Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Tracking the dynamics of single fused synaptic vesicle proteins from a single ribbon active zone in zebrafish retinal bipolar cells.

ABSTRACT: Clearance of fused synaptic vesicle components and availability of release sites are important determinants of recovery from short-term synaptic depression. However, the dynamics of release site clearance are not well established. This protocol illustrates single-molecule imaging of an exocytosis reporter, synaptophysin-pHluorin fusion protein (SypHy), by combining two-color laser scanning confocal microscopy with whole-cell patch-clamp recording of retinal bipolar cells from transgenic zebrafish that weakly express SypHy to track the dynamics of newly fused vesicle proteins at the active zone. For complete details on the use and execution of this profile, please refer to Vaithianathan et al. (2019).

SUBMITTER: Shrestha AP

PROVIDER: S-EPMC8783205 | biostudies-literature | 2022 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Tracking the dynamics of single fused synaptic vesicle proteins from a single ribbon active zone in zebrafish retinal bipolar cells.

Shrestha Abhishek P AP Vaithianathan Thirumalini T

STAR protocols 20220120 1

Clearance of fused synaptic vesicle components and availability of release sites are important determinants of recovery from short-term synaptic depression. However, the dynamics of release site clearance are not well established. This protocol illustrates single-molecule imaging of an exocytosis reporter, synaptophysin-pHluorin fusion protein (SypHy), by combining two-color laser scanning confocal microscopy with whole-cell patch-clamp recording of retinal bipolar cells from transgenic zebrafis ...[more]

PMID: 35098163

Similar Datasets

Tracking Newly Released Synaptic Vesicle Proteins at Ribbon Active Zones.

Project description:Clearance of synaptic vesicle proteins from active zones may be rate limiting for sustained neurotransmission. Issues of clearance are critical at ribbon synapses, which continually release neurotransmitters for prolonged periods of time. We used synaptophysin-pHluorin (SypHy) to visualize protein clearance from active zones in retinal bipolar cell ribbon synapses. Depolarizing voltage steps gave rise to small step-like changes in fluorescence likely indicating release of single SypHy molecules from fused synaptic vesicles near active zones. Temporal and spatial fluorescence profiles of individual responses were highly variable, but ensemble averages were well fit by clearance via free diffusion using Monte Carlo simulations. The rate of fluorescence decay of ensemble averages varied with the time and location of the fusion event, with clearance being most rapid at the onset of a stimulus when release rate is the highest.

| S-EPMC6598641 | biostudies-literature

Kif1a and intact microtubules maintain synaptic-vesicle populations at ribbon synapses in zebrafish hair cells.

Project description:Sensory hair cells of the inner ear utilize specialized ribbon synapses to transmit sensory stimuli to the central nervous system. This sensory transmission necessitates rapid and sustained neurotransmitter release, which relies on a large pool of synaptic vesicles at the hair-cell presynapse. Work in neurons has shown that kinesin motor proteins traffic synaptic material along microtubules to the presynapse, but how new synaptic material reaches the presynapse in hair cells is not known. We show that the kinesin motor protein Kif1a and an intact microtubule network are necessary to enrich synaptic vesicles at the presynapse in hair cells. We use genetics and pharmacology to disrupt Kif1a function and impair microtubule networks in hair cells of the zebrafish lateral-line system. We find that these manipulations decrease synaptic-vesicle populations at the presynapse in hair cells. Using electron microscopy, along with in vivo calcium imaging and electrophysiology, we show that a diminished supply of synaptic vesicles adversely affects ribbon-synapse function. Kif1a mutants exhibit dramatic reductions in spontaneous vesicle release and evoked postsynaptic calcium responses. Additionally, we find that kif1a mutants exhibit impaired rheotaxis, a behavior reliant on the ability of hair cells in the lateral line to respond to sustained flow stimuli. Overall, our results demonstrate that Kif1a-based microtubule transport is critical to enrich synaptic vesicles at the active zone in hair cells, a process that is vital for proper ribbon-synapse function.

| S-EPMC11188139 | biostudies-literature

Acute destruction of the synaptic ribbon reveals a role for the ribbon in vesicle priming.

