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Sphingolipids are important for the physicochemical properties of cellular membranes and deregulated in tumors. In human colon cancer tissue ceramide synthase (CerS) 4 and CerS5 are reduced which correlates with a reduced survival probability of late-stage colon cancer patients. Both enzymes are reduced after hypoxia in advanced colorectal cancer (CRC) cells (HCT-116, SW620) but not in non-metastatic CRC cells (SW480, Caco-2). Downregulation of CerS4 or CerS5 in advanced CRC cells enhanced tumor formation in nude mice and organoid growth in vitro. This was accompanied by an enhanced proliferation rate and metabolic changes leading to a shift towards the Warburg effect. In contrast, CerS4 or CerS5 depletion in Caco-2 cells reduced tumor growth in vivo. Lipidomic and proteomic analysis of membrane fractions revealed significant changes in tumor-promoting cellular pathways and cellular transporters. This study identifies CerS4 and CerS5 as prognostic markers for advanced colon cancer patients and provides a comprehensive overview about the associated cellular metabolic changes. We propose that the expression level of CerS4 and CerS5 in colon tumors could serve as a basis for decision-making for personalized treatment of advanced colon cancer patients. Trial registration: The study was accredited by the study board of the Deutsche Krebsgesellschaft (Registration No: St-D203, 2017/06/30, retrospectively registered)., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Klaus Scholich reports financial support was provided by German Research Foundation. Andreas Weigert reports financial support was provided by German Research Foundation. Bernhard Brune reports financial support was provided by German Research Foundation. Gerd Geisslinger reports financial support was provided by German Research Foundation. Ilka Wittig reports financial support was provided by German Research Foundation. Sabine Grosch reports financial support was provided by Fraunhofer Cluster of Excellence for Immune-Mediated Diseases. Sabine Grosch reports financial support was provided by German Research Foundation. Thomas Deller reports financial support was provided by Novartis AG., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

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.)

To better understand the role of sphingolipids in the multifactorial process of inflammatory bowel disease (IBD), we elucidated the role of CerS4 in colitis and colitis-associated cancer (CAC). For this, we utilized the azoxymethane/dextran sodium sulphate (AOM/DSS)-induced colitis model in global CerS4 knockout (CerS4 KO), intestinal epithelial (CerS4 Vil/Cre), or T-cell restricted knockout (CerS4 LCK/Cre) mice. CerS4 KO mice were highly sensitive to the toxic effect of AOM/DSS, leading to a high mortality rate. CerS4 Vil/Cre mice had smaller tumors than WT mice. In contrast, CerS4 LCK/Cre mice frequently suffered from pancolitis and developed more colon tumors. In vitro, CerS4-depleted CD8+ T-cells isolated from the thymi of CerS4 LCK/Cre mice showed impaired proliferation and prolonged cytokine production after stimulation in comparison with T-cells from WT mice. Depletion of CerS4 in human Jurkat T-cells led to a constitutively activated T-cell receptor and NF-κB signaling pathway. In conclusion, the deficiency of CerS4 in T-cells led to an enduring active status of these cells and prevents the resolution of inflammation, leading to a higher tumor burden in the CAC mouse model. In contrast, CerS4 deficiency in epithelial cells resulted in smaller colon tumors and seemed to be beneficial. The higher tumor incidence in CerS4 LCK/Cre mice and the toxic effect of AOM/DSS in CerS4 KO mice exhibited the importance of CerS4 in other tissues and revealed the complexity of general targeting CerS4.

