Changes In Membrane Capacitance Research Articles

On-chip electroporation and impedance spectroscopy of single-cells

We present an integrated microplatform for handling, electroporation and label free analysis of single mammalian cells in suspension. Accurate axial positioning of a cell flowing through a channel is accomplished through microfluidic control and flow direction reversal; electroporation is achieved by applying 2kVcm−1 at 50kHz through on-chip microelectrodes; label-free detection of changes in the cell undergoing electroporation is performed by means of electric impedance spectroscopy (EIS). Shuttling the cell back and forth allows either (a) assessment of a single cell at multiple points of time to evaluate dynamic processes or (b) increased quality of EIS results by averaging subsequent passages of the same cell through the measurement region. Electrical parameters are extracted from the measurement by fitting the impedance magnitude spectrum to an equivalent-circuit model of the microchannel and by using a three-shell model for the cell. The fitting procedure is shown to be robust and suggests cell swelling, exchange of intra- and extracellular liquids, and change of the cell membrane and nuclear envelope capacitance. Cell swelling was in agreement with bright-field micrographs, while the exchange of intra- and extracellular liquids has also been observed via established fluorescent markers. The quantification of cellular changes as well as of the changes in cellular dielectric properties induced by electroporation enables a better understanding and control of the cell electroporation process.

Preparation, characterization and study of nanofiltration composite membrane: Electrochemical and optical properties

The polystyrene based molybdate composite membranes were prepared by the sol–gel method by applying different pressures. XRD data shows that formation of nanocomposite and exhibit no other impurity phase. The membrane potential is found to be effected by both pressure and ion concentration. The effective charge density of these membranes was determined by TMS method and reveal dependence of membrane potential on physicochemical properties. The change in membrane capacitance and resistance values with the change in electrolyte concentration and applied frequency had been interpreted in terms of the charges produced in the electrical double layer at the membrane solution interface.

CFTR and Anoctamin 1 (ANO1) contribute to cAMP amplified exocytosis and insulin secretion in human and murine pancreatic beta-cells.

BackgroundMutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to the disease cystic fibrosis (CF). Although patients with CF often have disturbances in glucose metabolism including impaired insulin release, no previous studies have tested the hypothesis that CFTR has a biological function in pancreatic beta-cells.MethodsExperiments were performed on islets and single beta-cells from human donors and NMRI-mice. Detection of CFTR was investigated using PCR and confocal microscopy. Effects on insulin secretion were measured with radioimmunoassay (RIA). The patch-clamp technique was used to measure ion channel currents and calcium-dependent exocytosis (as changes in membrane capacitance) on single cells with high temporal resolution. Analysis of ultrastructure was done on transmission electron microscopy (TEM) images.ResultsWe detected the presence of CFTR and measured a small CFTR conductance in both human and mouse beta-cells. The augmentation of insulin secretion at 16.7 mM glucose by activation of CFTR by cAMP (forskolin (FSK) or GLP-1) was significantly inhibited when CFTR antagonists (GlyH-101 and/or CFTRinh-172) were added. Likewise, capacitance measurements demonstrated reduced cAMP-dependent exocytosis upon CFTR-inhibition, concomitant with a decreased number of docked insulin granules. Finally, our studies demonstrate that CFTR act upstream of the chloride channel Anoctamin 1 (ANO1; TMEM16A) in the regulation of cAMP- and glucose-stimulated insulin secretion.ConclusionOur work demonstrates a novel function for CFTR as a regulator of pancreatic beta-cell insulin secretion and exocytosis, and put forward a role for CFTR as regulator of ANO1 and downstream priming of insulin granules prior to fusion and release of insulin. The pronounced regulatory effect of CFTR on insulin secretion is consistent with impaired insulin secretion in patients with CF.

Rapid kinetics of endocytosis at rod photoreceptor synapses depends upon endocytic load and calcium.

