retention at presynaptic sites, and protein recycling. Here we discuss mechanisms that control VGCC abundance at synapses, highlighting findings from invertebrate and vertebrate models. [ABSTRACT FROM AUTHOR]

Neural activity-dependent synaptic plasticity is an important physiological phenomenon underlying environmental adaptation, memory and learning. However, its molecular basis, especially in presynaptic neurons, is not well understood. Previous studies have shown that the number of presynaptic active zones in the Drosophila melanogaster photoreceptor R8 is reversibly changed in an activitydependent manner. During reversible synaptic changes, both synaptic disassembly and assembly processes were observed. Although we have established a paradigm for screening molecules involved in synaptic stability and several genes have been identified, genes involved in stimulus-dependent synaptic assembly are still elusive. Therefore, the aim of this study was to identify genes regulating stimulus-dependent synaptic assembly in Drosophila using an automated synapse quantification system. To this end, we performed RNAi screening against 300 memory-defective, synapse-related or transmembrane molecules in photoreceptor R8 neurons. Candidate genes were narrowed down to 27 genes in the first screen using presynaptic protein aggregation as a sign of synaptic disassembly. In the second screen, we directly quantified the decreasing synapse number using a GFP-tagged presynaptic protein marker. We utilized custom-made image analysis software, which automatically locates synapses and counts their number along individual R8 axons, and identified cirl as a candidate gene responsible for synaptic assembly. Finally, we present a new model of stimulus-dependent synaptic assembly through the interaction of cirl and its possible ligand, ten-a. This study demonstrates the feasibility of using the automated synapse quantification system to explore activity-dependent synaptic plasticity in Drosophila R8 photoreceptors in order to identify molecules involved in stimulus-dependent synaptic assembly. [ABSTRACT FROM AUTHOR]

Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Given this functional significance, defining the processes that cooperate to establish AZ VGCC abundance is critical for understanding how these mechanisms set synaptic strength and how they might be regulated to control presynaptic plasticity. VGCC abundance at AZs involves multiple steps, including channel biosynthesis (transcription, translation, and trafficking through the endomembrane system), forward axonal trafficking and delivery to synaptic terminals, incorporation and retention at presynaptic sites, and protein recycling. Here we discuss mechanisms that control VGCC abundance at synapses, highlighting findings from invertebrate and vertebrate models.

Dopamine is an important modulator of cognition and movement. We recently found that evoked dopamine secretion is fast and relies on active zone-like release sites. Here, we used in vivo biotin identification (iBioID) proximity proteomics in mouse striatum to assess which proteins are present at these sites. Using three release site baits, we identified proteins that are enriched over the general dopamine axonal protein content, and they fell into several categories, including active zone, Ca2+ regulatory, and synaptic vesicle proteins. We also detected many proteins not previously associated with vesicular exocytosis. Knockout of the presynaptic organizer protein RIM strongly decreased the hit number obtained with iBioID, while Synaptotagmin-1 knockout did not. α-Synuclein, a protein linked to Parkinson’s disease, was enriched at release sites, and its enrichment was lost in both tested mutants. We conclude that RIM organizes scaffolded dopamine release sites and provide a proteomic assessment of the composition of these sites.

Ribbon synapses of cochlear inner hair cells (IHCs) are specialized to indefatigably transmit sound information at high rates. To understand the underlying mechanisms, structure-function analysis of the active zone (AZ) of these synapses is essential. Previous electron microscopy studies of synaptic vesicle (SV) dynamics at the IHC AZ used potassium stimulation, which limited the temporal resolution to minutes. Here, we established optogenetic IHC stimulation followed by quick freezing within milliseconds and electron tomography to study the ultrastructure of functional synapse states with good temporal resolution in mice. We characterized optogenetic IHC stimulation by patch-clamp recordings from IHCs and postsynaptic boutons revealing robust IHC depolarization and neurotransmitter release. Ultrastructurally, the number of docked SVs increased upon short (17–25 ms) and long (48–76 ms) light stimulation paradigms. We did not observe enlarged SVs or other morphological correlates of homotypic fusion events. Our results indicate a rapid recruitment of SVs to the docked state upon stimulation and suggest that univesicular release prevails as the quantal mechanism of exocytosis at IHC ribbon synapses.

