scholarly journals Light regulation of Ca2+ in the cone photoreceptor synaptic terminal

2008 ◽  
Vol 25 (5-6) ◽  
pp. 693-700 ◽  
Author(s):  
SUE-YEON CHOI ◽  
SKYLER JACKMAN ◽  
WALLACE B. THORESON ◽  
RICHARD H. KRAMER
Keyword(s):  
Membrane Potential ◽  
Hot Spots ◽  
Synaptic Vesicles ◽  
Light Regulation ◽  
Bright Light ◽  
Cone Photoreceptor ◽  
Two Photon ◽  
Ratiometric Imaging ◽  
High Level ◽  
Indicator Dyes

AbstractRetinal cones are depolarized in darkness, keeping voltage-gated Ca2+ channels open and sustaining exocytosis of synaptic vesicles. Light hyperpolarizes the membrane potential, closing Ca2+ channels and suppressing exocytosis. Here, we quantify the Ca2+ concentration in cone terminals, with Ca2+ indicator dyes. Two-photon ratiometric imaging of fura-2 shows that global Ca2+ averages ~360 nM in darkness and falls to ~190 nM in bright light. Depolarizing cones from their light to their dark membrane potential reveals hot spots of Ca2+ that co-label with a fluorescent probe for the synaptic ribbon protein ribeye, consistent with tight localization of Ca2+ channels near ribbons. Measurements with a low-affinity Ca2+ indicator show that the local Ca2+ concentration near the ribbon exceeds 4 μM in darkness. The high level of Ca2+ near the ribbon combined with previous estimates of the Ca2+ sensitivity of release leads to a predicted dark release rate that is much faster than observed, suggesting that the cone synapse operates in a maintained state of synaptic depression in darkness.

scholarly journals Light Dependence of Calcium and Membrane Potential Measured in Blowfly Photoreceptors In Vivo

1998 ◽  
Vol 112 (2) ◽  
pp. 113-124 ◽  
Author(s):  
Johannes Oberwinkler ◽  
Doekele G. Stavenga
Keyword(s):  
Membrane Potential ◽  
Light Intensity ◽  
Light Adaptation ◽  
Photoreceptor Cell ◽  
Dynamic Range ◽  
Bright Light ◽  
Molecular Probes ◽  
Calcium Indicator ◽  
Indicator Dyes

Light adaptation in insect photoreceptors is caused by an increase in the cytosolic Ca2+ concentration. To better understand this process, we measured the cytosolic Ca2+ concentration in vivo as a function of adapting light intensity in the white-eyed blowfly mutant chalky. We developed a technique to measure the cytosolic Ca2+ concentration under conditions as natural as possible. The calcium indicator dyes Oregon Green 1, 2, or 5N (Molecular Probes, Inc., Eugene, OR) were iontophoretically injected via an intracellular electrode into a photoreceptor cell in the intact eye; the same electrode was also used to measure the membrane potential. The blue-induced green fluorescence of these dyes could be monitored by making use of the optics of the facet lens and the rhabdomere waveguide. The use of the different Ca2+-sensitive dyes that possess different affinities for Ca2+ allowed the quantitative determination of the cytosolic Ca2+ concentration in the steady state. Determining the cytosolic Ca2+ concentration as a function of the adapting light intensity shows that the Ca2+ concentration is regulated in a graded fashion over the whole dynamic range where a photoreceptor cell can respond to light. When a photoreceptor is adapted to bright light, the cytosolic Ca2+ concentration reaches stable values higher than 10 μM. The data are consistent with the hypothesis that the logarithm of the increase in cytosolic Ca2+ concentration is linear with the logarithm of the light intensity. From the estimated values of the cytosolic Ca2+ concentration, we conclude that the Ca2+-buffering capacity is limited. The percentage of the Ca2+ influx that is buffered gradually decreases with increasing Ca2+ concentrations; at cytosolic Ca2+ concentration levels above 10 μM, buffering becomes minimal.

scholarly journals Individual synaptic vesicles mediate stimulated exocytosis from cochlear inner hair cells

2018 ◽  
Vol 115 (50) ◽  
pp. 12811-12816 ◽  
Author(s):  
Chad Paul Grabner ◽  
Tobias Moser
Keyword(s):  
Hair Cells ◽  
Synaptic Vesicles ◽  
Basolateral Membrane ◽  
Mean Amplitude ◽  
Auditory Pathway ◽  
Inner Hair Cell ◽  
Excitatory Postsynaptic Currents ◽  
High Level ◽  
High Degree ◽  
Channel Type