Project description:In vision, balance and hearing, sensory receptor cells translate sensory stimuli into electrical signals whose amplitude is graded with stimulus intensity. The output synapses of these sensory neurons must provide fast signaling to follow rapidly changing stimuli while also transmitting graded information covering a wide range of stimulus intensity and must be able to sustain this signaling for long time periods. To meet these demands, specialized machinery for transmitter release, the synaptic ribbon, has evolved at the synaptic outputs of these neurons. We found that acute disruption of synaptic ribbons by photodamage to the ribbon markedly reduced both sustained and transient components of neurotransmitter release in mouse bipolar cells and salamander cones without affecting the ultrastructure of the ribbon or its ability to localize synaptic vesicles to the active zone. Our results indicate that ribbons mediate both slow and fast signaling at sensory synapses and support an additional role for the synaptic ribbon in priming vesicles for exocytosis at active zones.

| S-EPMC3171202 | biostudies-literature

The mammalian rod synaptic ribbon is essential for Cav channel facilitation and ultrafast synaptic vesicle fusion.

Project description:Rod photoreceptors (PRs) use ribbon synapses to transmit visual information. To signal 'no light detected' they release glutamate continually to activate post-synaptic receptors. When light is detected glutamate release pauses. How a rod's individual ribbon enables this process was studied here by recording evoked changes in whole-cell membrane capacitance from wild-type and ribbonless (Ribeye-ko) mice. Wild-type rods filled with high (10 mM) or low (0.5 mM) concentrations of the Ca2+-buffer EGTA created a readily releasable pool (RRP) of 87 synaptic vesicles (SVs) that emptied as a single kinetic phase with a τ<0.4 ms. The lower concentration of EGTA accelerated Cav channel opening and facilitated release kinetics. In contrast, ribbonless rods created a much smaller RRP of 22 SVs, and they lacked Cav channel facilitation; however, Ca2+ channel-release coupling remained tight. These release deficits caused a sharp attenuation of rod-driven scotopic light responses. We conclude that the synaptic ribbon facilitates Ca2+-influx and establishes a large RRP of SVs.

| S-EPMC8594941 | biostudies-literature

Global Ca2+ signaling drives ribbon-independent synaptic transmission at rod bipolar cell synapses.

Project description:Ribbon-type presynaptic active zones are a hallmark of excitatory retinal synapses, and the ribbon organelle is thought to serve as the organizing point of the presynaptic active zone. Imaging of exocytosis from isolated retinal neurons, however, has revealed ectopic release (i.e., release away from ribbons) in significant quantities. Here, we demonstrate in an in vitro mouse retinal slice preparation that ribbon-independent release from rod bipolar cells activates postsynaptic AMPARs on AII amacrine cells. This form of release appears to draw on a unique, ribbon-independent, vesicle pool. Experimental, anatomical, and computational analyses indicate that it is elicited by a significant, global elevation of intraterminal [Ca(2+)] arising following local buffer saturation. Our observations support the conclusion that ribbon-independent release provides a read-out of the average behavior of all of the active zones in a rod bipolar cell's terminal.

| S-EPMC4004811 | biostudies-literature

Zebrafish Cacna1fa is required for cone photoreceptor function and synaptic ribbon formation.

Project description:Mutations in the human CACNA1F gene cause incomplete congenital stationary night blindness type 2 (CSNB2), a non-progressive, clinically heterogeneous retinal disorder. However, the molecular mechanisms underlying CSNB2 have not been fully explored. Here, we describe the positional cloning of a blind zebrafish mutant, wait until dark (wud), which encodes a zebrafish homolog of human CACNA1F. We identified two zebrafish cacna1f paralogs and showed that the cacna1fa transcript (the gene mutated in wud) is expressed exclusively in the photoreceptor layer. We demonstrated that Cacna1fa localizes at the photoreceptor synapse and is absent from wud mutants. Electroretinograms revealed abnormal cone photoreceptor responses from wud mutants, indicating a defect in synaptic transmission. Although there are no obvious morphological differences, we found that wud mutants lacked synaptic ribbons and that wud is essential for the development of synaptic ribbons. We found that Ribeye, the most prominent synaptic ribbon protein, was less abundant and mislocalized in adult wud mutants. In addition to cloning wud, we identified synaptojanin 1 (synj1) as the defective gene in slacker (slak), a blind mutant with floating synaptic ribbons. We determined that Cacna1fa was expressed in slak photoreceptors and that Synj1 was initially expressed wud photoreceptors, but was absent by 5 days postfertilization. Collectively, our data demonstrate that Cacna1fa is essential for cone photoreceptor function and synaptic ribbon formation and reveal a previously unknown yet critical role of L-type voltage-dependent calcium channels in the expression and/or distribution of synaptic ribbon proteins, providing a new model to study the clinical variability in human CSNB2 patients.

| S-EPMC4014194 | biostudies-literature

Ribbon synapses compute temporal contrast and encode luminance in retinal rod bipolar cells.

