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At least 1,333 records · Page 74Linked to original sources
PubMed2004

Releasing the peri-neuronal net to patch-clamp neurons in adult CNS.

The extracellular matrix of adult neural tissue contains chondroitin sulphated proteogylcans that form a dense peri-neuronal net surrounding the cell body and proximal dendrites of many neuronal classes. Development of the peri-neuronal net beyond approximately postnatal day 17 obscures visualization and often access by patch electrodes to neuronal membranes with the result that patch clamp recordings are most readily obtained from early postnatal animals. We describe a technique in which the surface tension of a sucrose-based medium promotes partial dissociation of thin tissue slices from adult tissue. Surface tension spreads the tissue and loosens the peri-neuronal net from neuronal membranes within minutes and in the absence of proteolytic enzymes. Furthermore, the extent of dissociation can be controlled so as to maintain the overall slice structure and allow identification of specific cell classes. Excellent structural preservation of neurons and dendrites can be obtained and full access by patch electrodes made possible for current- or voltage-clamp recordings in tissue well beyond the development of peri-neuronal nets. We demonstrate the feasibility of using this approach through patch recordings from neurons in the brainstem and cerebellum of adult gymnotiform fish and in deep cerebellar nuclei of rats as old as 6 months.

Animals↗
PubMed2000

Simultaneous patch-clamp recording and calcium imaging in a rhythmically active neuronal network in the brainstem slice preparation from mouse.

Intracellular calcium signals are critical for modulation of neuronal function, and also for pathophysiological states during human neurodegenerative disease, such as Morbus Alzheimer and amyotrophic lateral sclerosis (ALS). We investigated intracellular calcium signals in motoneurones of the nucleus hypoglossus from the mouse, which were maintained in a functionally intact state of rhythmic, respiratory-related activity. Simultaneous patch-clamp recordings and calcium imaging demonstrated that rhythmic inspiratory-related clusters of action potential (AP) discharges are paralleled by calcium oscillations both in somatic and dendritic compartments. Calcium oscillations resulted primarily from the AP-induced opening of voltage-dependent calcium channels in the soma and dendrites. Dendritic calcium transients differed from somatic responses in their kinetics, amplitude, voltage dependence and regulation of basal calcium levels. Based on a combination of infrared differential interference contrast optics, microfluorimetric calcium imaging and electrophysiological patch-clamp recordings, our results demonstrate that the brainstem slice preparation is an attractive model system to study the integration and superposition of calcium signals in a functionally intact neuronal net.

Action Potentials↗
PubMedDate not supplied

Confocal microscopic detection of potential-sensitive dyes used to reveal loss of voltage control during patch-clamp experiments.

We used a fast, fluorescent, potential-sensitive indicator (Di-8-ANEPPS) in combination with laser-scanning confocal microscopy in the line-scan mode (temporal resolution 500 Hz) to independently determine the transmembrane potential in voltage-clamped cells. While a linear relation between command voltage and Di-8-ANEPPS fluorescence was found in unexcitable Sf9 cells, pronounced nonlinearities were observed in cardiac myocytes. Comparison of the fluorescence records and current traces indicated that most of the observed nonlinearities could be attributed to voltage-escape during flow of membrane current. Voltage-escape during large membrane currents may lead to various experimental difficulties during voltage-clamp experiments. The voltage recording technique based on fluorescence was then used to compare the voltage-escape during flow of Na+ and Li+ ions via voltage-dependent (TTX sensitive) Na+ channels in cardiac myocytes. In these experiments, no significant differences in the degree of voltage-escape was found, suggesting that the two currents were similar in amplitude. In addition to the application presented in this paper, confocal microscopic detection of transmembrane potential with fluorescent dyes may be a useful technique for experiments in preparations that are difficult to impale with microelectrodes because of their small size.

Animals↗
PubMed1997

Improved preparation of Xenopus oocytes for patch-clamp recording.

We report a new technique for improving the efficiency of single-channel recording in the Xenopus oocyte expression system. Conventional methods of oocyte preparation (adequate for two-electrode voltage-clamp) often leave residual adhesions between the vitelline and plasma membranes. As a result, removal of the vitelline membrane for patch-clamp recording produces microscopic damage to the oocyte plasma membrane that interferes with the formation of high-resistance electrical seals. This problem has been alleviated by a three-phase method of oocyte preparation: (1) a strong initial enzymatic digestion using both collagenase and hyaluronidase, immediately followed by (2) exposure to hypertonic media and mechanical defolliculation, (3) pre-shrinkage and selection of only those oocytes displaying clear separation between vitelline and plasma membranes. Following this selection process, oocytes were returned to solutions of normal osmolarity, incubated overnight at 19 degrees C, and then injected with RNA for expression studies. Comparison of oocytes prepared by different methods indicated that use of these three steps significantly improved the success of high-resistance seal formation, without compromising oocyte survivability or the efficiency of channel expression.