IntroductionNeurotransmitter release at presynaptic active zones (AZs) requires concerted protein interactions within a dense 3D nano-hemisphere. Among the complex protein meshwork the (M)unc-13 family member Unc-13 of Drosophila melanogaster is essential for docking of synaptic vesicles and transmitter release.MethodsWe employ minos-mediated integration cassette (MiMIC)-based gene editing using GFSTF (EGFP-FlAsH-StrepII-TEV-3xFlag) to endogenously tag all annotated Drosophila Unc-13 isoforms enabling visualization of endogenous Unc-13 expression within the central and peripheral nervous system.Results and discussionElectrophysiological characterization using two-electrode voltage clamp (TEVC) reveals that evoked and spontaneous synaptic transmission remain unaffected in unc-13GFSTF 3rd instar larvae and acute presynaptic homeostatic potentiation (PHP) can be induced at control levels. Furthermore, multi-color structured-illumination shows precise co-localization of Unc-13GFSTF, Bruchpilot, and GluRIIA-receptor subunits within the synaptic mesoscale. Localization microscopy in combination with HDBSCAN algorithms detect Unc-13GFSTF subclusters that move toward the AZ center during PHP with unaltered Unc-13GFSTF protein levels.

Robust neural information transfer relies on a delicate molecular nano-architecture of chemical synapses. Neurotransmitter release is controlled by a specific arrangement of proteins within presynaptic active zones. How the specific presynaptic molecular architecture relates to postsynaptic organization and how synaptic nano-architecture is transsynaptically regulated to enable stable synaptic transmission remain enigmatic. Using time-gated stimulated emission-depletion microscopy at the Drosophila neuromuscular junction, we found that presynaptic nanorings formed by the active-zone scaffold Bruchpilot (Brp) align with postsynaptic glutamate receptor (GluR) rings. Individual rings harbor approximately four transsynaptically aligned Brp-GluR nanocolumns. Similar nanocolumn rings are formed by the presynaptic protein Unc13A and GluRs. Intriguingly, acute GluR impairment triggers transsynaptic nanocolumn formation on the minute timescale during homeostatic plasticity. We reveal distinct phases of structural transsynaptic homeostatic plasticity, with postsynaptic GluR reorganization preceding presynaptic Brp modulation. Finally, homeostatic control of transsynaptic nano-architecture and neurotransmitter release requires the auxiliary GluR subunit Neto. Thus, transsynaptic nanocolumn rings provide a substrate for rapid homeostatic stabilization of synaptic efficacy [ABSTRACT FROM AUTHOR]

Humans carrying the CORD7 (cone-rod dystrophy 7) mutation possess increased verbal IQ and working memory. This autosomal dominant syndrome is caused by the single-amino acid R844H exchange (human numbering) located in the 310 helix of the C2A domain of RIMS1/RIM1 (Rab3-interacting molecule 1). RIM is an evolutionarily conserved multi-domain protein and essential component of presynaptic active zones, which is centrally involved in fast, Ca2+-triggered neurotransmitter release. How the CORD7 mutation affects synaptic function has remained unclear thus far. Here, we established Drosophila melanogaster as a disease model for clarifying the effects of the CORD7 mutation on RIM function and synaptic vesicle release. To this end, using protein expression and X-ray crystallography, we solved the molecular structure of the Drosophila C2A domain at 1.92 Å resolution and by comparison to its mammalian homologue ascertained that the location of the CORD7 mutation is structurally conserved in fly RIM. Further, CRISPR/Cas9-assisted genomic engineering was employed for the generation of rim alleles encoding the R915H CORD7 exchange or R915E, R916E substitutions (fly numbering) to effect local charge reversal at the 310 helix. Through electrophysiological characterization by two-electrode voltage clamp and focal recordings we determined that the CORD7 mutation exerts a semi-dominant rather than a dominant effect on synaptic transmission resulting in faster, more efficient synaptic release and increased size of the readily releasable pool but decreased sensitivity for the fast calcium chelator BAPTA. In addition, the rim CORD7 allele increased the number of presynaptic active zones but left their nanoscopic organization unperturbed as revealed by super-resolution microscopy of the presynaptic scaffold protein Bruchpilot/ELKS/CAST. We conclude that the CORD7 mutation leads to tighter release coupling, an increased readily releasable pool size and more release sites thereby promoting more efficient synaptic transmitter release. These results strongly suggest that similar mechanisms may underlie the CORD7 disease phenotype in patients and that enhanced synaptic transmission may contribute to their increased cognitive abilities. [ABSTRACT FROM AUTHOR]