Spontaneous excitatory postsynaptic currents (sEPSCs) measured from the first synapse in the mammalian auditory pathway reach a large mean amplitude with a high level of variance (CV between 0.3 and 1). This has led some to propose that each inner hair cell (IHC) ribbon-type active zone (AZ), on average, releases ∼6 synaptic vesicles (SVs) per sEPSC in a coordinated manner. If true, then the predicted change in membrane capacitance (Cm) for such multivesicular fusion events would equate to ∼300 attofarads (aF). Here, we performed cell-attached Cm measurements to directly examine the size of fusion events at the basolateral membrane of IHCs where the AZs are located. The frequency of events depended on the membrane potential and the expression of Cav1.3, the principal Ca2+-channel type of IHCs. Fusion events averaged 40 aF, which equates to a normal-sized SV with an estimated diameter of 37 nm. The calculated SV volumes showed a high degree of variance (CV > 0.6). These results indicate that SVs fused individually with the plasma membrane during spontaneous and evoked release and SV volume may contribute more variability in EPSC amplitude than previously assumed.

scholarly journals Activity and cytosolic Na+ regulates synaptic vesicle endocytosis

2019 ◽  
Author(s):  
Yun Zhu ◽  
Dainan Li ◽  
Hai Huang
Keyword(s):  
Synaptic Transmission ◽  
High Frequency ◽  
Synaptic Vesicles ◽  
Glutamate Uptake ◽  
High Frequencies ◽  
Vesicle Recycling ◽  
Two Photon ◽  
Readily Releasable Pool ◽  
Content Change ◽  
Intracellular Ca

ABSTRACTRetrieval of synaptic vesicles via endocytosis is essential for maintaining sustained synaptic transmission, especially for neurons that fire action potentials at high frequencies. However, how activity regulates synaptic vesicles recycling is largely unknown. Here we report that Na+ substantially accumulated in the mouse calyx of Held terminals during repetitive high-frequency spiking. Elevated presynaptic Na+ accelerated both slow and rapid forms of endocytosis and facilitated endocytosis overshoot but did not affect the readily releasable pool size, Ca2+ influx, or exocytosis. To examine whether this facilitation of endocytosis is related to the Na+-dependent vesicular content change, we dialyzed increasing concentrations of glutamate into the presynaptic cytosol or blocked the vesicular glutamate uptake with bafilomycin and found the rate of endocytosis was not affected by regulating the glutamate content in the presynaptic terminal. Endocytosis is critically dependent on intracellular Ca2+, and the activity of Na+/Ca2+ exchanger (NCX) may be altered when the Na+ gradient is changed. However, neither NCX blocker nor change of extracellular Na+ concentration affected the endocytosis rate. Moreover, two-photon Ca2+ imaging showed that presynaptic Na+ did not affect the action potential-evoked intracellular Ca2+ transient and decay. Therefore, we revealed a novel mechanism of cytosolic Na+ in accelerating vesicle endocytosis. During high-frequency synaptic transmission, when large amounts of synaptic vesicles are fused, Na+ accumulated in terminals, facilitated vesicle recycling and sustained reliable synaptic transmission.

scholarly journals Optical Estimation of Absolute Membrane Potential Using One- and Two-Photon Fluorescence Lifetime Imaging Microscopy

2021 ◽  
Author(s):  
Julia R. Lazzari-Dean ◽  
Evan W. Miller
Keyword(s):  
Membrane Potential ◽  
Fluorescence Lifetime ◽  
Single Photon ◽  
Photon Counting ◽  
Fluorescence Lifetime Imaging ◽  
Single Photon Counting ◽  
Two Photon ◽  
Lifetime Imaging ◽  
Wide Range ◽  
Relative Measurements

AbstractBackgroundMembrane potential (Vmem) exerts physiological influence across a wide range of time and space scales. To study Vmem in these diverse contexts, it is essential to accurately record absolute values of Vmem, rather than solely relative measurements.Materials & MethodsWe use fluorescence lifetime imaging of a small molecule voltage sensitive dye (VF2.1.Cl) to estimate mV values of absolute membrane potential.ResultsWe test the consistency of VF2.1.Cl lifetime measurements performed on different single photon counting instruments and find that they are in striking agreement (differences of <0.5 ps/mV in the slope and <50 ps in the y-intercept). We also demonstrate that VF2.1.Cl lifetime reports absolute Vmem under two-photon (2P) illumination with better than 20 mV of Vmem resolution, a nearly 10-fold improvement over other lifetime-based methods.ConclusionsWe demonstrate that VF-FLIM is a robust and portable metric for Vmem across imaging platforms and under both one-photon and two-photon illumination. This work is a critical foundation for application of VF-FLIM to record absolute membrane potential signals in thick tissue.