Animals↗
PubMed1997

Laser-assisted patch clamping: a methodology.

Laser microsurgery can be used to perform both cell biological manipulations, such as targeted cell ablation, and molecular genetic manipulations, such as genetic transformation and chromosome dissection. In this report, we describe a laser microsurgical method that can be used either to ablate single cells or to ablate a small area (1-3 microns diameter) of the extracellular matrix. In plants and microorganisms, the extracellular matrix consists of the cell wall. While conventional patch clamping of these cells, as well as of many animal cells, requires enzymatic digestion of the extracellular matrix, we illustrate that laser microsurgery of a portion of the wall enables patch clamp access to the plasma membrane of higher plant cells remaining situated in their tissue environment. What follows is a detailed description of the construction and use of an economical laser microsurgery system, including procedures for single cell and targeted cell wall ablation. This methodology will be of interest to scientists wishing to perform cellular or subcellular ablation with a high degree of accuracy, or wishing to study how the extracellular matrix affects ion channel function.

Cell Fusion↗
PubMed1997

Ca2+-channel-dependent and -independent inhibition of exocytosis by extracellular ATP in voltage-clamped rat adrenal chromaffin cells.

Membrane currents and capacitance were measured to examine the effects of extracellular ATP on exocytosis in voltage-clamped rat adrenal chromaffin cells. ATP reversibly inhibited Ca2+ current (ICa) and exocytosis. The dependency of exocytosis on ICa evoked by 1-s depolarizations was determined. However, inhibition of exocytosis was 2.6 times larger than that estimated from the reduction of ICa, implying the existence of a Ca2+-channel-independent pathway. This inhibition did not rely on a further reduction of the intracellular Ca2+ concentration spike. ATP reduced the rate of exocytosis induced by clamping the intracellular Ca2+ concentration. Pertussis toxin blocked the inhibitory effects of ATP on ICa and exocytosis. Although RB-2, a P2Y antagonist, blocked the inhibitory effect of ATP on ICa, RB-2 itself produced large increase or decrease in membrane capacitance. Adenosine inhibited ICa via a pertussis-toxin-sensitive pathway but did not significantly inhibit exocytosis. Our data show that extracellular ATP inhibits exocytosis via inhibition of ICa by activation of a pertussis-toxin-sensitive G-protein linked to P2Y receptors. Furthermore, our data strongly suggest that ATP activates another pathway, which is also G-protein dependent and accounts for the majority of the inhibitory effect of ATP on exocytosis.

Adenosine Triphosphate↗
PubMed1998

Quantitative analysis of dual whole-cell voltage-clamp determination of gap junctional conductance.

The dual whole-cell voltage-clamp technique is used widely for determination of kinetics and conductance of gap junctions. The use of this technique may, however, occasion to considerable errors. We have analysed the errors in steady state junctional conductance measurements under different experimental conditions. The errors in measured junctional conductance induced by series resistance alone, and by series resistance in combination with membrane resistance, were quantified both theoretically and experimentally, on equivalent resistive circuits with known resistance values in a dual voltage-clamp setup. We present and analyse a method that accounts for series resistance and membrane resistance in the determination of true junctional conductance. This method requires that series resistance is determined during the experiment, and involves some calculations to determine membrane resistance. We demonstrate that correction for both membrane and series resistance reduces the error in measured junctional conductance to near zero, even when membrane resistances on both sides of the gap junction are as low as 20 MOmega and the (true) junctional conductance is as high as 100 nS.

Computer Simulation↗
PubMed1998

Small-volume and rapid extracellular solution exchange around Xenopus oocytes during voltage-clamp recordings.

A novel method for micro extracellular perfusion of Xenopus oocytes has been designed in order to minimise the amount of test solution required for drug applications during two-microelectrode voltage-clamp experiments. Voltage-clamp experiments on oocytes are performed in a glass-covered microbath without continuous perfusion. The microelectrodes access the oocytes via two small openings in the glass cover. Test solutions are applied to a funnel at the chamber inflow and the chamber volume is subsequently exchanged by operating a suction pump connected to the chamber outflow. Only 50 microl of drug- or neurotransmitter-containing solution is required for bath application. In addition, the new "funnel application technique" enables quick and timed applications of drugs or neurotransmitters to Xenopus oocytes within 200-400 ms.