Project description:Contrast is computed throughout the nervous system to encode changing inputs efficiently. The retina encodes luminance and contrast over a wide range of visual conditions and must adapt its responses to maintain sensitivity and to avoid saturation. We examined the means by which one type of adaptation allows individual synapses to compute contrast and encode luminance in biphasic responses to step changes in light levels. Light-evoked depletion of the readily releasable vesicle pool (RRP) at rod bipolar cell ribbon synapses in rat retina limited the dynamic range available to encode transient, but not sustained, responses, thereby allowing the transient and sustained components of release to compute temporal contrast and encode mean light levels, respectively. A release/replenishment model revealed that a single, homogeneous pool of synaptic vesicles is sufficient to generate this behavior and that a partial depletion of the RRP is the dominant mechanism for shaping the biphasic contrast/luminance response.

| S-EPMC3225507 | biostudies-literature

Unique dynamics and exocytosis properties of GABAergic synaptic vesicles revealed by three-dimensional single vesicle tracking.

Project description:Maintaining the balance between neuronal excitation and inhibition is essential for proper function of the central nervous system. Inhibitory synaptic transmission plays an important role in maintaining this balance. Although inhibitory transmission has higher kinetic demands compared to excitatory transmission, its properties are poorly understood. In particular, the dynamics and exocytosis of single inhibitory vesicles have not been investigated, due largely to both technical and practical limitations. Using a combination of quantum dots (QDs) conjugated to antibodies against the luminal domain of the vesicular GABA transporter to selectively label GABAergic (i.e., predominantly inhibitory) vesicles together with dual-focus imaging optics, we tracked the real-time three-dimensional position of single GABAergic vesicles up to the moment of exocytosis (i.e., fusion). Using three-dimensional trajectories, we found that GABAergic synaptic vesicles traveled a shorter distance prior to fusion and had a shorter time to fusion compared to synaptotagmin-1 (Syt1)-labeled vesicles, which were mostly from excitatory neurons. Moreover, our analysis revealed that GABAergic synaptic vesicles move more straightly to their release sites than Syt1-labeled vesicles. Finally, we found that GABAergic vesicles have a higher prevalence of kiss-and-run fusion than Syt1-labeled vesicles. These results indicate that inhibitory synaptic vesicles have a unique set of dynamics and exocytosis properties to support rapid synaptic inhibition, thereby maintaining a tightly regulated coordination between excitation and inhibition in the central nervous system.

| S-EPMC7936280 | biostudies-literature

Morphofunctional changes at the active zone during synaptic vesicle exocytosis.

Project description:Synaptic vesicle (SV) fusion with the plasma membrane (PM) proceeds through intermediate steps that remain poorly resolved. The effect of persistent high or low exocytosis activity on intermediate steps remains unknown. Using spray-mixing plunge-freezing cryo-electron tomography we observe events following synaptic stimulation at nanometer resolution in near-native samples. Our data suggest that during the stage that immediately follows stimulation, termed early fusion, PM and SV membrane curvature changes to establish a point contact. The next stage-late fusion-shows fusion pore opening and SV collapse. During early fusion, proximal tethered SVs form additional tethers with the PM and increase the inter-SV connector number. In the late-fusion stage, PM-proximal SVs lose their interconnections, allowing them to move toward the PM. Two SNAP-25 mutations, one arresting and one disinhibiting spontaneous release, cause connector loss. The disinhibiting mutation causes loss of membrane-proximal multiple-tethered SVs. Overall, tether formation and connector dissolution are triggered by stimulation and respond to spontaneous fusion rate manipulation. These morphological observations likely correspond to SV transition from one functional pool to another.

| S-EPMC10157379 | biostudies-literature

Clustered Ca2+ Channels Are Blocked by Synaptic Vesicle Proton Release at Mammalian Auditory Ribbon Synapses.

Project description:A Ca2+ current transient block (ICaTB) by protons occurs at some ribbon-type synapses after exocytosis, but this has not been observed at mammalian hair cells. Here we show that a robust ICaTB occurs at post-hearing mouse and gerbil inner hair cell (IHC) synapses, but not in immature IHC synapses, which contain non-compact active zones, where Ca2+ channels are loosely coupled to the release sites. Unlike ICaTB at other ribbon synapses, ICaTB in mammalian IHCs displays a surprising multi-peak structure that mirrors the EPSCs seen in paired recordings. Desynchronizing vesicular release with intracellular BAPTA or by deleting otoferlin, the Ca2+ sensor for exocytosis, greatly reduces ICaTB, whereas enhancing release synchronization by raising Ca2+ influx or temperature increases ICaTB. This suggests that ICaTB is produced by fast multivesicular proton-release events. We propose that ICaTB may function as a submillisecond feedback mechanism contributing to the auditory nerve's fast spike adaptation during sound stimulation.

| S-EPMC6365105 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data