Two-photon fluorescent Zn2+ probe for ratiometric imaging and biosensing of Zn2+ in living cells and larval zebrafish

2020 ◽  
Vol 148 ◽  
pp. 111666 ◽  
Author(s):  
Wanying Li ◽  
Zhichao Liu ◽  
Bingqing Fang ◽  
Ming Jin ◽  
Yang Tian
Keyword(s):  
Living Cells ◽  
Two Photon ◽  
Larval Zebrafish ◽  
Ratiometric Imaging

A New Pre-Embedding Immunogold Method That Permits to Obtain a Very High Signal with a Very Good Ultrastucture

2001 ◽  
Vol 7 (S2) ◽  
pp. 1044-1045
Author(s):  
G. Grondin ◽  
A.R. Bcaudoin
Keyword(s):  
New Technologies ◽  
Immunofluorescence Microscopy ◽  
High Signal ◽  
Sodium Metaperiodate ◽  
Two Photon ◽  
Cacodylate Buffer ◽  
Ultrastructure Preservation ◽  
High Level ◽  
Very High

The ultimate goal of the immunocytochemistry is to get the best signal with the lowest background while preserving ultrastructure. Unfortunaly too often the experimentator faces a compromise between density of the signal and ultrastructure preservation (1). with the advent of hight resolution confocal light microscopy, two photon imaging and other new technologies there is a need to provide the best immunocytochemical complementary results with the high resolution of the electron microscope. But often for exemple the high level of labelling observed by immunofluorescence microscopy is not matched by immunocytochemistry (2). One approach to obtain comparable levels of labelling is to use comparable protocols. We propose here such a new immunogold method. to illustrate our point we used a very convenient biological material, the adherent cells grown on 35 mm plastic culture dishes. The following procedure was applied : Confluent endothelial cells were fixed in situ for 3 hours with the following freshly prepared and filtered solution : 1 % 1-lysine, 4% paraformaldehyde, 0.04% glutaraldehyde, 0.25% sodium metaperiodate in 0.04M sodium cacodylate buffer, pH 7.4.

A water-soluble two-photon fluorescence chemosensor for ratiometric imaging of mitochondrial viscosity in living cells

2016 ◽  
Vol 4 (35) ◽  
pp. 5907-5912 ◽  
Author(s):  
Meng Zhao ◽  
Yingzhong Zhu ◽  
Jian Su ◽  
Qian Geng ◽  
Xiaohe Tian ◽  
...  
Keyword(s):  
Living Cells ◽  
Water Soluble ◽  
Two Photon ◽  
Ratiometric Imaging ◽  
Two Photon Fluorescence ◽  
Photon Fluorescence

We report a novel water-soluble ratiometric TPEF chemosensor EIN that is specifically responsive and singularly sensitive to mitochondria viscosity in living cells.

Bright and two-photon active red fluorescent dyes that selectively move back and forth between the mitochondria and nucleus upon changing the mitochondrial membrane potential

2018 ◽  
Vol 6 (45) ◽  
pp. 7396-7401 ◽  
Author(s):  
Hitomi Seki ◽  
Shozo Onishi ◽  
Naoya Asamura ◽  
Yasutaka Suzuki ◽  
Jun Kawamata ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Mitochondrial Membrane Potential ◽  
Mitochondrial Membrane ◽  
Fluorescent Dyes ◽  
Two Photon

Pyrene-based two-photon active and bright red emitters that localize between the mitochondria and nucleus in response to changes in the mitochondrial membrane potential.

scholarly journals Photochromic meta-diamides for optical modulation of ligand activity and neuron function

2020 ◽  
Vol 19 (6) ◽  
pp. 854-857
Author(s):  
Cuncun Zhou ◽  
Yunfan Ji ◽  
Liping Ren ◽  
Xusheng Shao
Keyword(s):  
Membrane Potential ◽  
Aedes Albopictus ◽  
Light Regulation ◽  
Optical Modulation ◽  
Biological Functions ◽  
Dum Neurons ◽  
Neuron Function ◽  
Ligand Activity

In order to achieve light regulation of biological functions, a series of photoswitchable azobenzene-based meta-diamide analogues were synthesized. One of the ABMDAs can lead to activity changes towards Aedes albopictus larvae upon photoisomerization and enables optical modulation of membrane potential of DUM neurons.

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