Acetylcholine↗
PubMed1998

Pharmacological properties of slow anion currents in intact guard cells of Arabidopsis. Application of the discontinuous single-electrode voltage-clamp to different species.

More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K+ currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K+ channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca2+. In the presence of K+ channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 microM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals.

Anions↗
PubMed1995

Patch-clamping in brain slices: synaptic transmission from ATP to long-term potentiation.

Application of patch-clamp techniques to brain slices has resulted in an enormous increase in the resolution of synaptic currents in mammalian central neurones. This improved resolution has allowed direct observation of miniature and evoked synaptic currents, leading, in agreement with other findings, to the conclusion that the quantal size of synaptic currents in the brain is limited by the number of postsynaptic receptors. Possible explanations for the skewed miniature distribution, observed at all fast central synapses are discussed, with reference to anatomical observations. As a result, a model is proposed which is consistent with much of the apparently contradictory data on the induction and maintenance of long-term potentiation. In addition to the study of mechanisms of synaptic transmission, improved resolution provided by the patch-clamp technique has allowed resolution of synaptic currents, in the brain, mediated by ATP. The role of ATP as a central neurotransmitter is discussed in terms of the above findings.

Adenosine Triphosphate↗
PubMed1994

Combining patch-clamp and optical methods in brain slices.

Combining patch-clamp and optical imaging techniques in brain slices offers several advantages for physiological studies of nerve cells. Numerous practical considerations weigh heavily in this design of an apparatus suitable for such combined measurements. These considerations include the thickness of the slices, the type of microscope to be used for imaging and the kind of optical signal to be measured. A system that combine optical and patch-clamp methods can be modified readily to permit studies of intracellular and extracellular signaling pathways via flash photolysis of caged compounds.

Animals↗
PubMedDate not supplied

A supercharger for single electrode voltage and current clamping.

The theory and several circuits for supercharging is described for single micro and patch electrode voltage and current clamping. Supercharging is of use where the charging time of the cell membrane capacity results in the membrane potential change to considerably lag behind the command potential. This membrane capacity charging time is dependent on the series electrode resistance, the membrane resistance and capacity. Thus although amplifier voltage response times may be in microseconds, the membrane potential response to a step command potential often requires several milliseconds. Supercharging overcomes this difficulty by accelerating the membrane capacity charging time by applying a large initial and brief supercharging or 'booster' potential or current, to the command signal. Supercharging can improve membrane potential rise times by more than a hundred and thus bring membrane potentials to a stationary state well under a millisecond. Early changes in membrane properties are thus more readily observed during voltage and current clamping without being masked by capacitive currents. This technique also provides for the measurement of membrane time constants. The circuitry described is a simple add-on to the command signal pathway, is easy to adjust and provides the appropriate supercharging potentials for sequential step command signals of different amplitudes.

Electrodes↗
PubMed1992

Influence of sympatho-adrenal system on insulin sensitivity using the euglycemic clamp technique.

The aim of the present study was to further investigate the role of the adrenergic system on insulin action using the euglycemic clamp technique. Whole-body glucose metabolism (GM) was calculated as the glucose infusion rate for maintaining euglycemia under insulin infusion and used as an indicator of insulin sensitivity. Euglycemic clamps were performed in adrenodemedullated, epinephrine-treated, phentolamine-treated (alpha-blockade), propranolol-treated (beta-blockade), and epinephrine plus phentolamine and/or propranolol-treated rats. The following results were obtained at an insulin level of approximately 80 mU/l. GM in adrenodemedullated rats (13.97 +/- 0.98 mg/kg/min) was significantly higher than that of the control rats (10.26 +/- 0.50 mg/kg/min, P less than 0.01). GM in epinephrine-treated rats (1.7 mg/kg body weight/h) was 2.12 +/- 0.49 mg/kg/min (P less than 0.001 vs. control). Dose-response curves for phentolamine and propranolol established maximally effective doses (3.0 mg and 12 mg/kg body weight/h, respectively). Using these doses, GM in epinephrine plus phentolamine-treated rats (4.90 +/- 0.39 mg/kg/min) was significantly higher than that of epinephrine alone and GM in epinephrine and propranolol-treated rats (4.49 +/- 0.47 mg/kg/min) was also significantly higher than that of the epinephrine alone. GM in the epinephrine plus both propranolol and phentolamine (5.94 +/- 0.45 mg/kg/min) was significantly higher than that of the epinephrine alone, but not different from either treatment alone and was not additive. Neither phentolamine alone (9.48 +/- 1.45 mg/kg/min), propranolol alone (10.36 +/- 0.55 mg/kg/min) or the combination of blockades (11.14 +/- 0.65 mg/kg/min) had any effect on GM.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗
PubMed1996

