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Pre- and postsynaptic forms of long-term potentiation (LTP) are candidate synaptic mechanisms underlying learning and memory. At layer 5 pyramidal neurons, LTP increases the initial synaptic strength but also short-term depression during high-frequency transmission. This classical form of presynaptic LTP has been referred to as redistribution of synaptic efficacy. However, the underlying mechanisms remain unclear. We therefore performed whole-cell recordings from layer 5 pyramidal neurons in acute cortical slices of rats and analyzed presynaptic function before and after LTP induction by paired pre- and postsynaptic neuronal activity. LTP was successfully induced in about half of the synaptic connections tested and resulted in increased synaptic short-term depression during high-frequency transmission and a decelerated recovery from short-term depression due to an increased fraction of a slow recovery component. Analysis with a recently established sequential two-step vesicle priming model indicates an increase in the abundance of fully-primed and slowly-recovering vesicles. A systematic analysis of short-term plasticity and synapse-to-synapse variability of synaptic strength at various types of synapses revealed that stronger synapses generally recover more slowly from synaptic short-term depression. Finally, pharmacological stimulation of the cyclic adenosine monophosphate and diacylglycerol signaling pathways, which are both known to promote synaptic vesicle priming, mimicked LTP and slowed the recovery from short-term depression. Our data thus demonstrate that LTP at layer 5 pyramidal neurons increases synaptic strength primarily by enlarging a subpool of fully-primed slowly-recovering vesicles.

The composition of voltage-gated Ca 2+ channel (Ca v ) subtypes that gate action potential (AP)-evoked release changes during the development of mammalian CNS synapses. Ca v 2.2 and Ca v 2.3 lose their function in gating-evoked release during postnatal synapse maturation. In mature boutons, Ca v 2.1 currents provide the almost exclusive trigger for evoked release, and Ca v 2.3 currents are required for the induction of presynaptic long-term potentiation. However, the functional significance of Ca v 2.2 remained elusive in mature boutons, although they remain present at active zones and continue contributing significantly to presynaptic Ca 2+ influx. Here, we addressed the functional significance of Ca v 2.2 and Ca v 2.3 at mature parallel-fiber (PF) to Purkinje neuron synapses of mice of either sex. These synapses are known to exhibit the corresponding developmental Ca v subtype changes in gating release. We addressed two hypotheses, namely that Ca v 2.2 and Ca v 2.3 are involved in triggering spontaneous glutamate release and that they are engaged in vesicle recruitment during repetitive evoked release. We found that spontaneous miniature release is Ca 2+ dependent. However, experiments with Ca v subtype-specific blockers excluded the spontaneous opening of Ca v s as the Ca 2+ source for spontaneous glutamate release. Thus, neither Ca v 2.2 nor Ca v 2.3 controls spontaneous release from PF boutons. Furthermore, vesicle recruitment during brief bursts of APs was also independent of Ca 2+ influx through Ca v 2.2 and Ca v 2.3. However, Ca v 2.2, but not Ca v 2.3, currents significantly boosted vesicle recruitment during sustained high-frequency synaptic transmission. Thus, in mature PF boutons Ca v 2.2 channels are specifically required to sustain synaptic transmission during prolonged neuronal activity. SIGNIFICANCE STATEMENT At young CNS synapses, action potential-evoked release is gated via three subtypes of voltage-gated Ca 2+ channels: Ca v 2.1, Ca v 2.2, and Ca v 2.3. During postnatal maturation, Ca v 2.2 and Ca v 2.3 lose their function in gating evoked release, such that at mature synapses Ca v 2.1 provides the almost exclusive source for triggering evoked release. Ca v 2.3 currents are required for the induction of presynaptic long-term potentiation. However, the function of the still abundant Ca v 2.2 in mature boutons remained largely elusive. Here, we studied mature cerebellar parallel-fiber synapses and found that Ca v 2.2 does not control spontaneous release. However, Ca 2+ influx through Ca v 2.2 significantly boosted vesicle recruitment during trains of action potentials. Thus, Ca v 2.2 in mature parallel-fiber boutons participate in sustaining synaptic transmission during prolonged activity., (Copyright © 2023 the authors.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The efficacy of neocortical synapses to transmit during bursts of action potentials (APs) increases during development but the underlying mechanisms are largely unclear. We investigated synaptic efficacy at synapses between layer 5 pyramidal neurons (L5PNs) during development, using paired recordings, presynaptic two-photon Ca 2+ imaging, and numerical simulations. Our data confirm a developmental increase in paired-pulse ratios (PPRs). Independent of age, Ca 2+ imaging revealed no AP invasion failures and linear summation of presynaptic Ca 2+ transients, making differences in Ca 2+ signaling an unlikely reason for developmental changes in PPR. Cumulative excitatory postsynaptic current (EPSC) amplitudes indicate that neither the size of the readily-releasable pool (RRP) nor replenishment rates were different between age groups, while the time-courses of depression differed significantly. At young synapses, EPSCs depressed rapidly to near steady-state during the first four APs, and synaptic failures (F syn ) increased from 0 to 30%. At mature synapses this drop was significantly slower and strongly biphasic, such that near steady-state depression was reached not before 18 APs with F syn remaining between 0 and 5%. While young synapses reliably transmitted during pairs of APs, albeit with strong depression, mature synapses maintained near 100% transfer efficacy with significantly less depression during high-frequency bursts of APs. Our analysis indicates that at mature synapses a replenishment pool (RepP) is responsible for their high efficacy during bursting activity, while this RepP is functionally immature at young synapses. Hence, our data provide evidence that the functional maturation of a RepP underlies increasing synaptic efficacy during the development of an excitatory cortical synapse., (Copyright © 2020 Bornschein, Brachtendorf and Schmidt.)