BNT162b2, a nucleoside-modified mRNA formulated in lipid nanoparticles that encodes the SARS-CoV-2 spike glycoprotein (S) stabilized in its prefusion conformation, has demonstrated 95% efficacy in preventing COVID-19 1 . Here we extend a previous phase-I/II trial report 2 by presenting data on the immune response induced by BNT162b2 prime-boost vaccination from an additional phase-I/II trial in healthy adults (18-55 years old). BNT162b2 elicited strong antibody responses: at one week after the boost, SARS-CoV-2 serum geometric mean 50% neutralizing titres were up to 3.3-fold above those observed in samples from individuals who had recovered from COVID-19. Sera elicited by BNT162b2 neutralized 22 pseudoviruses bearing the S of different SARS-CoV-2 variants. Most participants had a strong response of IFNγ + or IL-2 + CD8 + and CD4 + T helper type 1 cells, which was detectable throughout the full observation period of nine weeks following the boost. Using peptide-MHC multimer technology, we identified several BNT162b2-induced epitopes that were presented by frequent MHC alleles and conserved in mutant strains. One week after the boost, epitope-specific CD8 + T cells of the early-differentiated effector-memory phenotype comprised 0.02-2.92% of total circulating CD8 + T cells and were detectable (0.01-0.28%) eight weeks later. In summary, BNT162b2 elicits an adaptive humoral and poly-specific cellular immune response against epitopes that are conserved in a broad range of variants, at well-tolerated doses., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Pruning, the elimination of excess synapses is a phenomenon of fundamental importance for correct wiring of the central nervous system. The establishment of the cerebellar climbing fiber (CF)-to-Purkinje cell (PC) synapse provides a suitable model to study pruning and pruning-relevant processes during early postnatal development. Until now, the role of microglia in pruning remains under intense investigation. Here, we analyzed migration of microglia into the cerebellar cortex during early postnatal development and their possible contribution to the elimination of CF-to-PC synapses. Microglia enrich in the PC layer at pruning-relevant time points giving rise to the possibility that microglia are actively involved in synaptic pruning. We investigated the contribution of microglial fractalkine (CX 3 CR1) signaling during postnatal development using genetic ablation of the CX 3 CR1 receptor and an in-depth histological analysis of the cerebellar cortex. We found an aberrant migration of microglia into the granule and the molecular layer. By electrophysiological analysis, we show that defective fractalkine signaling and the associated migration deficits neither affect the pruning of excess CFs nor the development of functional parallel fiber and inhibitory synapses with PCs. These findings indicate that CX 3 CR1 signaling is not mandatory for correct cerebellar circuit formation. MAIN POINTS: Ablation of CX 3 CR1 results in a transient migration defect in cerebellar microglia. CX 3 CR1 is not required for functional pruning of cerebellar climbing fibers. Functional inhibitory and parallel fiber synapse development with Purkinje cells is undisturbed in CX 3 CR1-deficient mice., (© 2020 The Authors. Glia published by Wiley Periodicals, Inc.)

An effective vaccine is needed to halt the spread of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic. Recently, we reported safety, tolerability and antibody response data from an ongoing placebo-controlled, observer-blinded phase I/II coronavirus disease 2019 (COVID-19) vaccine trial with BNT162b1, a lipid nanoparticle-formulated nucleoside-modified mRNA that encodes the receptor binding domain (RBD) of the SARS-CoV-2 spike protein 1 . Here we present antibody and T cell responses after vaccination with BNT162b1 from a second, non-randomized open-label phase I/II trial in healthy adults, 18-55 years of age. Two doses of 1-50 μg of BNT162b1 elicited robust CD4 + and CD8 + T cell responses and strong antibody responses, with RBD-binding IgG concentrations clearly above those seen in serum from a cohort of individuals who had recovered from COVID-19. Geometric mean titres of SARS-CoV-2 serum-neutralizing antibodies on day 43 were 0.7-fold (1-μg dose) to 3.5-fold (50-μg dose) those of the recovered individuals. Immune sera broadly neutralized pseudoviruses with diverse SARS-CoV-2 spike variants. Most participants had T helper type 1 (T H 1)-skewed T cell immune responses with RBD-specific CD8 + and CD4 + T cell expansion. Interferon-γ was produced by a large fraction of RBD-specific CD8 + and CD4 + T cells. The robust RBD-specific antibody, T cell and favourable cytokine responses induced by the BNT162b1 mRNA vaccine suggest that it has the potential to protect against COVID-19 through multiple beneficial mechanisms.

Ceramide synthase 5 is one of six enzymes that catalyze the production of ceramides from sphingosine or sphinganine. Ceramides are important components of cell membranes and act as signaling molecules. Previously it has been shown that ceramide synthase 6 and 2 influence colitis in several animal models with sometimes opposite effects. Here, we investigated the disease course of dextran sodium sulfate-induced acute colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer in mice with global ceramide synthase 5 knockout (CerS5-ko) or with ceramide synthase 5 knockout restricted to the colon epithelium (CerS5fl/fl VilCre). We monitored disease development and analyzed colon barrier function as well as the immune cell status in these mice. CerS5-ko mice but not CerS5fl/fl-VilCre mice were more susceptible to acute and chronic inflammation. However, the cell barrier function of colon epithelial cells was not disturbed by downregulation of ceramide synthase 5. Instead, untreated CerS5-ko mice displayed reduced numbers of CD3 + immune cells in the spleen, colon, and blood, especially of intraepithelial CD8 + T-cells, which was not obvious in CerS5fl/fl Vil Cre mice. Reduced T-cell number in colon tissue of CerS5-ko mice was accompanied by a reduced expression of IL-1β, IFNγ, and IL-4. In vitro investigations revealed that knockdown of ceramide synthase 5 in T-cells impaired T-cell activation. In summary, we show that CerS5-ko mice were more susceptible to dextran sodium sulfate-induced colitis and azoxymethane/dextran sodium sulfate-induced colitis-associated colon cancer. A reduced number of T-cells in the colon epithelium that was already the case in untreated CerS5-ko mice might have contributed to this effect.

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.)

