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author:("manado, Leon")
1.  Zebrafish Tg(7.2mab21l2:EGFP)ucd2 Transgenics Reveal a Unique Population of Retinal Amacrine Cells 
Purpose
Amacrine cells constitute a diverse, yet poorly characterized, cell population in the inner retina. Here, the authors sought to characterize the morphology, molecular physiology, and electrophysiology of a subpopulation of EGFP-expressing retinal amacrine cells identified in a novel zebrafish transgenic line.
Methods
After 7.2 kb of the zebrafish mab21l2 promoter was cloned upstream of EGFP, it was used to create the Tg(7.2mab21l2:EGFP)ucd2 transgenic line. Transgenic EGFP expression was analyzed by fluorescence microscopy in whole mount embryos, followed by detailed analysis of EGFP-expressing amacrine cells using fluorescence microscopy, immunohistochemistry, and electrophysiology.
Results
A 7.2-kb fragment of the mab21l2 promoter region is sufficient to drive transgene expression in the developing lens and tectum. Intriguingly, EGFP was also observed in differentiated amacrine cells. EGFP-labeled amacrine cells in Tg(7.2mab21l2:EGFP)ucd2 constitute a novel GABA- and glycine-negative amacrine subpopulation. Morphologically, EGFP-expressing cells stratify in sublamina 1 to 2 (type 1 OFF) or sublamina 3 to 4 (type 1 ON) or branch diffusely (type 2). Electrophysiologically, these cells segregate into amacrine cells with somas in the vitreal part of the INL and linear responses to current injection or, alternatively, amacrine cells with somas proximal to the IPL and active oscillatory voltage signals.
Conclusions
The novel transgenic line Tg(7.2mab21l2:EGFP) ucd2 uncovers a unique subpopulation of retinal amacrine cells.
doi:10.1167/iovs.10-5376
PMCID: PMC3925879  PMID: 21051702
2.  Endophilin Drives the Fast Mode of Vesicle Retrieval in a Ribbon Synapse 
Compensatory endocytosis of exocytosed membrane and recycling of synaptic vesicle components is essential for sustained synaptic transmission at nerve terminals. At the ribbon-type synapse of retinal bipolar cells, manipulations expected to inhibit the interactions of the clathrin adaptor protein complex (AP2) affect only the slow phase of endocytosis (τ = 10–15 s), leading to the conclusion that fast endocytosis (τ = 1–2 s) occurs by a mechanism that differs from the classical pathway of clathrin-coated vesicle retrieval from the plasma membrane. Here we investigate the role of endophilin in endocytosis at this ribbon synapse. Endophilin A1 is a synaptically enriched N-BAR domain-containing protein, suggested to function in clathrin-mediated endocytosis. Internal dialysis of the synaptic terminal with dominant-negative endophilin A1 lacking its linker and Src homology 3 (SH3) domain inhibited the fast mode of endocytosis, while slow endocytosis continued. Dialysis of a peptide that binds endophilin SH3 domain also decreased fast retrieval. Electron microscopy indicated that fast endocytosis occurred by retrieval of small vesicles in most instances. These results indicate that endophilin is involved in fast retrieval of synaptic vesicles occurring by a mechanism that can be distinguished from the classical pathway involving clathrin-AP2 interactions.
doi:10.1523/JNEUROSCI.6223-09.2011
PMCID: PMC3926091  PMID: 21653855
3.  Synaptic mechanisms of adaptation and sensitization in the retina 
Nature neuroscience  2013;16(7):934-941.