Coupling distances between Ca 2+ channels and release sensors regulate vesicular release probability (p v ). Tight coupling is thought to provide a framework for high p v and loose coupling for high plasticity at low p v . At synapses investigated during development, coupling distances decrease, thereby increasing p v and transmission fidelity. We find that neocortical high-fidelity synapses deviate from these rules. Paired recordings from pyramidal neurons with "slow" and "fast" Ca 2+ chelators combined with experimentally constrained simulations suggest that coupling tightens significantly during development. However, fluctuation analysis revealed that neither p v (∼0.63) nor the number of release sites (∼8) changes concomitantly. Moreover, the amplitude and time course of presynaptic Ca 2+ transients are not different between age groups. These results are explained by high-p v release sites with Ca 2+ microdomains in young synapses and nanodomains in mature synapses. Thus, at neocortical synapses, a developmental reorganization of the active zone leaves p v unaffected, emphasizing developmental and functional synaptic diversity., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Ca 2+ concentrations drop rapidly over a distance of a few tens of nanometers from an open voltage-gated Ca 2+ channel (Ca v ), thereby, generating a spatially steep and temporally short-lived Ca 2+ gradient that triggers exocytosis of a neurotransmitter filled synaptic vesicle. These non-steady state conditions make the Ca 2+ -binding kinetics of the Ca 2+ sensors for release and their spatial coupling to the Ca v s important parameters of synaptic efficacy. In the mammalian central nervous system, the main release sensors linking action potential mediated Ca 2+ influx to synchronous release are Synaptotagmin (Syt) 1 and 2. We review here quantitative work focusing on the Ca 2+ kinetics of Syt2-mediated release. At present similar quantitative detail is lacking for Syt1-mediated release. In addition to triggering release, Ca 2+ remaining bound to Syt after the first of two successive high-frequency activations was found to be capable of facilitating release during the second activation. More recently, the Ca 2+ sensor Syt7 was identified as additional facilitation sensor. We further review how several recent functional studies provided quantitative insights into the spatial topographical relationships between Syts and Ca v s and identified mechanisms regulating the sensor-to-channel coupling distances at presynaptic active zones. Most synapses analyzed in matured cortical structures were found to operate at tight, nanodomain coupling. For fast signaling synapses a developmental switch from loose, microdomain to tight, nanodomain coupling was found. The protein Septin5 has been known for some time as a developmentally down-regulated "inhibitor" of tight coupling, while Munc13-3 was found only recently to function as a developmentally up-regulated mediator of tight coupling. On the other hand, a highly plastic synapse was found to operate at loose coupling in the matured hippocampus. Together these findings suggest that the coupling topography and its regulation is a specificity of the type of synapse. However, to definitely draw such conclusion our knowledge of functional active zone topographies of different types of synapses in different areas of the mammalian brain is too incomplete.

Spatial relationships between Ca v channels and release sensors at active zones (AZs) are a major determinant of synaptic fidelity. They are regulated developmentally, but the underlying molecular mechanisms are largely unclear. Here, we show that Munc13-3 regulates the density of Ca v 2.1 and Ca v 2.2 channels, alters the localization of Ca v 2.1, and is required for the development of tight, nanodomain coupling at parallel-fiber AZs. We combined EGTA application and Ca 2+ -channel pharmacology in electrophysiological and two-photon Ca 2+ imaging experiments with quantitative freeze-fracture immunoelectron microscopy and mathematical modeling. We found that a normally occurring developmental shift from release being dominated by Ca 2+ influx through Ca v 2.1 and Ca v 2.2 channels with domain overlap and loose coupling (microdomains) to a nanodomain Ca v 2.1 to sensor coupling is impaired in Munc13-3-deficient synapses. Thus, at AZs lacking Munc13-3, release remained triggered by Ca v 2.1 and Ca v 2.2 microdomains, suggesting a critical role of Munc13-3 in the formation of release sites with calcium channel nanodomains., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Diminished parasympathetic reactivity is a physiological feature of posttraumatic stress disorder (PTSD). The objective of this study was to compare female PTSD patients with non-traumatized healthy women with respect to autonomic cardiovascular regulation during exposure to two stressors. Hospitalized PTSD patients (n = 52) and controls (n = 37) completed standardized laboratory-based stress testing including a mental arithmetic test and a standardized audiotape recording of a crying infant. Controls and PTSD patients both showed a significantly increased heart rate and reduced pre-ejection period from baseline rest to the arithmetic stressor. However, as judged from nonsignificant changes in baroreflex sensitivity, parasympathetic activation caused by the crying infant stressor was blunted in PTSD patients as compared to healthy individuals. Under the crying infant condition, a vagal dominance was observed only in controls, and not in PTSD patients. Our data demonstrate that, in PTSD patients, diminished parasympathetic reactivity is not restricted to trauma-related events but can also be observed in response to a social stimulus such as listening to a crying infant. This finding suggests that the altered vagal reactivity in PTSD patients reflects the physiological consequences of a generally disturbed autonomous response to emotionally relevant stressors.