Release from rods is triggered by the opening of L-type Ca2+ channels that lie beneath synaptic ribbons. After exocytosis, vesicles are retrieved by compensatory endocytosis. Previous work showed that endocytosis is dynamin-dependent in rods but dynamin-independent in cones. We hypothesized that fast endocytosis in rods may also differ from cones in its dependence upon the amount of Ca2+ influx and/or endocytic load. We measured exocytosis and endocytosis from membrane capacitance (C m) changes evoked by depolarizing steps in voltage clamped rods from tiger salamander retinal slices. Similar to cones, the time constant for endocytosis in rods was quite fast, averaging <200 ms. We manipulated Ca2+ influx and the amount of vesicle release by altering the duration and voltage of depolarizing steps. Unlike cones, endocytosis kinetics in rods slowed after increasing Ca2+ channel activation with longer step durations or more strongly depolarized voltage steps. Endocytosis kinetics also slowed as Ca2+ buffering was decreased by replacing BAPTA (10 or 1 mM) with the slower Ca2+ buffer EGTA (5 or 0.5 mM) in the pipette solution. These data provide further evidence that endocytosis mechanisms differ in rods and cones and suggest that endocytosis in rods is regulated by both endocytic load and local Ca2+ levels.

Intramembrane Cavitation as a Predictive Bio-Piezoelectric Mechanism for Ultrasonic Brain Stimulation

Low-intensity ultrasonic waves can remotely and nondestructively excite central nervous system (CNS) neurons. While diverse applications for this effect are already emerging, the biophysical transduction mechanism underlying this excitation remains unclear. Recently, we suggested that ultrasound-induced intramembrane cavitation within the bilayer membrane could underlie the biomechanics of a range of observed acoustic bioeffects. In this paper, we show that, in CNS neurons, ultrasound-induced cavitation of these nanometric bilayer sonophores can induce a complex mechanoelectrical interplay leading to excitation, primarily through the effect of currents induced by membrane capacitance changes. Our model explains the basic features of CNS acoustostimulation and predicts how the experimentally observed efficacy of mouse motor cortical ultrasonic stimulation depends on stimulation parameters. These results support the hypothesis that neuronal intramembrane piezoelectricity underlies ultrasound-induced neurostimulation, and suggest that other interactions between the nervous system and pressure waves or perturbations could be explained by this new mode of biological piezoelectric transduction.

Molecular Mechanisms of Endocytosis and Exocytosis in Yeast Studied by High-Resolution Membrane Capacitance Measurements

Exo- and endocytosis are crucial in numerous physiological processes in eukaryotic cells, such as polar growth, cell division, motility or signaling. Endo- as well as exocytotic events result in small changes of the cell surface area, which can be resolved in electrophysiological recordings in the form of discrete steps in membrane capacitance. By applying high resolution recordings with yeast protoplasts, we were able to resolve discrete changes in membrane capacitance mainly in the range 0.2-1.2fF, corresponding to fusion and fission of single vesicles of 80-250nm. Analysis of exo- and endocytosis in the yeast sec6-4 mutant, which is characterized by a temperature dependent block of exocytosis, revealed low fission/fusion activity at room temperature. Incubation at the restrictive temperature of 37°C for 3h and shifting to the permissive temperature of 25°C resulted in a transient increase in endo-/exocytotic activity of up to 10-fold. This study demonstrates that patch clamp capacitance measurements in yeast provide a novel in vivo tool to examine the molecular mechanisms of endo-/exocytosis in this model organism. The recordings gain information in real time on the geometry of endo-/exocytotic vesicles, on the kinetics of the events and their dependence on signaling cascades and key proteins.