The G protein-coupled estrogen receptor 1 (GPER1) is involved in the regulation of physiological processes such as cellular growth and proliferation, but also in pathophysiological processes such as tumor development. The role of GPER1 in breast cancer is contradictory. Therefore, we investigated the influence of GPER1 overexpression on cellular processes in MCF-7 breast cancer cells. GPER1 overexpression leads to a cell cycle arrest in the G1 phase, induction of autophagy and reduced proliferation. Reduced proliferation was accompanied by a reduced basal respiration and reduced glycolysis rate in GPER1 overexpressing cells. This is presumably ascribable to mitophagy induction following GPER1 overexpression. However, GPER1 overexpressing cells were less sensitive against doxorubicin as compared to control cells. In previous work we showed the effect of transient GPER1 overexpression on the synthesis of several ceramide synthases (CerS) thereby influencing the sphingolipid pathway. Therefore, we investigated CerS expression and sphingolipid level in stable GPER1 overexpressing and control cells. Stable GPER1 overexpression strongly reduced CerS4, CerS5 and CerS6 promoter activity and CerS5 and CerS6 mRNA expression, whereas CerS2 mRNA expression was upregulated. The GPER1 effect on CerS5 promoter is mediated by GSK-3β signaling. In addition, other enzymes of the sphingolipid pathway were upregulated. Our study provides new insights into the role of GPER1 and the activated sphingolipid pathways and how GPER1 may influence cellular processes such as cancer cell survival following chemotherapy. Further studies are needed to investigate the molecular mechanisms leading to these cellular effects. Finding new therapeutic targets for modulating specifically GPER1 in breast tumors may improve endocrine breast cancer therapy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The postnatal development of cerebellar climbing fiber (CF) to Purkinje neuron (PN) synapses is characterized by a substantial pruning during the first 3 weeks after birth, switching from multiple- to single-CF innervation. Previous studies suggested that CF maturation is governed by bidirectional changes of synaptic plasticity. The strengthening of surviving "winner" CFs, which translocate from the PN soma to the dendrite, is thought to be guided by long-term potentiation (LTP), while weakening of to-be-eliminated "loser" CFs, which remain on the soma, was proposed to be due to long-term depression (LTD). However, there are conflicting results from previous studies, whether or not strengthening of winner and weakening of loser CFs during postnatal development is accompanied by changes in short-term plasticity and, thus, whether pre- or postsynaptic forms of LTD and LTP are operational. We, therefore, analyzed the developmental profile of paired-pulse depression (PPD) in "weak" and "strong" CFs in 3-21-day old Igsf9 -eGFP mice, which allow visual identification of GFP-labeled CFs. We found that in 3-8-day old mice strong CFs are marked by a stronger PPD compared to weak CFs. Surprisingly, PPD of strong CFs eases during maturation, while PPD in weak CFs remains unchanged. This easing of PPD is neither due to changes in presynaptic influx-release coupling nor to an increased saturation of postsynaptic receptors. Thus, our results imply that synaptic contacts of CFs show distinct features of PPD depending on their affiliation to winner or loser CFs and depending on their somatic or dendritic location.