Sensory systems continually adjust the way stimuli are processed. What are the circuit mechanisms underlying this plasticity? We investigated how synapses in the retina of zebrafish adjust to changes in the temporal contrast of a visual stimulus by imaging activity in vivo. Following an increase in contrast, bipolar cell synapses with strong initial responses depressed, whereas synapses with weak initial responses facilitated. Depression and facilitation predominated in different strata of the inner retina, where bipolar cell output was anticorrelated with the activity of amacrine cell synapses providing inhibitory feedback. Pharmacological block of GABAergic feedback converted facilitating bipolar cell synapses into depressing ones. These results indicate that depression intrinsic to bipolar cell synapses causes adaptation of the ganglion cell response to contrast, whereas depression in amacrine cell synapses causes sensitization. Distinct microcircuits segregating to different layers of the retina can cause simultaneous increases or decreases in the gain of neural responses.
doi:10.1038/nn.3408
PMCID: PMC3924174  PMID: 23685718
4.  New light on photon detection 
The Journal of Physiology  2012;590(Pt 16):3641-3642.
doi:10.1113/jphysiol.2012.237040
PMCID: PMC3476622  PMID: 22904363
5.  Olfactory Stimulation Selectively Modulates the OFF Pathway in the Retina of Zebrafish 
Neuron  2013;79(1):97-110.
Summary
Cross-modal regulation of visual performance by olfactory stimuli begins in the retina, where dopaminergic interneurons receive projections from the olfactory bulb. However, we do not understand how olfactory stimuli alter the processing of visual signals within the retina. We investigated this question by in vivo imaging activity in transgenic zebrafish expressing SyGCaMP2 in bipolar cell terminals and GCaMP3.5 in ganglion cells. The food-related amino acid methionine reduced the gain and increased sensitivity of responses to luminance and contrast transmitted through OFF bipolar cells but not ON. The effects of olfactory stimulus were blocked by inhibiting dopamine uptake and release. Activation of dopamine receptors increased the gain of synaptic transmission in vivo and potentiated synaptic calcium currents in isolated bipolar cells. These results indicate that olfactory stimuli alter the sensitivity of the retina through the dopaminergic regulation of presynaptic calcium channels that control the gain of synaptic transmission through OFF bipolar cells.
Highlights
•Olfactory stimuli regulate transmission of signals through retinal bipolar cells•Modulation of synaptic gain and sensitivity occur in OFF bipolar cells but not ON•An inhibitor of dopamine uptake blocks odor-induced changes in synaptic gain•Dopamine potentiates presynaptic calcium channels in isolated bipolar cells
Esposti et al. show that olfactory stimulation selectively modulates synaptic transmission from retinal OFF bipolar cells in zebrafish. Dopamine plays a key role in this cross modal interaction by acting on the presynaptic calcium channels.
doi:10.1016/j.neuron.2013.05.001
PMCID: PMC3710973  PMID: 23849198
6.  Optimization of a GCaMP calcium indicator for neural activity imaging 
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Recent efforts in protein engineering have significantly increased the performance of GECIs. The state-of-the art single-wavelength GECI, GCaMP3, has been deployed in a number of model organisms and can reliably detect three or more action potentials (APs) in short bursts in several systems in vivo. Through protein structure determination, targeted mutagenesis, high-throughput screening, and a battery of in vitro assays, we have increased the dynamic range of GCaMP3 by several-fold, creating a family of “GCaMP5” sensors. We tested GCaMP5s in several systems: cultured neurons and astrocytes, mouse retina, and in vivo in Caenorhabditis chemosensory neurons, Drosophila larval neuromuscular junction and adult antennal lobe, zebrafish retina and tectum, and mouse visual cortex. Signal-to-noise ratio was improved by at least 2–3-fold. In the visual cortex, two GCaMP5 variants detected twice as many visual stimulus-responsive cells as GCaMP3. By combining in vivo imaging with electrophysiology we show that GCaMP5 fluorescence provides a more reliable measure of neuronal activity than its predecessor GCaMP3. GCaMP5 allows more sensitive detection of neural activity in vivo and may find widespread applications for cellular imaging in general.