Screening the Insertion of Families of Bioactive Microbial Metabolites into Tethered Bilayer Lipid Membranes (TBLMS)

While it is known that many microbial metabolites can permeabilize phospholipid membranes, it is not easy to find quantitative comparisons across large numbers of such metabolites. Such comparisons are of use in screening likely candidates for ongoing research into the development of new and improved antibiotics. We have studied the effects of 30 microbial metabolites on the electrical conductivity of a tethered bilayer lipid membrane comprising two standard phospholipid components. The technique allows rapid quantification of the activity of these compounds. As well as the electrical conductivity, we have analyzed the changes in membrane capacitance by the insertion of metabolites which reports on the change of membrane thickness, as well as the introduction of water molecules into the membrane. Using a standard 10 µM concentration for each of the 30 metabolites, we rank them according to their impact on membrane conduction in PBS solution (Fig 1). We also demonstrate that by determining the change in conduction (ΔG) against the change in capacitance (ΔC) caused by membrane insertion it is possible to determine the complexation properties of the metabolites as they insert into the membrane.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The Role of Doc2B in Depolarization-Evoked and G Protein-Coupled Receptor Modulated Exocytosis in Mouse Chromaffin Cells

Previously we demonstrated that activation of Gq protein-coupled H1 histamine receptors potentiates stimulus-coupled exocytosis in bovine chromaffin cells, despite inhibiting Ca2+ influx through voltage-gated calcium channels (VGCCs). This potentiation is restricted to the immediately releasable pool (IRP) of vesicles coupled to VGCCs, and requires the priming protein Munc13-1. Doc2B is a calcium-binding protein, known to interact with Munc13 to promote exocytosis; whether it plays a role in agonist-regulated exocytosis is unknown. To address this question, we isolated chromaffin cells from Doc2B (+/+), (+/-) and (-/-) mice and examined the effects of PLC-coupled GPCR activation on depolarization-evoked exocytosis.We found that under control stimulus conditions, depolarization-evoked exocytosis, measured as changes in membrane capacitance, was not significantly different between Doc2B (+/+), (+/-) and (-/-) cells; similarly no differences were found between the sizes of the releasable vesicle pools (IRP, RRP, SRP). Equimolar replacement of extracellular Ca2+ by Ba2+ also revealed no significant differences in the synchronous and asynchronous phases of Ba2+-evoked exocytosis in Doc2B (+/+), (+/-) or (-/-) cells. Finally, although more than 80% of cells, independent of their genotype, responded to histamine with a reduction of Ca2+ influx, potentiation of exocytosis was surprisingly modest (2-fold) and, even in Doc2 (+/+) cells rarely observed (5/17 cells, (+/-): 5/24 cells, (-/-): 2/18). This is in stark contrast to bovine cells, where 80% of the cells show a 5-fold potentiation of exocytosis. Importantly, Doc2B (+/+), (+/-) and (-/-) cells responded to the DAG analog PMA with potentiation. Taken together, the results suggest that there are profound species differences in the coupling between GqPCRs and the exocytotic machinery in chromaffin cells and that Doc2B is not required for agonist-dependent potentiation of exocytosis in mice.

Membrane Capacitance Changes due to Temperature Increase in Rat Cardiac Myocytes

The effect of infrared laser stimulation of excitable tissue was explained by the rise of temperature that increases the cell membrane capacitance, which causes cell depolarization and action potential activation (Shapiro et al., Nat. Commun. 3:736, 2012). As in cardiac myocytes the relation of cell capacitance and temperature is not well known, we studied the dependence of membrane capacitance on the bath temperature under steady-state conditions in isolated rat ventricular and atrial myocytes. Individual myocytes were whole-cell patch clamped and the membrane capacitance was recorded by the high-resolution square wave stimulation method (Novak and Zahradnik, Ann. Biomed. Eng. 34:1201, 2006). The bath temperature was increased by means of a temperature controller at a rate of 0.1°C/s from about 23°C to about 33°C. Experiments on 6 atrial and 3 ventricular myocytes provided an average increase of the cell capacitance by 0.30 ± 0.02 %/°C that corresponds to 0.17 ± 0.03 pF/°C for an average myocyte of 56.6 ± 9.9 pF capacitance (mean ± SEM). The increase of capacitance was proportional to the cell capacitance (R=0.81). There were no significant differences between atrial and ventricular myocytes in the temperature sensitivity of membrane capacitance. The temperature increase caused no mechanical activity or changes in the shape of myocytes. These data are in quantitative agreement with the data obtained on HEK cells and bilayers (Shapiro et al., Nat. Commun. 3:736, 2012) and confirm the effect of temperature on cell capacitance in the case of cardiac myocytes, thus provide an independent support for the theory of optical stimulation of excitable tissue.Supported by APVV-0721-10 and by VEGA 2/0203/11 and VEGA 2/0197/11.

