scholarly journals Cell dialysis by sharp electrodes can cause nonphysiological changes in neuron properties

2015 ◽  
Vol 114 (2) ◽  
pp. 1255-1271 ◽  
Author(s):  
Scott L. Hooper ◽  
Jeffrey B. Thuma ◽  
Christoph Guschlbauer ◽  
Joachim Schmidt ◽  
Ansgar Büschges
Keyword(s):  
Membrane Potential ◽  
Outward Current ◽  
Input Resistance ◽  
Time Dependent ◽  
Current Time ◽  
Ion Concentrations ◽  
Electrode Penetration ◽  
Hyperpolarization Activated Current ◽  
Property Changes ◽  
Glucose Electrode

We recorded from lobster and leech neurons with two sharp electrodes filled with solutions often used with these preparations (lobster: 0.6 M K2SO4 or 2.5 M KAc; leech: 4 M KAc), with solutions approximately matching neuron cytoplasm ion concentrations, and with 6.5 M KAc (lobster, leech) and 0.6 M KAc (lobster). We measured membrane potential, input resistance, and transient and sustained depolarization-activated outward current amplitudes in leech and these neuron properties and hyperpolarization-activated current time constant in lobster, every 10 min for 60 min after electrode penetration. Neuron properties varied with electrode fill. For fills with molarities ≥2.5 M, neuron properties also varied strongly with time after electrode penetration. Depending on the property being examined, these variations could be large. In leech, cell size also increased with noncytoplasmic fills. The changes in neuron properties could be due to the ions being injected from the electrodes during current injection. We tested this possibility in lobster with the 2.5 M KAc electrode fill by making measurements only 10 and 60 min after penetration. Neuron properties still changed, although the changes were less extreme. Making measurements every 2 min showed that the time-dependent variations in neuron properties occurred in concert with each other. Neuron property changes with high molarity electrode-fill solutions were great enough to decrease neuron firing strongly. An experiment with 14C-glucose electrode fill confirmed earlier work showing substantial leak from sharp electrodes. Sharp electrode work should thus be performed with cytoplasm-matched electrode fills.

Ca2+-activated Cl− current in cultured myenteric neurons from murine proximal colon

2003 ◽  
Vol 284 (4) ◽  
pp. C839-C847 ◽  
Author(s):  
Sok Han Kang ◽  
Pieter Vanden Berghe ◽  
Terence K. Smith
Keyword(s):  
Membrane Potential ◽  
Channel Blocker ◽  
Reversal Potential ◽  
Outward Current ◽  
Proximal Colon ◽  
Time Dependent ◽  
Equilibrium Potential ◽  
Resting Membrane Potential ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp

Whole cell patch-clamp recordings were made from cultured myenteric neurons taken from murine proximal colon. The micropipette contained Cs+ to remove K+ currents. Depolarization elicited a slowly activating time-dependent outward current ( I tdo), whereas repolarization was followed by a slowly deactivating tail current ( I tail). I tdo and I tail were present in ∼70% of neurons. We identified these currents as Cl− currents ( I Cl), because changing the transmembrane Cl− gradient altered the measured reversal potential ( E rev) of both I tdo and I tail with that for I tailshifted close to the calculated Cl− equilibrium potential ( E Cl). I Cl are Ca2+-activated Cl− current [ I Cl(Ca)] because they were Ca2+dependent. E Cl, which was measured from the E rev of I Cl(Ca) using a gramicidin perforated patch, was −33 mV. This value is more positive than the resting membrane potential (−56.3 ± 2.7 mV), suggesting myenteric neurons accumulate intracellular Cl−. ω-Conotoxin GIVA [0.3 μM; N-type Ca2+ channel blocker] and niflumic acid [10 μM; known I Cl(Ca) blocker], decreased the I Cl(Ca). In conclusion, these neurons have I Cl(Ca) that are activated by Ca2+entry through N-type Ca2+ channels. These currents likely regulate postspike frequency adaptation.