doi:10.1523/JNEUROSCI.2601-12.2012
PMCID: PMC3482105  PMID: 23035093
GCaMP5; GCaMP3; GECI; genetically encoded calcium indicator; functional imaging
7.  Genetically encoded calcium indicators for multi-color neural activity imaging and combination with optogenetics 
Genetically encoded calcium indicators (GECIs) are powerful tools for systems neuroscience. Here we describe red, single-wavelength GECIs, “RCaMPs,” engineered from circular permutation of the thermostable red fluorescent protein mRuby. High-resolution crystal structures of mRuby, the red sensor RCaMP, and the recently published red GECI R-GECO1 give insight into the chromophore environments of the Ca2+-bound state of the sensors and the engineered protein domain interfaces of the different indicators. We characterized the biophysical properties and performance of RCaMP sensors in vitro and in vivo in Caenorhabditis elegans, Drosophila larvae, and larval zebrafish. Further, we demonstrate 2-color calcium imaging both within the same cell (registering mitochondrial and somatic [Ca2+]) and between two populations of cells: neurons and astrocytes. Finally, we perform integrated optogenetics experiments, wherein neural activation via channelrhodopsin-2 (ChR2) or a red-shifted variant, and activity imaging via RCaMP or GCaMP, are conducted simultaneously, with the ChR2/RCaMP pair providing independently addressable spectral channels. Using this paradigm, we measure calcium responses of naturalistic and ChR2-evoked muscle contractions in vivo in crawling C. elegans. We systematically compare the RCaMP sensors to R-GECO1, in terms of action potential-evoked fluorescence increases in neurons, photobleaching, and photoswitching. R-GECO1 displays higher Ca2+ affinity and larger dynamic range than RCaMP, but exhibits significant photoactivation with blue and green light, suggesting that integrated channelrhodopsin-based optogenetics using R-GECO1 may be subject to artifact. Finally, we create and test blue, cyan, and yellow variants engineered from GCaMP by rational design. This engineered set of chromatic variants facilitates new experiments in functional imaging and optogenetics.
doi:10.3389/fnmol.2013.00002
PMCID: PMC3586699  PMID: 23459413
calcium imaging; genetically encoded calcium indicator; multi-color imaging; protein engineering; optogenetics
8.  Regulation of thalamocortical axon branching by BDNF and synaptic vesicle cycling 
During development, axons form branches in response to extracellular molecules. Little is known about the underlying molecular mechanisms. Here, we investigate how neurotrophin-induced axon branching is related to synaptic vesicle cycling for thalamocortical axons. The exogenous application of brain-derived neurotrophic factor (BDNF) markedly increased axon branching in thalamocortical co-cultures, while removal of endogenous BDNF reduced branching. Over-expression of a C-terminal fragment of AP180 that inhibits clathrin-mediated endocytosis affected the laminar distribution and the number of branch points. A dominant-negative synaptotagmin mutant that selectively targets synaptic vesicle cycling, strongly suppressed axon branching. Moreover, axons expressing the mutant synaptotagmin were resistant to the branch-promoting effect of BDNF. These results suggest that synaptic vesicle cycling might regulate BDNF induced branching during the development of the axonal arbor.
doi:10.3389/fncir.2013.00202
PMCID: PMC3868945  PMID: 24391549
axon; branching; neurotrophin; synapse; endocytosis; thalamus; neocortex; development
9.  Clathrin is required for the function of the mitotic spindle 
Nature  2005;434(7037):1152-1157.