Charaterization of Calcium Current and Exocytosis in Zebrafish Lateral Line Hair Cells During Development

Zebrafish offer a genetically manipulable system for studying hair cell signal transduction and exocytosis, but little is known about the physiological properties of hair cells in the developing zebrafish. To study these properties, we have developed an in vivo voltage clamp preparation that allows one to record from hair cells in the lateral line organs of living zebrafish. Here, we report the physiological properties of the hair cells of the zebrafish lateral line at 4 to 12 days post-fertilization (dpf). Hair cells were voltage clamped using a two-sine wave technique to characterize the calcium current and membrane capacitance change during exocytosis. In response to step depolarizations, hair cells exhibited maxima calcium current (8.6 +/-0.97pA) at −10mV beginning at 4-8 dpf, which were accompanied by near-linear increases in capacitance (117.4+/-32fF/s). At approximately 9-12 dpf, a shift in the I-V relationship of the calcium current occurs and hair cells begin to exhibit dramatically larger calcium current (18.3+/- 2.9 pA)) with peak current exhibited at 0-20 mV .The exocytosis (169+/-21 fF/s) also increased at −10mV. These results indicate a dramatic developmental switch in hair cell properties in the second week of life for zebrafish.

Insights into monitoring changes in the viable cell density and cell physiology using scanning, multi-frequency dielectric spectroscopy

• Dielectric spectroscopy (DS) is now the most common method for estimating the in situ live cell concentration in animal cell culture. • DS and traditional offline methods for cell counting based on Trypan Blue correlate well during the growth phases but with some cell lines, deviations are observed during the late growth phase. • Scanning multi-frequency DS can detect the physiological changes of the cells during the death phase of the culture including changes in cell size, membrane capacitance and internal conductivity [1-3]. • The concept of using the Area Ratio Algorithm (ARA) looks to be a relatively simple and promising method for providing on-line cell counts that correlate well with traditional methods for the complete cell growth cycle.

Dynamic Response of Model Lipid Membranes to Ultrasonic Radiation Force

Low-intensity ultrasound can modulate action potential firing in neurons in vitro and in vivo. It has been suggested that this effect is mediated by mechanical interactions of ultrasound with neural cell membranes. We investigated whether these proposed interactions could be reproduced for further study in a synthetic lipid bilayer system. We measured the response of protein-free model membranes to low-intensity ultrasound using electrophysiology and laser Doppler vibrometry. We find that ultrasonic radiation force causes oscillation and displacement of lipid membranes, resulting in small (<1%) changes in membrane area and capacitance. Under voltage-clamp, the changes in capacitance manifest as capacitive currents with an exponentially decaying sinusoidal time course. The membrane oscillation can be modeled as a fluid dynamic response to a step change in pressure caused by ultrasonic radiation force, which disrupts the balance of forces between bilayer tension and hydrostatic pressure. We also investigated the origin of the radiation force acting on the bilayer. Part of the radiation force results from the reflection of the ultrasound from the solution/air interface above the bilayer (an effect that is specific to our experimental configuration) but part appears to reflect a direct interaction of ultrasound with the bilayer, related to either acoustic streaming or scattering of sound by the bilayer. Based on these results, we conclude that synthetic lipid bilayers can be used to study the effects of ultrasound on cell membranes and membrane proteins.

Synthesis, characterization and influence of electrolyte solutions on electrical properties of organic–inorganic composite membrane

In this work various electrical properties of titanium molybdate composite membrane had been discussed. The membrane was characterized by X-ray diffraction, scanning electron microscopy, particle size analysis and Infra-red spectroscopy. The effective fixed charge density of the membrane was determined by TMS method and it showed the dependence of membrane potential on, the charge on the membrane matrix, porosity and size of permeating ions. The change in membrane capacitance and resistance values with the change in electrolyte concentration and applied frequency had been interpreted in terms of the charges produced in the electrical double layer at the membrane solution interface. The magnitude of membrane capacitance had been found to be dependent on the capacitance of the double layer. In higher frequency range the impedance data evaluated on the basis of simple equivalent electrical circuit model and had been found to follow theoretical prediction. Other parameters such as transport number, distribution coefficient and charge effectiveness were also calculated.