Properties of potassium currents in Purkinje cells of failing human hearts

2002 ◽  
Vol 283 (6) ◽  
pp. H2495-H2503 ◽  
Author(s):  
Wei Han ◽  
Liming Zhang ◽  
Gernot Schram ◽  
Stanley Nattel
Keyword(s):  
Purkinje Cells ◽  
Ventricular Myocytes ◽  
Outward Current ◽  
Ionic Currents ◽  
Time Dependent ◽  
Transient Outward Current ◽  
Whole Cell Patch Clamp ◽  
Cardiac Purkinje Fibers ◽  
Cardiac Purkinje Cells ◽  
Hyperpolarization Activated Current

Cardiac Purkinje fibers play an important role in cardiac arrhythmias, but no information is available about ionic currents in human cardiac Purkinje cells (PCs). PCs and midmyocardial ventricular myocytes (VMs) were isolated from explanted human hearts. K+ currents were evaluated at 37°C with whole cell patch clamp. PCs had clear inward rectifier K+current ( I K1), with a density not significantly different from VMs between −110 and −20 mV. A Cs+-sensitive, time-dependent hyperpolarization-activated current was measurable negative to −60 mV. Transient outward current ( I to) density was smaller, but end pulse sustained current ( I sus) was larger, in PCs vs. VMs. I to recovery was substantially slower in PCs, leading to strong frequency dependence. Unlike VM I to, which was unaffected by 10 mM tetraethylammonium, Purkinje I to was strongly inhibited by tetraethylammonium, and Purkinje I to was 10-fold more sensitive to 4-aminopyridine than VM. PC I sus was also reduced strongly by 10 mM tetraethylammonium. In conclusion, human PCs demonstrate a prominent I K1, a time-dependent hyperpolarization-activated current, and an I towith pharmacological sensitivity and recovery kinetics different from those in the atrium or ventricle and compatible with a different molecular basis.

scholarly journals Permeant ion effects on the gating kinetics of the type L potassium channel in mouse lymphocytes.

1991 ◽  
Vol 97 (6) ◽  
pp. 1251-1278 ◽  
Author(s):  
M S Shapiro ◽  
T E DeCoursey
Keyword(s):  
Mole Fraction ◽  
Outward Current ◽  
K Channel ◽  
Current Time ◽  
Ion Concentrations ◽  
Gating Kinetics ◽  
Outer Vestibule ◽  
Ion Species ◽  
Kinetics Of ◽  
External K

Permeant ion species was found to profoundly affect the gating kinetics of type l K+ currents in mouse T lymphocytes studied with the whole-cell or on-cell patch gigaohm-seal techniques. Replacing external K+ with Rb+ (as the sole monovalent cation, at 160 mM) shifted the peak conductance voltage (g-V) relation by approximately 20 mV to more negative potentials, while NH4+ shifted the g-V curve by 15 mV to more positive potentials. Deactivation (the tail current time constant, tau tail) was slowed by an average of 14-fold at -70 mV in external Rb+, by approximately 8-fold in Cs+, and by a factor of two to three in NH4+. Changing the external K+ concentration, [K+]o, from 4.5 to 160 mM or [Rb+]o from 10 to 160 mM had no effect on tau tail. With all the internal K+ replaced by Rb+ or Cs+ and either isotonic Rb+ or K+ in the bath, tau tail was indistinguishable from that with K+ in the cell. With the exception of NH4+, activation time constants were insensitive to permeant ion species. These results indicate that external permeant ions have stronger effects than internal permeant ions, suggesting an external modulatory site that influences K+ channel gating. However, in bi-ionic experiments with reduced external permeant ion concentrations, tau tail was sensitive to the direction of current flow, indicating that the modulatory site is either within the permeation pathway or in the outer vestibule of the channel. The latter interpretation implies that outward current through an open type l K+ channel significantly alters local ion concentrations at the modulatory site in the outer vestibule, and consequently at the mouth of the channel. Experiments with mixtures of K+ and Rb+ in the external solution reveal that deactivation kinetics are minimally affected by addition of Rb+ until the Rb+ mole fraction approaches unity. This relationship between mole fraction and tau tail, together with the concentration independence of tau tail, was hard to reconcile with simple models in which occupancy of a site within the permeation pathway prevents channel closing, but is consistent with a model in which a permeant ion binding site in the outer vestibule modulates gating depending on the species of ion occupying the site. A description of the ionic selectivity of the type l K+ channel is presented in the companion paper (Shapiro and DeCoursey, 1991b).

scholarly journals MOTOR NEURONES OF THE CRAYFISH WALKING SYSTEM POSSESS TEA+-REVEALED REGENERATIVE ELECTRICAL PROPERTIES

1994 ◽  
Vol 188 (1) ◽  
pp. 339-345
Author(s):  
D Cattaert ◽  
A Araque ◽  
W Buno ◽  
F Clarac
Keyword(s):  
Membrane Potential ◽  
Outward Current ◽  
Rhythmic Activity ◽  
Input Resistance ◽  
Stomatogastric Ganglion ◽  
Resting Membrane Potential ◽  
Delayed Rectifier ◽  
Muscarinic Agonist ◽  
M Current ◽  
Motor Neurones