Clathrin has an established function in the generation of vesicles that transfer membrane and proteins around the cell1-4. The formation of clathrin-coated vesicles occurs continuously in non-dividing cells5, but is shut down during mitosis6, when clathrin concentrates at the spindle apparatus7,8. Here we show that clathrin stabilises fibres of the mitotic spindle to aid congression of chromsomes. Clathrin bound the spindle directlyvia the N-terminal domain of clathrin heavy chain (CHC). Depletion of CHC using RNA interference prolonged mitosis; kinetochore fibres were destabilised leading to defective congression of chromosomes to the metaphase plate and persistent activation of the spindle checkpoint. Normal mitosis was rescued by clathrin triskelia but not the N-terminal domain of CHC indicating that stabilisation of kinetochore fibres was dependent on the unique structure of clathrin. The importance of clathrin for normal mitosis may be relevant to understanding human cancers that involve gene fusions of clathrin heavy chain.
doi:10.1038/nature03502
PMCID: PMC3492753  PMID: 15858577
clathrin; mitosis; mitotic spindle; endocytosis; cancer; cell division
10.  Trimerization is important for the function of clathrin at the mitotic spindle 
Journal of cell science  2006;119(Pt 19):4071-4078.
Summary
Clathrin is a triskelion consisting of three heavy chains each with an associated light chain. During mitosis, clathrin contributes to kinetochore fibre stability. As the N-terminal domain at the foot of each leg can bind to the mitotic spindle, we proposed previously a “bridge hypothesis” wherein clathrin acts as a brace between two or three microtubules within a kinetochore fibre to increase fibre stability. Here, we have tested this hypothesis by replacing endogenous clathrin heavy chain in human cells with a panel of clathrin constructs. Mutants designed to abolish trimerization were unable to rescue the mitotic defects caused by depletion of endogenous clathrin. In contrast, stunted triskelia with contracted legs could partially rescue normal mitosis. These results indicate that the key structural features of clathrin that are necessary for its function in mitosis are a trimeric molecule with a spindle interaction domain at each end, supporting the “bridge hypothesis” for clathrin function in mitosis.
doi:10.1242/jcs.03192
PMCID: PMC3475310  PMID: 16968737
Clathrin; mitosis; endocytosis; RNAi
11.  Imaging pHluorin-based probes at hippocampal synapses 
Accurate measurement of synaptic vesicle exocytosis and endocytosis is crucial to understanding the molecular basis of synaptic transmission. The fusion of a pH-sensitive GFP (pHluorin) to various synaptic vesicle proteins has allowed the study of synaptic vesicle recycling in real-time. Two such probes, synaptopHluorin and sypHy have been imaged at synapses of hippocampal neurons in culture. The combination of these reporters with techniques for molecular interference, such as RNAi allows for the study of molecules involved in synaptic vesicle recycling. Here we describe methods for the culture and transfection of hippocampal neurons, imaging of pHluorin-based probes at synapses and analysis of pHluorin signals down to the resolution of individual synaptic vesicles.
PMCID: PMC3474192  PMID: 19066036
Hippocampal synapses; neurons; synaptic vesicle; sypHy; synaptopHluorin; endocytosis; exocytosis; imaging
12.  Clathrin-mediated endocytosis at the synaptic terminal: bridging the gap between physiology and molecules 
Traffic (Copenhagen, Denmark)  2010;11(12):1489-1497.
It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non-neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding of these processes will require molecular events to be manipulated while observing endocytosis in living synapses. Here we review recent work that seeks to bridge the gap between physiology and molecules to unravel the endocytic machinery operating at the synaptic terminal.
doi:10.1111/j.1600-0854.2010.01104.x
PMCID: PMC3371399  PMID: 20633242
Synaptic vesicle; endocytosis; clathrin; pHluorin; adaptors
13.  Encoding of Luminance and Contrast by Linear and Nonlinear Synapses in the Retina 
Neuron  2012;73(4-2):758-773.
Summary
Understanding how neural circuits transmit information is technically challenging because the neural code is contained in the activity of large numbers of neurons and synapses. Here, we use genetically encoded reporters to image synaptic transmission across a population of sensory neurons—bipolar cells in the retina of live zebrafish. We demonstrate that the luminance sensitivities of these synapses varies over 104 with a log-normal distribution. About half the synapses made by ON and OFF cells alter their polarity of transmission as a function of luminance to generate a triphasic tuning curve with distinct maxima and minima. These nonlinear synapses signal temporal contrast with greater sensitivity than linear ones. Triphasic tuning curves increase the dynamic range over which bipolar cells signal light and improve the efficiency with which luminance information is transmitted. The most efficient synapses signaled luminance using just 1 synaptic vesicle per second per distinguishable gray level.