Elimination rate of fat emulsion particles from plasma in Japanese subjects as determined by a triglyceride clamp technique.

The elimination rate of emulsion triglyceride (TG) from plasma was investigated in Japanese subjects by a plasma TG clamp technique. Two different studies were performed. In Study 1, a lipid emulsion (20% long-chain triglyceride emulsion: LCT) was infused into a healthy research associate to achieve a certain concentration of TG in the plasma. Thereafter, the infusion rate was adjusted to maintain the chosen concentrations of TG in plasma (namely, 4-5 mmol/L, 3-4 mmol/L, and approximately 2 mmol/L) over a period of 160 min by measuring plasma TG concentrations at 10-min intervals. Concentrations of TG in plasma were clamped within 2.13 +/- 0.13 mmol/L by an infusion rate of 0.10 g.kg-1.h-1, within 3.34 +/- 0.20 mmol/L by an infusion rate of 0.14 g.kg-1.h-1, and within 4.46 +/- 0.22 mmol/L by an infusion rate of 0.11 g.kg-1.h-1. The mean rate of infusion of emulsified TG that had maintained the steady concentrations of TG in plasma was limited to the very narrow range of 0.12 +/- 0.02 g of TG.kg-1.h-1 regardless of the chosen concentration of TG in plasma. Concentrations of nonesterified fatty acids (NEFA) also remained at a fixed level of 1.378 +/- 0.103 mEq/L regardless of the chosen concentration of TG in the plasma. Study 2 was undertaken to determine whether plasma TG concentration reached a plateau during a period when emulsion TG was infused into three different subjects at a rate of 0.12 g.kg-1.h-1. The plasma TG concentrations were steady at a level of 2.04 +/- 0.32 mmol/L, and the plasma NEFA concentrations remained at a fixed level of 1.33 +/- 0.13 mEq/L, over a period of 160 min after 50-min priming infusion. These results indicate that the plasma TG elimination rate was limited to the narrow range of 0.12 +/- 0.02 g.kg-1.h-1 when the fat emulsion was infused into Japanese subjects in a steady state. However, the plasma TG elimination rate in Japanese subjects appeared to be lower than that of Europeans. This may be due to a difference in lipoprotein lipase activity caused by different dietary habits, namely, a lower fat intake.

Adult↗
PubMed1994

Modified method for the performance of glucose insulin clamp studies in conscious rats.

A modified method for performance of insulin-glucose clamp studies in rats was developed via catheterization of the tail vessels, after preconditioning of animals to limited restraint. The procedure is performed in conscious animals under local anesthesia and employs a specially designed foam rubber jacket which allows the animal mobility of the limbs and forward vision. In addition, a table utilizing a belt system allows easy positioning of the animal in the left and right lateral and supine positions during surgery. After initial development of the procedure, its use in 123 animals is reported. Line placement was successfully achieved in all cases with insignificant blood loss or morbidity and zero mortality. We note that 11% of animals did not complete the subsequent insulin-glucose clamp study due to either one of the vascular cannulae leaving the vessel (one animal), venous rupture (12 animals), or cannula blockage unrelated to surgical technique (one animal). Studies on Wistar Kyoto, Spontaneously Hypertensive, and Sprague-Dawley rats showed a fall in catecholamines after animals were replaced in cages, with stabilization within 30 min. In comparison to traditional techniques, this method is, therefore, proposed as a less traumatic and rapid way of performing infusion studies in conscious rats with a high success rate and minimization of loss of animal life due to procedural problems.

Anesthesia↗
PubMed2006

Detection of SEB gene by bilayer lipid membranes nucleic acid biosensor supported by modified patch-clamp pipette electrode.