In crustaceans, some motor neurones (MNs) have been shown to be part of the central pattern generator in the stomatogastric system (Harris-Warrick et al. 1992; Moulins, 1990), the swimmeret system (Heitler, 1978) or the walking system (Chrachri and Clarac, 1990). These MNs induce changes in the central rhythm when depolarized and are conditional oscillators in the stomatogastric ganglion. Moreover, in the walking system, rhythmic activity can be triggered by muscarinic cholinergic agonists (Chrachri and Clarac, 1987). We have recently analyzed the role of muscarinic receptors in crayfish walking leg MNs (D. Cattaert and A. Araque, in preparation) and demonstrated that oxotremorine, a muscarinic agonist, evoked long-lasting depolarizing responses associated with an increased input resistance. The outward current blocked by oxotremorine is likely to be carried by K+, as is the case for the M current (IM) in vertebrates (Brown and Adams, 1980). In most neurones, K+ conductances play a principal role in maintaining the membrane potential at rest: for example, IM is active at the resting membrane potential, thus contributing to its maintenance, and the 'delayed-rectifier' (IK) assists the fast repolarization after an action potential. Some K+ conductances are Ca2+-dependent (IK,Ca) and are activated by an increase in internal Ca2+ concentration. In such cases, Ca2+ currents may result in hyperpolarization of the neurone through activation of IK,Ca. In opposition to these K+ currents, the direct effect of Na+ and Ca2+ conductances is to depolarize the neurone. For example, the persistant Na+ current (INap) that is responsible for the slow subthreshold depolarization termed slow pre-potentials (Gestrelius et al. 1983; Leung and Yim, 1991) participates in the formation of pacemaker depolarization (Barrio et al. 1991) and generates plateau-type responses in control conditions (Barrio et al. 1991; Llinas and Sugimori, 1980). Similarly Ca2+ or non-specific (Na+/Ca2+) conductances generate such events in Aplysia californica burster neurones (Adams and Benson, 1985), crustacean cardiac ganglion (Tazaki and Cooke, 1990), insect neurones (Hancox and Pitman, 1991) and crustacean stomatogastric ganglion (Kiehn and Harris-Warrick, 1992). Since crustacean MNs can participate in rhythm production, such depolarizing conductances may exist in most of them and may contribute to the long-lasting MN depolarizations and spike bursts present during locomotion.

scholarly journals EPSPs in rat neocortical neurons in vitro. I. Electrophysiological evidence for two distinct EPSPs

1989 ◽  
Vol 61 (3) ◽  
pp. 607-620 ◽  
Author(s):  
B. Sutor ◽  
J. J. Hablitz
Keyword(s):  
Membrane Potential ◽  
Outward Current ◽  
Pyramidal Cells ◽  
Input Resistance ◽  
Membrane Current ◽  
Inward Rectification ◽  
Resting Membrane Potential ◽  
Bipolar Electrode ◽  
Postsynaptic Potentials ◽  
Current Pulses

1. To investigate excitatory postsynaptic potentials (EPSPs), intracellular recordings were performed in layer II/III neurons of the rat medial frontal cortex. The average resting membrane potential of the neurons was more than -75 mV and their average input resistance was greater than 20 M omega. The amplitudes of the action potentials evoked by injection of depolarizing current pulses were greater than 100 mV. The electrophysiological properties of the neurons recorded were similar to those of regular-spiking pyramidal cells. 2. Current-voltage relationships, determined by injecting inward and outward current pulses, displayed considerable inward rectification in both the depolarizing and hyperpolarizing directions. The steady-state input resistance increased with depolarization and decreased with hyperpolarization, concomitant with increases and decreases, respectively, in the membrane time constant. 3. Postsynaptic potentials were evoked by electrical stimulation via a bipolar electrode positioned in layer IV of the neocortex. Stimulus-response relationships, determined by gradually increasing the stimulus intensity, were consistent among the population of neurons examined. A short-latency EPSP [early EPSP (eEPSP)] was the response with the lowest threshold. Amplitudes of the eEPSP ranged from 4 to 8 mV. Following a hyperpolarization of the membrane potential, the amplitude of the eEPSP decreased. Upon depolarization, a slight increase in amplitude and duration was observed, accompanied by a significant increase in time to peak. 4. The membrane current underlying the eEPSP (eEPSC) was measured using the single-electrode voltage-clamp method. The amplitude of the eEPSC was apparently independent of the membrane potential in 8 of 12 neurons tested. In the other 4 neurons, the amplitude of the eEPSC increased with hyperpolarization and decreased with depolarization. 5. Higher stimulus intensities evoked, in addition to the eEPSP, a delayed EPSP [late EPSP (lEPSP)] in greater than 90% of the neurons tested. The amplitude of the lEPSP ranged from 12 to 20 mV, and the latency varied between 20 and 60 ms. The amplitude of the lEPSP varied with membrane potential, decreasing with depolarization and increasing following hyperpolarization. The membrane current underlying the lEPSP (lEPSC) displayed a similar voltage dependence. 6. At stimulus intensities that led to the activation of inhibitory postsynaptic potentials (IPSPs), the lEPSP was no longer observed.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of visceral primary afferent neurons in the nodose ganglion of the rabbit