Synaptic heterogeneity is a hallmark of nervous systems that enables complex and adaptable communication in neural circuits. To understand circuit function, it is thus critical to determine the factors that contribute to the functional diversity of synapses. We investigated the contributions of voltage-gated calcium channel (VGCC) abundance, spatial organization, and subunit composition to synapse diversity among and between synapses formed by two closely related Drosophila glutamatergic motor neurons with distinct neurotransmitter release probabilities (P r ). Surprisingly, VGCC levels are highly predictive of heterogeneous P r among individual synapses of either low- or high-P r inputs, but not between inputs. We find that the same number of VGCCs are more densely organized at high-P r synapses, consistent with tighter VGCC-synaptic vesicle coupling. We generated endogenously tagged lines to investigate VGCC subunits in vivo and found that the α2δ-3 subunit Straightjacket along with the CAST/ELKS active zone (AZ) protein Bruchpilot, both key regulators of VGCCs, are less abundant at high-P r inputs, yet positively correlate with P r among synapses formed by either input. Consistently, both Straightjacket and Bruchpilot levels are dynamically increased across AZs of both inputs when neurotransmitter release is potentiated to maintain stable communication following glutamate receptor inhibition. Together, these findings suggest a model in which VGCC and AZ protein abundance intersects with input-specific spatial and molecular organization to shape the functional diversity of synapses., Competing Interests: AM, SG, AD, JR, KO No competing interests declared, (© 2023, Medeiros et al.)

Active zones consist of protein scaffolds that are tightly attached to the presynaptic plasma membrane. They dock and prime synaptic vesicles, couple them to voltage-gated Ca2+ channels, and direct neurotransmitter release toward postsynaptic receptor domains. Simultaneous RIM + ELKS ablation disrupts these scaffolds, abolishes vesicle docking, and removes active zone-targeted Munc13, but some vesicles remain releasable. To assess whether this enduring vesicular fusogenicity is mediated by non-active zone-anchored Munc13 or is Munc13-independent, we ablated Munc13-1 and Munc13-2 in addition to RIM + ELKS in mouse hippocampal neurons. The hextuple knockout synapses lacked docked vesicles, but other ultrastructural features were near-normal despite the strong genetic manipulation. Removing Munc13 in addition to RIM + ELKS impaired action potential-evoked vesicle fusion more strongly than RIM + ELKS knockout by further decreasing the releasable vesicle pool. Hence, Munc13 can support some fusogenicity without RIM and ELKS, and presynaptic recruitment of Munc13, even without active zone anchoring, suffices to generate some fusion-competent vesicles.

A goal of modern neuroscience involves understanding how connections in the brain form and function. Such a knowledge is essential to inform how defects in the exquisite complexity of nervous system growth influence neurological disease. Studies of the nervous system in the fruit fly Drosophila melanogaster enabled the discovery of a wealth of molecular and genetic mechanisms underlying development of synapses--the specialized cell-to-cell connections that comprise the essential substrate for information flow and processing in the nervous system. For years, the major driver of knowledge was the neuromuscular junction due to its ease of examination. Analogous studies in the central nervous system lagged due to a lack of genetic accessibility of specific neuron classes, synaptic labels compatible with cell-typespecific access, and high resolution, quantitative imaging strategies. However, understanding how central synapses form remains a prerequisite to understanding brain development. In the last decade, a host of new tools and techniques extended genetic studies of synapse organization into central circuits to enhance our understanding of synapse formation, organization, and maturation. In this review, we consider the current state-of-the-field. We first discuss the tools, technologies, and strategies developed to visualize and quantify synapses in vivo in genetically identifiable neurons of the Drosophila central nervous system. Second, we explore how these tools enabled a clearer understanding of synaptic development and organization in the fly brain and the underlying molecular mechanisms of synapse formation. These studies establish the fly as a powerful in vivo genetic model that offers novel insights into neural development. [ABSTRACT FROM AUTHOR]