Highlights
► Bipolar cell synapses display luminance sensitivities varying over 104 ► Transmission from ON and OFF cells can switch polarity ► Nonlinear synapses signal luminance more efficiently than linear ones ► Nonlinear synapses display higher contrast-sensitivity than linear ones
How does the visual system extract information from visual scenes with wide variations in luminance? Odermatt et al. found that bipolar cells have varying sensitivity to luminance and extend the range of luminances that they signal by generating responses with different polarities.
doi:10.1016/j.neuron.2011.12.023
PMCID: PMC3314971  PMID: 22365549
14.  In vivo evidence that retinal bipolar cells generate spikes modulated by light 
Nature neuroscience  2011;14(8):951-952.
Retinal bipolar cells have been assumed to generate purely graded responses to light. To test this idea we imaged the presynaptic calcium transient in live zebrafish. We found that ON, OFF, transient and sustained bipolar cells are all capable of generating fast “all-or-none” calcium transients modulated by visual stimulation.
doi:10.1038/nn.2841
PMCID: PMC3232443  PMID: 21706020
15.  Spikes in Retinal Bipolar Cells Phase-Lock to Visual Stimuli with Millisecond Precision 
Current Biology  2011;21(22):1859-1869.
Summary
Background
The conversion of an analog stimulus into the digital form of spikes is a fundamental step in encoding sensory information. Here, we investigate this transformation in the visual system of fish by in vivo calcium imaging and electrophysiology of retinal bipolar cells, which have been assumed to be purely graded neurons.
Results
Synapses of all major classes of retinal bipolar cell encode visual information by using a combination of spikes and graded signals. Spikes are triggered within the synaptic terminal and, although sparse, phase-lock to a stimulus with a jitter as low as 2–3 ms. Spikes in bipolar cells encode a visual stimulus less reliably than spikes in ganglion cells but with similar temporal precision. The spike-generating mechanism does not alter the temporal filtering of a stimulus compared with the generator potential. The amplitude of the graded component of the presynaptic calcium signal can vary in time, and small fluctuations in resting membrane potential alter spike frequency and even switch spiking on and off.
Conclusions
In the retina of fish, the millisecond precision of spike coding begins in the synaptic terminal of bipolar cells. This neural compartment regulates the frequency of digital signals transmitted to the inner retina as well as the strength of graded signals.
Graphical Abstract
Highlights
► The spike code of vision begins in retinal bipolar cells ► Spikes in bipolar cells phase-lock to visual stimuli with millisecond precision ► Spiking and graded calcium signals can switch on and off at individual synapses ► Spikes in bipolar cells encode a stimulus less reliably than spikes in ganglion cells
doi:10.1016/j.cub.2011.09.042
PMCID: PMC3235547  PMID: 22055291
16.  Homeostatic synaptic plasticity through changes in presynaptic calcium influx 
Chronic perturbations of electrical activity within neural circuits lead to compensatory changes in synaptic strength collectively termed homeostatic synaptic plasticity. The post-synaptic mechanisms underlying these modifications have been characterized in some detail, but the presynaptic mechanisms that alter the efficiency of evoked neurotransmitter release are less clear. To investigate the role of presynaptic calcium influx, we have combined the use of two fluorescent proteins in cultured hippocampal neurons: a calcium reporter localized to synaptic vesicles, SyGCaMP2, and a reporter of vesicle fusion, SypHy. We find that a decrease in the activity of the network causes an increase in the amount of calcium entering the synaptic bouton in response to an action potential and an increase in the probability of vesicle fusion. Homeostatic changes in release probability varied as the third power of calcium influx. These results indicate that changes in the number and/or function of presynaptic calcium channels are major determinants of homeostatic changes in synaptic strength.
doi:10.1523/JNEUROSCI.6636-10.2011
PMCID: PMC3124754  PMID: 21593333
homeostatic plasticity; calcium; synapse; vesicle; GCaMP2
17.  A genetically-encoded reporter of synaptic activity in vivo 
Nature methods  2009;6(12):883-889.