Otoferlin Couples to Clathrin-Mediated Endocytosis in Mature Cochlear Inner Hair Cells

The encoding of auditory information with indefatigable precision requires efficient resupply of vesicles at inner hair cell (IHC) ribbon synapses. Otoferlin, a transmembrane protein responsible for deafness in DFNB9 families, has been postulated to act as a calcium sensor for exocytosis as well as to be involved in rapid vesicle replenishment of IHCs. However, the molecular basis of vesicle recycling in IHCs is largely unknown. In the present study, we used high-resolution liquid chromatography coupled with mass spectrometry to copurify otoferlin interaction partners in the mammalian cochlea. We identified multiple subunits of the adaptor protein complex AP-2 (CLAP), an essential component of clathrin-mediated endocytosis, as binding partners of otoferlin in rats and mice. The interaction between otoferlin and AP-2 was confirmed by coimmunoprecipitation. We also found that AP-2 interacts with myosin VI, another otoferlin binding partner important for clathrin-mediated endocytosis (CME). The expression of AP-2 in IHCs was verified by reverse transcription PCR. Confocal microscopy experiments revealed that the expression of AP-2 and its colocalization with otoferlin is confined to mature IHCs. When CME was inhibited by blocking dynamin action, real-time changes in membrane capacitance showed impaired synaptic vesicle replenishment in mature but not immature IHCs. We suggest that an otoferlin-AP-2 interaction drives Ca(2+)- and stimulus-dependent compensating CME in mature IHCs.

The V499G/Y501H Mutation Impairs Fast Motor Kinetics of Prestin and Has Significance for Defining Functional Independence of Individual Prestin Subunits

Outer hair cells (OHCs) are a mammalian innovation for mechanically amplifying sound energy to overcome the viscous damping of the cochlear partition. Although the voltage-dependent OHC membrane motor, prestin, has been demonstrated to be essential for mammalian cochlear amplification, the molecular mechanism by which prestin converts electrical energy into mechanical displacement/force remains elusive. Identifying mutations that alter the motor function of prestin provides vital information for unraveling the energy transduction mechanism of prestin. We show that the V499G/Y501H mutation does not deprive prestin of its voltage-induced motor activity, but it does significantly impair the fast motor kinetics and voltage operating range. Furthermore, mutagenesis studies suggest that Val-499 is the primary site responsible for these changes. We also show that V499G/Y501H prestin forms heteromers with wild-type prestin and that the fast motor kinetics of wild-type prestin is not affected by heteromer formation with V499G/Y501H prestin. These results suggest that prestin subunits are individually functional within a given multimer.

Systematic dielectrophoretic analysis of the Ara C induced NB4 cell apoptosis combined with gene expression profiling

Dielectrophoresis (DEP) can be used to noninvasively measure the dielectric state of the cell, and this data can be used to monitor cell health or apoptosis. In this study, we followed events associated with cytosine arabinoside (Ara-C)-induced apoptosis in NB4 cells using DEP analysis. Our data showed that the membrane capacitance of NB4 cells decreases from 9.42 to 7.63 mF/m2 in the first 2 hours following treatment with Ara-C, and that this decreased capacitance persists for >12 hours. Additionally, cytoplasmic conductivity decreases from 0.217 to 0.190 S/m within 2 hours of Ara-C treatment; this level is maintained for a short period of time before decreasing. We also investigated these events molecularly at the level of gene expression using microarray analysis and showed that the expression of genes related to membrane capacitance and cytoplasmic conductivity change dramatically as early as 2 hours post-Ara-C treatment, and further demonstrated a temporal relationship between the dielectric properties and key events in apoptosis. This study, integrating physical electrical properties of the cell membrane and cytoplasm with those of conductivity-related gene networks, provides new insights into the molecular mechanisms underlying the initiation of apoptosis, establishing a systematic foundation for DEP application in follow-up drug screening and development of medicines for treating leukemia.