Within 1 millisecond of action potential arrival at presynaptic terminals voltage–gated Ca2+ channels open. The Ca2+ channels are linked to synaptic vesicles which are tethered by active zone proteins. Ca2+ entrance into the active zone triggers: (1) the fusion of the vesicle and exocytosis, (2) the replenishment of the active zone with vesicles for incoming exocytosis, and (3) various types of endocytosis for vesicle reuse, dependent on the pattern of firing. These time-dependent vesicle dynamics are controlled by presynaptic Ca2+ sensor proteins, regulating active zone scaffold proteins, fusion machinery proteins, motor proteins, endocytic proteins, several enzymes, and even Ca2+ channels, following the decay of Ca2+ concentration after the action potential. Here, I summarize the Ca2+-dependent protein controls of synchronous and asynchronous vesicle release, rapid replenishment of the active zone, endocytosis, and short-term plasticity within 100 msec after the action potential. Furthermore, I discuss the contribution of active zone proteins to presynaptic plasticity and to homeostatic readjustment during and after intense activity, in addition to activity-dependent endocytosis. [ABSTRACT FROM AUTHOR]

The mouse neuromuscular junction (NMJ) has long been used as a model synapse for the study of neurotransmission in both healthy and disease states of the NMJ. Neurotransmission from these neuromuscular nerve terminals occurs at highly organized structures called active zones (AZs). Within AZs, the relationships between the voltage-gated calcium channels and docked synaptic vesicles govern the probability of acetylcholine release during single action potentials, and the short-term plasticity characteristics during short, high frequency trains of action potentials. Understanding these relationships is important not only for healthy synapses, but also to better understand the pathophysiology of neuromuscular diseases. In particular, we are interested in Lambert-Eaton myasthenic syndrome (LEMS), an autoimmune disorder in which neurotransmitter release from the NMJ decreases, leading to severe muscle weakness. In LEMS, the reduced neurotransmission is traditionally thought to be caused by the antibody-mediated removal of presynaptic voltage-gated calcium channels. However, recent experimental data and AZ computer simulations have predicted that a disruption in the normally highly organized active zone structure, and perhaps autoantibodies to other presynaptic proteins, contribute significantly to pathological effects in the active zone and the characteristics of chemical transmitters. [ABSTRACT FROM AUTHOR]

Voltage-gated Ca2+ channels (VGCCs) mediate Ca2+ influx to trigger neurotransmitter release at specialized presynaptic sites termed active zones (AZs). The abundance of VGCCs at AZs regulates neurotransmitter release probability (Pr), a key presynaptic determinant of synaptic strength. Although biosynthesis, delivery, and recycling cooperate to establish AZ VGCC abundance, experimentally isolating these distinct regulatory processes has been difficult. Here, we describe how the AZ levels of cacophony (Cac), the sole VGCC-mediating synaptic transmission in Drosophila, are determined. We also analyzed the relationship between Cac, the conserved VGCC regulatory subunit α2δ, and the core AZ scaffold protein Bruchpilot (BRP) in establishing a functional AZ. We find that Cac and BRP are independently regulated at growing AZs, as Cac is dispensable for AZ formation and structural maturation, and BRP abundance is not limiting for Cac accumulation. Additionally, AZs stop accumulating Cac after an initial growth phase, whereas BRP levels continue to increase given extended developmental time. AZ Cac is also buffered against moderate increases or decreases in biosynthesis, whereas BRP lacks this buffering. To probe mechanisms that determine AZ Cac abundance, intravital FRAP and Cac photoconversion were used to separately measure delivery and turnover at individual AZs over a multi-day period. Cac delivery occurs broadly across the AZ population, correlates with AZ size, and is rate-limited by α2δ. Although Cac does not undergo significant lateral transfer between neighboring AZs over the course of development, Cac removal from AZs does occur and is promoted by new Cac delivery, generating a cap on Cac accumulation at mature AZs. Together, these findings reveal how Cac biosynthesis, synaptic delivery, and recycling set the abundance of VGCCs at individual AZs throughout synapse development and maintenance.

