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Activity-dependent modulation of endocytosis by calmodulin at a large central synapse.

ABSTRACT: Although Ca(2+)/calmodulin has been suggested to play a role during endocytosis, it remains unknown if binding of Ca(2+) to calmodulin is essential for initiating endocytosis or if this interaction only has a modulatory effect on endocytosis. In this study, using time-resolved capacitance measurements at the rat calyx of Held synapse, the role of calmodulin in endocytosis was examined. Our results demonstrate that blocking calmodulin with an inhibitory peptide, which interfers with the binding of calmodulin to downstream targets, slowed the rate of endocytosis, but only when accompanied by high Ca(2+) influx. In response to a short train of action potential-like stimulation, blocking calmodulin had no effect on endocytosis. Furthermore, we have identified conditions in which inhibition of calmodulin fails to affect the rate of endocytosis, but nevertheless retards recruitment of synaptic vesicles to the fast-releasing vesicle pool responsible for synchronous release. The results indicate that calmodulin facilitates endocytosis in an activity-dependent manner but is not mandatory for endocytosis, and suggest that calmodulin modulates an endocytotic intermediate process, which in turn affects synaptic vesicle recruitment and membrane fission.

SUBMITTER: Yao L

PROVIDER: S-EPMC3252931 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Activity-dependent acceleration of endocytosis at a central synapse.

Project description:Accumulated evidence indicates the existence of rapid and slow endocytosis at many synapses. It has been proposed that rapid endocytosis is activated by intense stimulation when vesicle recycling needs to be speeded up to supply vesicles at hippocampal synapses. However, the evidence, as obtained with imaging techniques, which are somewhat indirect in indicating rapid endocytosis, is controversial. Furthermore, a slower time course of endocytosis is often found after more intense nerve activity, casting doubt on the role of rapid endocytosis at synapses. Here, we addressed this issue at a mammalian central synapse, the calyx of Held, using a capacitance measurement technique that provides a higher time resolution than imaging techniques. We found that rapid endocytosis with a time constant of approximately 1-2 s was activated during intense nerve activity. Reducing the presynaptic calcium current or buffering the intracellular calcium with EGTA significantly inhibited rapid endocytosis, suggesting that calcium triggers rapid endocytosis. During intense stimulation, rapid endocytosis retrieved up to approximately eight vesicles per second per active zone, approximately eightfold larger than reported in the hippocampus, and thus played a dominant role during and within 3 s after intense stimulation. Slow endocytosis became dominant 3 s after intense stimulation likely because of the fall of the intracellular calcium level that deactivated rapid endocytosis. These results underscore the importance of calcium-triggered rapid endocytosis, which offers the nerve terminal the plasticity to speed up vesicle cycling during intense nerve activity.

| S-EPMC1378116 | biostudies-literature

Activity-dependent modulation of synapse-regulating genes in astrocytes.

Project description:Astrocytes regulate the formation and function of neuronal synapses via multiple signals; however, what controls regional and temporal expression of these signals during development is unknown. We determined the expression profile of astrocyte synapse-regulating genes in the developing mouse visual cortex, identifying astrocyte signals that show differential temporal and layer-enriched expression. These patterns are not intrinsic to astrocytes, but regulated by visually evoked neuronal activity, as they are absent in mice lacking glutamate release from thalamocortical terminals. Consequently, synapses remain immature. Expression of synapse-regulating genes and synaptic development is also altered when astrocyte signaling is blunted by diminishing calcium release from astrocyte stores. Single-nucleus RNA sequencing identified groups of astrocytic genes regulated by neuronal and astrocyte activity, and a cassette of genes that show layer-specific enrichment. Thus, the development of cortical circuits requires coordinated signaling between astrocytes and neurons, highlighting astrocytes as a target to manipulate in neurodevelopmental disorders.

| S-EPMC8497060 | biostudies-literature

Activity-dependent modulation of synapse-regulating genes in astrocytes

Project description:Analysis of astrocyte heterogeneity in the developing mouse visual cortex, and identification of genes regulated by neuronal and astrocyte activity

2021-10-07 | GSE163775 | GEO

Essential function of alpha-calcium/calmodulin-dependent protein kinase II in neurotransmitter release at a glutamatergic central synapse.

Project description:A significant fraction of the total calciumcalmodulin-dependent protein kinase II (CaMKII) activity in neurons is associated with synaptic connections and is present in nerve terminals, thus suggesting a role for CaMKII in neurotransmitter release. To determine whether CaMKII regulates neurotransmitter release, we generated and analyzed knockout mice in which the dominant alpha-isoform of CaMKII was specifically deleted from the presynaptic side of the CA3-CA1 hippocampal synapse. Conditional CA3 alpha-CaMKII knockout mice exhibited an unchanged basal probability of neurotransmitter release at CA3-CA1 synapses but showed a significant enhancement in the activity-dependent increase in probability of release during repetitive presynaptic stimulation, as was shown with the analysis of unitary synaptic currents. These data indicate that alpha-CaMKII serves as a negative activity-dependent regulator of neurotransmitter release at hippocampal synapses and maintains synapses in an optimal range of release probabilities necessary for normal synaptic operation.

| S-EPMC153083 | biostudies-literature

Activity-dependent bulk endocytosis and clathrin-dependent endocytosis replenish specific synaptic vesicle pools in central nerve terminals.