The Epithelial Sodium Channel (ENaC) Establishes a Trafficking Vesicle Pool Responsible for Its Regulation

The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na+ transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,β,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.

Bidirectional regulation of adrenal catecholamine release by prostaglandin E2

Catecholamine secretion from adrenal chromaffin cells is elevated in response to adverse physiological conditions. Similarly, systemic immune challenge leads to intra‐adrenal inflammatory signaling and a resulting induction of synthetic enzymes for prostaglandin E2 (PGE2). In the present study we investigated the functional impact of PGE2 on calcium signaling and catecholamine release from mouse adrenal chromaffin cells. PGE2 potently (EC50 = 5.5 nM) inhibited Cav2 voltage‐gated calcium currents (ICa), while having no effect on resting intracellular [Ca2+] or the peak amplitude of nicotinic acetylcholine receptor currents. The inhibition of ICa is mediated by pertussis toxin‐sensitive G proteins, and reversed by a strongly depolarizing voltage step, characteristic of direct G protein βγ subunit binding to the channel. PGE2 acts primarily through a family of cognate GPCRs (EP1‐EP4). We detected mRNA for all four EP receptors in mouse adrenal gland, but using selective pharmacological tools and EP receptor knockout mice we show the inhibition of ICa is mediated by EP3 receptors. Monitoring changes in membrane capacitance in response to brief depolarizations, we show Ca2+‐ dependent exocytosis is reduced in parallel with ICa in wild‐type mice. Conversely, PGE2 potentiates exocytosis in chromaffin cells isolated from EP3 receptor knockout mice, revealing a signaling modality distinct from the inhibitory EP3 pathway. Using carbon fiber amperometry, we find the potentiation of exocytosis is also apparent in wild type cells during sustained depolarizations mimicking acute stress. Our data support a model in which distinct EP receptor subtypes mediate activity‐dependent, bidirectional modulation of catecholamine release by PGE2. This work is supported by the American Heart Association [11GRNT7890031] and the National Institutes of Health [R01 NS052446].

Patch-clamp Capacitance Measurements and Ca&lt;sup&gt;2+&lt;/sup&gt; Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 μm diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca(2+) currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABA(A) and GABA(C) receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied. The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

The Role of Caveolae and Membrane Conformation in Load-Induced Ventricular Conduction Slowing

Mechanical load has been shown to alter cardiac electrophysiology, but the underlying mechanisms are still not completely understood. Conduction slowing has been observed during increased ventricular load in the isolated heart and has been associated with changes in intercellular coupling and membrane capacitance. The goal of this work was to investigate the effect of increased ventricular pressure on cardiomyocyte membrane conformation and caveolar structure, and the role of these changes in conduction slowing associated with load. Murine hearts were isolated, retrogradely perfused, and pressure loaded through a cannula inserted into the left ventricular cavity. In the pressure loaded wild-type heart, electron microscopy revealed a tautening of excess membrane folds, fewer sub-sarcolemmal caveolae, and an apparent opening of the neck of sarcolemmal caveolae compared to the unloaded state. Optical mapping revealed a significant 15-18% slowing of conduction velocity with load in both the directions of fastest and slowest conduction (CVmax and CVmin, respectively). Hearts isolated from caveolin-3 deficient mice have no caveolae on the cardiomyocyte membranes, and control hearts treated with methyl-β-cyclodextrin (MβCD) have a diminished number of caveolar structures, as assessed by electron microscopy. These hearts lacked changes in sub-sarcolemmal caveolae and caveolar structure with pressure load, and conduction slowing was attenuated. Cardiomyocyte caveolar localization and conformation change with increased ventricular load, and this alteration appears to play an important role in load-induced conduction slowing, possibly by contributing to changes in effective membrane capacitance.