The general mechanism of calcium-triggered chemical transmitter release from neuronal synapses has been intensely studied, is well-known, and highly conserved between species and synapses across the nervous system. However, the structural and functional details within each transmitter release site (or active zone) are difficult to study in living tissue using current experimental approaches owing to the small spatial compartment within the synapse where exocytosis occurs with a very rapid time course. Therefore, computer simulations offer the opportunity to explore these microphysiological environments of the synapse at nanometer spatial scales and on a sub-microsecond timescale. Because biological reactions and physiological processes at synapses occur under conditions where stochastic behavior is dominant, simulation approaches must be driven by such stochastic processes. MCell provides a powerful simulation approach that employs particle-based stochastic simulation tools to study presynaptic processes in realistic and complex (3D) geometries using optimized Monte Carlo algorithms to track finite numbers of molecules as they diffuse and interact in a complex cellular space with other molecules in solution and on surfaces (representing membranes, channels and binding sites). In this review we discuss MCell-based spatially realistic models of the mammalian and frog neuromuscular active zones that were developed to study presynaptic mechanisms that control transmitter release. In particular, these models focus on the role of presynaptic voltage-gated calcium channels, calcium sensors that control the probability of synaptic vesicle fusion, and the effects of action potential waveform shape on presynaptic calcium entry. With the development of these models, they can now be used in the future to predict disease-induced changes to the active zone, and the effects of candidate therapeutic approaches.

To achieve the functional polarization that underlies brain computation, neurons sort protein material into distinct compartments. Ion channel composition, for example, differs between axons and dendrites, but the molecular determinants for their polarized trafficking remain obscure. Here, we identify mechanisms that target voltage-gated Ca2+ channels (Ca V s) to distinct subcellular compartments. In hippocampal neurons, Ca V 2s trigger neurotransmitter release at the presynaptic active zone, and Ca V 1s localize somatodendritically. After knockout of all three Ca V 2s, expression of Ca V 2.1, but not Ca V 1.3, restores neurotransmitter release. We find that chimeric Ca V 1.3s with Ca V 2.1 intracellular C-termini localize to the active zone, mediate synaptic vesicle exocytosis, and render release sensitive to Ca V 1 blockers. This dominant targeting function of the Ca V 2.1 C-terminus requires the first EF hand in its proximal segment, and replacement of the Ca V 2.1 C-terminus with that of Ca V 1.3 abolishes Ca V 2.1 active zone localization and function. We conclude that Ca V intracellular C-termini mediate compartment-specific targeting. [Display omitted] • Ca V 2.1s, but not Ca V 1.3s, are at the active zone and trigger neurotransmitter release • Swapping the Ca V 2.1 C-terminus onto Ca V 1.3 confers active zone targeting to Ca V 1.3 • Ca V 1.3 with the Ca V 2.1 C-terminus mediates release that is L-type blocker sensitive • The first EF hand in the C-terminus is required for Ca V 2.1 active zone targeting Voltage-gated Ca2+ channel subtypes are differentially sorted into the soma, dendrites, and axon of a neuron. Chin et al. find that the intracellular C-termini of Ca V 1 and Ca V 2 channels are key determinants for compartment targeting and report an essential trafficking role for the first EF hand of Ca V 2.1. [ABSTRACT FROM AUTHOR]

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using – in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses. [ABSTRACT FROM AUTHOR]

Efficient and reliable neurotransmission requires precise coupling between action potentials (APs), Ca2+ entry and neurotransmitter release. However, Ca2+ requirements for release, including the number of channels required, their subtypes, and their location with respect to primed vesicles, remains to be precisely defined for central synapses. Indeed, Ca2+ entry may occur through small numbers or even single open Ca2+ channels, but these questions remain largely unexplored in simple active zone (AZ) synapses common in the nervous system, and key to addressing Ca2+ channel and synaptic dysfunction underlying numerous neurologic and neuropsychiatric disorders. Here, we present single channel analysis of evoked AZ Ca2+ entry, using cell-attached patch clamp and lattice lightsheet microscopy (LLSM), resolving small channel numbers evoking Ca2+ entry following depolarization, at single AZs in individual central lamprey reticulospinal presynaptic terminals from male and females. We show a small pool (mean of 23) of Ca2+ channels at each terminal, comprising N-(CaV2.2), P/Q-(CaV2.1), and R-(CaV2.3) subtypes, available to gate neurotransmitter release. Significantly, of this pool only one to seven channels (mean of 4) open on depolarization. High temporal fidelity lattice light-sheet imaging reveals AP-evoked Ca2+ transients exhibiting quantal amplitude variations of 0-6 event sizes between individual APs and stochastic variation of precise locations of Ca2+ entry within the AZ. Further, total Ca2+ channel numbers at each AZ correlate to the number of presynaptic primed synaptic vesicles. Dispersion of channel openings across the AZ and the similar number of primed vesicles and channels indicate that Ca2+ entry via as few as one channel may trigger neurotransmitter release. [ABSTRACT FROM AUTHOR]