Project description:Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high- and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

| S-EPMC2889610 | biostudies-other

Cholesterol regulates multiple forms of vesicle endocytosis at a mammalian central synapse.

Project description:Endocytosis in synapses sustains neurotransmission by recycling vesicle membrane and maintaining the homeostasis of synaptic membrane. A role of membrane cholesterol in synaptic endocytosis remains controversial because of conflicting observations, technical limitations in previous studies, and potential interference from non-specific effects after cholesterol manipulation. Furthermore, it remains unclear whether cholesterol participates in distinct forms of endocytosis that function under different activity levels. In this study, applying the whole-cell membrane capacitance measurement to monitor endocytosis in real time at the rat calyx of Held terminals, we found that disrupting cholesterol with dialysis of cholesterol oxidase or methyl-β-cyclodextrin impaired three different forms of endocytosis, including slow endocytosis, rapid endocytosis, and endocytosis of the retrievable membrane that exists at the surface before stimulation. The effects were observed when disruption of cholesterol was mild enough not to change Ca(2+) channel current or vesicle exocytosis, indicative of stringent cholesterol requirement in synaptic endocytosis. Extracting cholesterol with high concentrations of methyl-β-cyclodextrin reduced exocytosis, mainly by decreasing the readily releasable pool and the vesicle replenishment after readily releasable pool depletion. Our study suggests that cholesterol is an important, universal regulator in multiple forms of vesicle endocytosis at mammalian central synapses.

| S-EPMC4490978 | biostudies-literature

Activity-dependent modulation of synapse-regulating genes in astrocytes

Project description:Activity-dependent modulation of synapse-regulating genes in astrocytes

| PRJNA687513 | ENA

Selective saturation of slow endocytosis at a giant glutamatergic central synapse lacking dynamin 1.

Project description:Exocytosis of synaptic vesicles is rapidly followed by compensatory plasma membrane endocytosis. The efficiency of endocytosis varies with experimental conditions, but the molecular basis for this control remains poorly understood. Here, the function of dynamin 1, the neuron-specific member of a family of GTPases implicated in vesicle fission, was investigated with high temporal resolution via membrane capacitance measurements at the calyx of Held, a giant glutamatergic synapse. Endocytosis at dynamin 1 KO calyces was the same as in wild type after weak stimuli, consistent with the nearly normal ultrastructure of mutant synapses. However, following stronger stimuli, the speed of slow endocytosis, but not of other forms of endocytosis, failed to scale with the increased endocytic load. Thus, high level expression of dynamin 1 is essential to allow the slow, clathrin-mediated endocytosis, which accounts for the bulk of the endocytic response, to operate efficiently over a wide range of activity.

| S-EPMC2579887 | biostudies-literature

Rapid bulk endocytosis and its kinetics of fission pore closure at a central synapse.

Project description:Upon exocytosis, fused vesicles must be retrieved for recycling. One route of retrieval is to generate endosome-like structures, from which small vesicles bud off. Endosome-like structures are widely thought to be generated slowly ( approximately 1 min) from the plasma membrane, a process called bulk endocytosis. Although the concept of bulk endocytosis seems established, the kinetic evidence showing the instant of the bulk membrane fission at synapses is still missing. The present work provides this missing piece of evidence at a calyx-type synapse. We used the capacitance measurement technique, which offers a high time resolution ( approximately 1 ms) to resolve the fission process. The instant of bulk membrane fission was reflected as a brief downward capacitance shift (DCS) of approximately 20-500 fF (mean = 123 fF) with 10-90% decay time of approximately 30-500 ms. At least 8.6-11.0% of exocytosed vesicles were retrieved by DCSs. During a DCS, the decrease in the fission pore conductance was detected, from which we found that the fission pore diameter decreased at approximately 39 nm/s. This provided the measurement of the rate of fission during bulk endocytosis at synapses. The DCS frequency peaked (approximately equal to 0.021 Hz) in <10 s after stimulation and decayed with a half time <20 s, indicating that the time course of bulk endocytosis is much faster than previously estimated with low time-resolution techniques. Our results also suggest that bulk endocytosis was composed of two kinetically different steps: the DCS that reflected the fission process and the time between stimulation and the DCS, during which membrane invagination led to the fission pore formation.

| S-EPMC1891249 | biostudies-literature

The role of calcium/calmodulin-activated calcineurin in rapid and slow endocytosis at central synapses.

Project description:Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses-slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knock-out of calcineurin. These manipulations significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7- to 10-d-old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These results suggest that calcium influx during nerve firing activates calcium/calmodulin-dependent calcineurin, which controls the speed of both rapid and slow endocytosis at synapses by dephosphorylating endocytic proteins. The calcium/calmodulin/calcineurin signaling pathway may underlie regulation of endocytosis by nerve activity and calcium as reported at many synapses over the last several decades.

| S-EPMC2954493 | biostudies-literature

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