Paired-pulse facilitation (PPF) is a dynamic enhancement of transmitter release considered crucial in CNS information processing. The mechanisms of PPF remain controversial and may differ between synapses. Endogenous Ca(2+) buffers such as parvalbumin (PV) and calbindin-D28k (CB) are regarded as important modulators of PPF, with PV acting as an anti-facilitating buffer while saturation of CB can promote PPF. We analysed transmitter release and PPF at intracortical, recurrent Purkinje neuron (PN) to PN synapses, which show PPF during high-frequency activation (200 Hz) and strongly express both PV and CB. We quantified presynaptic Ca(2+) dynamics and quantal release parameters in wild-type (WT), and CB and PV deficient mice. Lack of CB resulted in increased volume averaged presynaptic Ca(2+) amplitudes and in increased release probability, while loss of PV had no significant effect on these parameters. Unexpectedly, none of the buffers significantly influenced PPF, indicating that neither CB saturation nor residual free Ca(2+) ([Ca(2+)]res) was the main determinant of PPF. Experimentally constrained, numerical simulations of Ca(2+)-dependent release were used to estimate the contributions of [Ca(2+)]res, CB, PV, calmodulin (CaM), immobile buffer fractions and Ca(2+) remaining bound to the release sensor after the first of two action potentials ('active Ca(2+)') to PPF. This analysis indicates that PPF at PN-PN synapses does not result from either buffer saturation or [Ca(2+)]res but rather from slow Ca(2+) unbinding from the release sensor.

Astrocytes operate in close spatial relationship to other cells including neurons. Structural interaction is controlled by a dynamic interplay between actin-based cell motility and contact formation via cell-cell and cell-extracellular matrix adhesions. A central player in the control of cell adhesion is the cytoskeletal adaptor protein Vinculin. Incorporation of Vinculin affects mechanical properties and turnover of cell adhesion sites. To study the in vivo function of Vinculin in astrocytes, a mouse line with astrocyte specific and inducible deletion of vinculin was generated. Deletion of vinculin decreased the expression of the glial acidic fibrillary protein (GFAP) in Bergmann glial cells in the cerebellum. In addition, localization of GFAP to Bergmann glial endfeet was disturbed, indicating a role for vinculin in controlling its expression and localization. In contrast, vimentin expression, morphology, activation state and polarity of the targeted cells as well as the localization of the extracellular matrix protein laminin was not compromised. Furthermore, stab wound lesions were performed in the cerebellar cortex. In both wildtype and vinculin knockout mice GFAP expression was upregulated in Bergmann glial cells of the lesioned area with no differences observed between genotypes in expression and localization of GFAP. These results propose a selective requirement for vinculin in cellular events related to cell adhesion in vivo. As in vitro data suggested a major role for vinculin in the control of the cytoskeletal connection affecting mechanical stability and cell motility, our data add a note of caution to the extrapolation of in vitro data to in vivo function., (Copyright © 2013 Wiley Periodicals, Inc.)

The coupling distance between presynaptic Ca(2+) influx and the sensor for vesicular transmitter release determines speed and reliability of synaptic transmission. Nanodomain coupling (<100 nm) favors fidelity and is employed by synapses specialized for escape reflexes and by inhibitory synapses involved in synchronizing fast network oscillations. Cortical glutamatergic synapses seem to forgo the benefits of tight coupling, yet quantitative detail is lacking. The reduced transmission fidelity of loose coupling, however, raises the question whether it is indeed a general characteristic of cortical synapses. Here we analyzed excitatory parallel fiber to Purkinje cell synapses, major processing sites for sensory information and well suited for analysis because they typically harbor only a single active zone. We quantified the coupling distance by combining multiprobability fluctuation analyses, presynaptic Ca(2+) imaging, and reaction-diffusion simulations in wild-type and calretinin-deficient mice. We found a coupling distance of <30 nm at these synapses, much shorter than at any other glutamatergic cortical synapse investigated to date. Our results suggest that nanodomain coupling is a general characteristic of conventional cortical synapses involved in high-frequency transmission, allowing for dense gray matter packing and cost-effective neurotransmission., (Copyright © 2013 Elsevier Ltd. All rights reserved.)