Cognitive semantics, which has gained acceleration in the last ten years, is a point of view that sees individual’s perception and cognitive level as variables in meaning production process. Utilizing theories in psychology, one of the most important concepts put forward in the aforesaid discipline is active zones, which argues that meaning is a production related to conceptualization and perception. Active zone constructions in adjectives can be discussed as a cognitive structure that helps to display under what conditions and how the cognitive mappings of the adjective and the described noun can vary. In this study, it is aimed to reveal the dynamics in active zone constructions in Turkish shape adjectives in terms of the adjective yuvarlak. Within this perspective, co-occurrence appearances of the research topic adjective are obtained through Turkish National Corpus v3 and active zone constructions are analysed with content analysis method.

The presynaptic active zone (AZ) of chemical synapses is a highly dynamic compartment where synaptic vesicle fusion and neurotransmitter release take place. During evolution the AZ was optimized for speed, accuracy, and reliability of chemical synaptic transmission in combination with miniaturization and plasticity. Single-molecule localization microscopy (SMLM) offers nanometer spatial resolution as well as information about copy number, localization, and orientation of proteins of interest in AZs. This type of imaging allows quantifications of activity dependent AZ reorganizations, e.g., in the context of presynaptic homeostatic potentiation. In combination with high-pressure freezing and optogenetic or electrical stimulation AZs can be imaged with millisecond temporal resolution during synaptic activity. Therefore SMLM allows the determination of key parameters in the complex spatial environment of AZs, necessary for next generation simulations of chemical synapses with realistic protein arrangements.

Both paralogs of the calcium-dependent activator protein for secretion (CAPS) are required for exocytosis of synaptic vesicles (SVs) and large dense core vesicles (LDCVs). Despite approximately 80% sequence identity, CAPS1 and CAPS2 have distinct functions in promoting exocytosis of SVs and LDCVs in dorsal root ganglion (DRG) neurons. However, the molecular mechanisms underlying these differences remain enigmatic. In this study, we applied high- and super-resolution imaging techniques to systematically assess the subcellular localization of CAPS paralogs in DRG neurons deficient in both CAPS1 and CAPS2. CAPS1 was found to be more enriched at the synapses. Using – in-depth sequence analysis, we identified a unique CAPS1 N-terminal sequence, which we introduced into CAPS2. This CAPS1/2 chimera reproduced the pre-synaptic localization of CAPS1 and partially rescued synaptic transmission in neurons devoid of CAPS1 and CAPS2. Using immunoprecipitation combined with mass spectrometry, we identified CAPS1-specific interaction partners that could be responsible for its pre-synaptic enrichment. Taken together, these data suggest an important role of the CAPS1-N terminus in the localization of the protein at pre-synapses.

Traditional regulation methods of active sites have successfully optimized the performance of electrocatalysts, but seem unable to achieve further breakthrough in the catalytic activity. Unlike the conventional viewpoint of focusing on single active site, the concept of local microstructure active zone is more comprehensive and new methods to regulate the reaction zone for electrocatalytic reactions are developed accordingly. The local microstructure active zone refers to the zone with high catalytic activity formed by the interaction between active atoms and neighboring coordination atoms as well as the surrounding environment. Instead of the traditional single active atom site, the active zone is more suitable for the actual electrochemical reaction process. According to this concept, the activity of electrocatalysts can be coordinated by multiple active atoms. This strategy is beneficial for understanding the relationship between material, structure, and catalysis, which realizes the design and synthesis of high‐performance electrocatalysts. This review provides the research progress of this strategy for electrocatalytic reactions, with emphasis on their applications in oxygen evolution reaction, urea oxidation reaction, and carbon dioxide reduction. [ABSTRACT FROM AUTHOR]