This work reports a kind of novel bilayer lipid membranes (BLMs) nucleic acid biosensor supported by modified patch-clamp pipette electrode was developed to detect staphylococcus enterotoxins B (SEB) gene. BLMs were formed within 15 min and able to be operated at least 24 h. Hydrophobic dodecane tail (C12) modified 18 bp single-stranded DNA (ssDNA) probe was immobilized on BLMs. The electrochemical currents versus the different concentration of ssDNA probe immobilized on BLMs indicated linearly correlation. The BLMs nucleic acid biosensor was fabricated by selecting the ssDNA probe as the signal sensing element with the concentration of 273.65 ng/mL. The electrochemical performance of the biosensor for the detection of SEB was investigated. The result showed that linear relationship was found between the current and ln(concentration) from 20 to 5000 ng/mL and the detection limit was 20 ng/mL. In addition, the biosensor was specific response to SEB gene and showed no significant current alteration in electrolyte which containing no SEB gene. Finally, Atom Force Microscope (AFM) images could be observed and used to evaluate the superficial microstructure of BLMs, ssDNA immobilized on BLMs and BLMs after hybridization. The BLMs nucleic acid biosensor supported by modified patch-clamp pipette electrode will become a highly sensitive, rapid, selective analytical tool for detection of Staphylococcus aureus, which produce SEB.

Biosensing Techniques↗
PubMed2005

Calbindin-D28k content and firing pattern of hippocampal granule cells in amygdala-kindled rats: a perforated patch-clamp study.

The dentate gyrus is believed to play an important pathophysiological role during experimentally induced kindling. In this study, we investigated whether an altered content of the calcium binding protein calbindin-D(28k) or an increased intrinsic excitability of hippocampal granule cells contribute to the induction of the kindling phenomenon. We determined the firing pattern of granule cells in hippocampal slices using perforated patch-clamp recordings in current clamp mode. The expression of calbindin-D(28k) and glutamic acid decarboxylase (GAD(67)) by granule cells was analyzed immunohistochemically. Rats developed secondarily generalized limbic seizures within approximately 11 days of twice-daily stimulation of the amygdala. As reported for other kindling paradigms, this protocol induced a clear up-regulation of GAD(67) in granule cells, indicating their involvement in the induced neuronal activity. However, when comparing kindled and control rats, we could not detect any differences in intrinsic excitability: Firing frequency, after-hyperpolarisations, action potentials, input resistance and membrane potentials were nearly identical between both groups. Furthermore, we did not observe any differences in the calbindin-D(28k) immunoreactivity between groups. In every slice, virtually all granule cells were found to be strongly calbindin-D(28k) positive, and there was no apparent reduction in the general level of calbindin-D(28k) expression. We conclude that changes in intrinsic membrane properties or in the calbindin-D(28k) content of granule cells are not necessary for the development of amygdala kindling.

Action Potentials↗
PubMed2006

Firing pattern of rat hippocampal neurons: a perforated patch clamp study.

To test whether the slow afterhyperpolarization (sAHP) underlies the filter function of hippocampal granule cells (GCs), we compared the sAHP and spike frequency adaptation between granule cells and CA3 pyramidal cells (PCs) in hippocampal slices employing gramicidin perforated patch clamp recordings to best preserve the physiological cytoplasmic constitution. sAHPs were evoked in GCs and PCs with trains of action potentials in current clamp mode and showed comparable kinetics in both types of cells. The threshold frequency (500 ms firing) triggering a detectable sAHP was approximately 10 Hz and approximately 3 Hz in GCs and PCs, respectively. Half maximal sAHPs were reached at 30 Hz and 8 Hz in GCs and PCs, respectively. Maximal amplitude of sAHPs in GCs amounted to approximately 3.5 mV, was approximately 2-fold smaller than in PCs and could not be further increased with higher firing frequencies. The time course of sAHP activation was investigated with 50 Hz trains of action potentials applied for increasing durations. In both types of cells, the sAHP amplitude increased with a time constant of approximately 400 ms. Nevertheless, sAHP never exceeded 4 mV in GCs but rose to approximately 12 mV in PCs when cells fired for 3 s. The repetitive firing pattern of GCs and PCs was compared by injecting current amplitudes adjusted to provoke an initial firing frequency of 50 Hz. In GCs firing frequency declined slower (tau = 229 ms) and leveled off at a higher tonic firing frequency (28 Hz) when compared to PCs (tau = 126 ms, 18 Hz). We conclude that the intrinsic excitability of GCs cannot be primarily regulated by the sAHP. The sAHP in GCs is minimal most likely due to a small sAHP-channel density as well as to a more rigid control of intracellular Ca(2+) levels.

Animals↗