To image synaptic activity within neural circuits, we have tethered the genetically-encoded calcium indicator (GECI) GCaMP2 to synaptic vesicles by fusion to synaptophysin. The resulting reporter, SyGCaMP2, detects the electrical activity of neurons with two advantages over existing cytoplasmic GECIs: the locations of synapses are identified and the reporter displays a linear response over a wider range of spike frequencies. Simulations and experimental measurements indicate that linearity arises because SyGCaMP2 samples the brief calcium transient passing through the presynaptic compartment close to voltage-sensitive calcium channels rather than changes in bulk calcium concentration. In vivo imaging in zebrafish demonstrates that SyGCaMP2 can assess electrical activity in conventional synapses of spiking neurons in the optic tectum as well as graded voltage signals transmitted by ribbon synapses of retinal bipolar cells. Localizing a GECI to synaptic terminals provides a strategy for monitoring activity across large groups of neurons at the level of individual synapses.
doi:10.1038/nmeth.1399
PMCID: PMC2859341  PMID: 19898484
synapse; calcium; action potential; fluorescent reporter; hippocampal neuron; GCaMP2; zebrafish; retina; tectum
18.  Computational processing of optical measurements of neuronal and synaptic activity in networks 
Journal of Neuroscience Methods  2010;188(1):141-150.
Imaging of optical reporters of neural activity across large populations of neurones is a widely used approach for investigating the function of neural circuits in slices and in vivo. Major challenges in analysing such experiments include the automatic identification of neurones and synapses, extraction of dynamic signals, and assessing the temporal and spatial relationships between active units in relation to the gross structure of the circuit. We have developed an integrated set of software tools, named SARFIA, by which these aspects of dynamic imaging experiments can be analysed semi-automatically. Key features are image-based detection of structures of interest using the Laplace operator, determining the positions of units in a layered network, clustering algorithms to classify units with similar functional responses, and a database to store, exchange and analyse results across experiments. We demonstrate the use of these tools to analyse synaptic activity in the retina of live zebrafish by multi-photon imaging of SyGCaMP2, a genetically encoded synaptically localised calcium reporter. By simultaneously recording activity across tens of bipolar cell terminals distributed throughout the IPL we made a functional map of the ON and OFF signalling channels and found that these were only partially separated. The automated detection of signals across many neurones in the retina allowed the reliable detection of small populations of neurones generating “ectopic” signals in the “ON” and “OFF” sublaminae. This software should be generally applicable for the analysis of dynamic imaging experiments across hundreds of responding units.
doi:10.1016/j.jneumeth.2010.01.033
PMCID: PMC2849931  PMID: 20152860
Calcium; Fluorescent reporter; Image analysis; Retina; SyGCaMP2; Software; Synapse; Zebrafish
19.  Reversible binding and rapid diffusion of proteins in complex with inositol lipids serves to coordinate free movement with spatial information 
The Journal of Cell Biology  2009;184(2):297-308.