Drug resistance is one major reason for failure of cancer therapy. In the past 10 years, evidence emerged showing that ceramides of specific chain length, generated by six different ceramide synthases (CerS), are deregulated in different cancer types thereby influencing chemosensitivity. In this review we sum up the cellular mechanisms regulated by CerS and the respective ceramides of specific chain length contributing to chemoresistance and how we can interfere with these mechanisms to overcome drug resistance by targeting CerS. We compile an overview of the different cellular effects influenced by CerS in dependency of the used drug and cancer type. Finally, the potential of CerS as new drug targets in chemotherapy or as biomarkers for the prediction of therapeutic response rates is discussed., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Resistance against chemotherapy is a life-threatening complication in colon cancer therapy. To increase response rate, new additional targets that contribute to chemoresistance are still needed to be explored. Ceramides, which belong to the group of sphingolipids, are well-known regulators of cell death and survival, respectively. Here, we show that in human wild-type ( wt ) p53 HCT-116 colon cancer cells treatment with oxaliplatin or 5-fluorouracil (5-FU) leads to a strong increase in ceramide synthase 5 (CerS5) expression and C 16:0 -ceramide levels, which was not shown in HCT-116 lacking p53 expression (HCT-116 p53 -/- ). The increase in CerS5 expression occurs by stabilizing CerS5 mRNA at the 3'-UTR. By contrast, in the p53-deficient cells CerS2 expression and CerS2-related C 24:0 - and C 24:1 -ceramide levels were elevated which is possibly related to enhanced polyadenylation of the CerS2 transcript in these cells. Stable knockdown of CerS5 expression using CerS5-targeting shRNA led to an increased sensitivity of HCT-116 p53 wt cells, but not of p53 -/- cells, to oxaliplatin and 5-FU. Enhanced sensitivity was accompanied by an inhibition of autophagy and inhibition of mitochondrial respiration in these cells. However, knockdown of CerS2 had no significant effects on chemosensitivity of both cell lines. In conclusion, in p53 wt colon cancer cells chemosensitivity against oxaliplatin or 5-FU could be enhanced by downregulation of CerS5 expression leading to reduced autophagy and mitochondrial respiration., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: The inferior olive (IO) innervates the cerebellum forming synapses in the deep cerebellar nuclei (DCN) and the cerebellar cortex. Beside the well-known exception of synapses on Purkinje neurons, synapses between IO efferents and other neuronal targets have not been studied intensively, mostly due to the technical challenge of unequivocally identifying IO efferents in electrophysiological experiments., New Method: We describe the transgenic mouse line Igsf9-eGFP, which expresses GFP in IO neurons, as a suitable tool for studying IO efferents using live imaging, immunohistochemistry and electrophysiology., Results: In the Igsf9-eGFP line, GFP-positive neurons are found in all IO subnuclei. Their efferents show the expected trajectories innervating the DCN and, as climbing fibers (CFs), the cerebellar cortex. In the DCN the dentate nucleus shows the strongest innervation, and, within the cerebellar cortex, CFs show a rostral-to-caudal gradient. GFP-positive CFs undergo a normal postnatal maturation, and evoke normal synaptic responses in Purkinje neurons and DCN neurons., Comparison With Existing Methods: IO efferents have been labelled via anterograde labelling, viral transfection and in transgenic mouse lines. The latter approach does not require stereotactic injections. However, available mouse lines show only a sparse GFP expression, harbor GFP-positive axons of other cerebellar fibers, or have not been characterized in detail., Conclusions: The Igsf9-eGFP line is characterized by a moderate density of GFP-positive IO efferents, which can be visually targeted for extracellular stimulation with micrometer precision. The mouse line will allow studying fiber-specific responses in all neurons targeted by the IO., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Endogenous Ca(2+)-binding proteins affect synaptic transmitter release and short-term plasticity (STP) by buffering presynaptic Ca(2+) signals. At parallel-fiber (PF)-to-Purkinje neuron (PN) synapses in the cerebellar cortex loss of calretinin (CR), the major buffer at PF terminals, results in increased presynaptic Ca(2+) transients and an almost doubling of the initial vesicular releases probability (p r). Surprisingly, however, it has been reported that loss of CR from PF synapses does not alter paired-pulse facilitation (PPF), while it affects presynaptic Ca(2+) signals as well as p r. Here, we addressed this puzzling observation by analyzing the frequency- and Ca(2+)-dependence of PPF at unitary PF-to-PN synapses of wild-type (WT) and CR-deficient (CR(-/-)) mice using paired recordings and computer simulations. Our analysis revealed that PPF in CR(-/-) is indeed smaller than in the WT, to a degree, however, that indicates that rapid vesicle replenishment and recruitment of additional release sites dominate the synaptic efficacy of the second response. These Ca(2+)-driven processes operate more effectively in the absence of CR, thereby, explaining the preservation of robust PPF in the mutants.

Ceramide synthases (CerS) are important enzymes of the sphingolipid pathway, responsible for the production of ceramides with distinct chain lengths. In human breast cancer tissue, we detected a significant increase in CerS4 and CerS6 mRNA in estrogen receptor positive (ER+) cancer tissue. To clarify the molecular mechanism of this upregulation, we cloned CerS2, -4, -5 and CerS6 promoter and 3'-UTR fragments into luciferase reporter gene plasmids and determined luciferase activity in MCF-7 (ERα/β) and MDA-MB-231 (ERβ) cells after 17β-estradiol treatment. Only the activities of CerS4 and CerS5 promoter Luc constructs, as well as CerS2- and CerS5-3'-UTR Luc constructs increased after estradiol treatment in MCF-7 cells, and this could be inhibited by the anti-estrogen fulvestrant. Co-transfection with the G protein-coupled estrogen receptor 1 (GPER1) also enhanced CerS2, CerS4 and CerS6 promoter activity whereas CerS5 promoter activity was inhibited in both cell lines. Promoter deletion and mutation constructs from CerS4 and CerS5 promoters revealed that estradiol and GPER1 mediate their effects on both promoters by activating AP-1, most likely through dimerization of c-Jun and c-Fos. At least we could show, that cell proliferation induced by estradiol could be blocked by co-treatment with Fumonisin B1, indicating that upregulation of CerS in breast cancer cells by estrogen is important for cell proliferation and possibly tumor development. In conclusion, our data highlight transcriptional and posttranscriptional mechanisms regulating CerS expression in human cells which provide the basis for further studies investigating the regulation of CerS expression and ceramide synthesis after diverse stimuli in physiological and pathophysiological processess., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

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.)