Polyphosphoinositol lipids convey spatial information partly by their interactions with cellular proteins within defined domains. However, these interactions are prevented when the lipids' head groups are masked by the recruitment of cytosolic effector proteins, whereas these effectors must also have sufficient mobility to maximize functional interactions. To investigate quantitatively how these conflicting functional needs are optimized, we used different fluorescence recovery after photobleaching techniques to investigate inositol lipid–effector protein kinetics in terms of the real-time dissociation from, and diffusion within, the plasma membrane. We find that the protein–lipid complexes retain a relatively rapid (∼0.1–1 µm2/s) diffusion coefficient in the membrane, likely dominated by protein–protein interactions, but the limited time scale (seconds) of these complexes, dictated principally by lipid–protein interactions, limits their range of action to a few microns. Moreover, our data reveal that GAP1IP4BP, a protein that binds PtdIns(4,5)P2 and PtdIns(3,4,5)P3 in vitro with similar affinity, is able to “read” PtdIns(3,4,5)P3 signals in terms of an elongated residence time at the membrane.
doi:10.1083/jcb.200809073
PMCID: PMC2654307  PMID: 19153221
21.  The mouth of a dense-core vesicle opens and closes in a concerted action regulated by calcium and amphiphysin 
The Journal of Cell Biology  2008;182(5):1017-1028.
Secretion of hormones and peptides by neuroendocrine cells occurs through fast and slow modes of vesicle fusion but the mechanics of these processes are not understood. We used interference reflection microscopy to monitor deformations of the membrane surface and found that both modes of fusion involve the tightly coupled dilation and constriction of the vesicle. The rate of opening is calcium dependent and occurs rapidly at concentrations <5 μM. The fast mode of fusion is blocked selectively by a truncation mutant of amphiphysin. Vesicles do not collapse when fusion is triggered by strontium, rather they remain locked open and membrane scission is blocked. In contrast, constriction of the vesicle opening continues when endocytosis is blocked by inhibiting the function of dynamin. Thus, fast and slow modes of fusion involve similar membrane deformations and vesicle closure can be uncoupled from membrane scission. Regulation of these processes by calcium and amphiphysin may provide a mechanism for controlling the release of vesicle contents.
doi:10.1083/jcb.200807034
PMCID: PMC2528570  PMID: 18779374
22.  Calcium and Protein Kinase C Regulate the Actin Cytoskeleton in the Synaptic Terminal of Retinal Bipolar Cells  
The Journal of Cell Biology  1998;143(6):1661-1672.
The organization of filamentous actin (F-actin) in the synaptic pedicle of depolarizing bipolar cells from the goldfish retina was studied using fluorescently labeled phalloidin. The amount of F-actin in the synaptic pedicle relative to the cell body increased from a ratio of 1.6 ± 0.1 in the dark to 2.1 ± 0.1 after exposure to light. Light also caused the retraction of spinules and processes elaborated by the synaptic pedicle in the dark.
Isolated bipolar cells were used to characterize the factors affecting the actin cytoskeleton. When the electrical effect of light was mimicked by depolarization in 50 mM K+, the actin network in the synaptic pedicle extended up to 2.5 μm from the plasma membrane. Formation of F-actin occurred on the time scale of minutes and required Ca2+ influx through L-type Ca2+ channels. Phorbol esters that activate protein kinase C (PKC) accelerated growth of F-actin. Agents that inhibit PKC hindered F-actin growth in response to Ca2+ influx and accelerated F-actin breakdown on removal of Ca2+.
To test whether activity-dependent changes in the organization of F-actin might regulate exocytosis or endocytosis, vesicles were labeled with the fluorescent membrane marker FM1-43. Disruption of F-actin with cytochalasin D did not affect the continuous cycle of exocytosis and endocytosis that was stimulated by maintained depolarization, nor the spatial distribution of recycled vesicles within the synaptic terminal. We suggest that the actions of Ca2+ and PKC on the organization of F-actin regulate the morphology of the synaptic pedicle under varying light conditions.
PMCID: PMC2132988  PMID: 9852158
actin; calcium; protein kinase C; synapse; vesicle
23.  New light on photon detection 
The Journal of Physiology  2012;590(16):3641-3642.
doi:10.1113/jphysiol.2012.237040
PMCID: PMC3476622  PMID: 22904363

Results 1-23 (23)