1985 ◽  
Vol 54 (2) ◽  
pp. 245-260 ◽  
Author(s):  
C. E. Stansfeld ◽  
D. I. Wallis
Keyword(s):  
Action Potentials ◽  
Input Resistance ◽  
Nodose Ganglion ◽  
Time Dependent ◽  
Membrane Properties ◽  
Resting Potential ◽  
Inward Rectification ◽  
C Cells ◽  
Axonal Conduction ◽  
A Cells

The active and passive membrane properties of rabbit nodose ganglion cells and their responsiveness to depolarizing agents have been examined in vitro. Neurons with an axonal conduction velocity of less than 3 m/s were classified as C-cells and the remainder as A-cells. Mean axonal conduction velocities of A- and C-cells were 16.4 m/s and 0.99 m/s, respectively. A-cells had action potentials of brief duration (1.16 ms), high rate of rise (385 V/s), an overshoot of 23 mV, and relatively high spike following frequency (SFF). C-cells typically had action potentials with a "humped" configuration (duration 2.51 ms), lower rate of rise (255 V/s), an overshoot of 28.6 mV, an after potential of longer duration than A-cells, and relatively low SFF. Eight of 15 A-cells whose axons conducted at less than 10 m/s had action potentials of longer duration with a humped configuration; these were termed Ah-cells. They formed about 10% of cells whose axons conducted above 2.5 m/s. The soma action potential of A-cells was blocked by tetrodotoxin (TTX), but that of 6/11 C-cells was unaffected by TTX. Typically, A-cells showed strong delayed (outward) rectification on passage of depolarizing current through the soma membrane and time-dependent (inward) rectification on inward current passage. Input resistance was thus highly sensitive to membrane potential close to rest. In C-cells, delayed rectification was not marked, and slight time-dependent rectification occurred in only 3 of 25 cells; I/V curves were normally linear over the range: resting potential to 40 mV more negative. Data on Ah-cells were incomplete, but in our sample of eight cells time-dependent rectification was absent or mild. C-cells had a higher input resistance and a higher neuronal capacitance than A-cells. In a proportion of A-cells, RN was low at resting potential (5 M omega) but increased as the membrane was hyperpolarized by a few millivolts. A-cells were depolarized by GABA but were normally unaffected by 5-HT or DMPP. C-cells were depolarized by GABA in a similar manner to A-cells but also responded strongly to 5-HT; 53/66 gave a depolarizing response, and 3/66, a hyperpolarizing response. Of C-cells, 75% gave a depolarizing response to DMPP.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of ouabain on background and voltage-dependent currents in canine colonic myocytes

1990 ◽  
Vol 259 (3) ◽  
pp. C402-C408 ◽  
Author(s):  
E. P. Burke ◽  
K. M. Sanders
Keyword(s):  
Membrane Potential ◽  
Potential Gradient ◽  
Circular Muscle ◽  
Input Resistance ◽  
Pump Current ◽  
Muscle Layer ◽  
Membrane Properties ◽  
Resting Potential ◽  
Voltage Dependent ◽  
In Cells

Previous studies have suggested that the membrane potential gradient across the circular muscle layer of the canine proximal colon is due to a gradient in the contribution of the Na(+)-K(+)-ATPase. Cells at the submucosal border generate approximately 35 mV of pump potential, whereas at the myenteric border the pump contributes very little to resting potential. Results from experiments in intact muscles in which the pump is blocked are somewhat difficult to interpret because of possible effects of pump inhibitors on membrane conductances. Therefore, we studied isolated colonic myocytes to test the effects of ouabain on passive membrane properties and voltage-dependent currents. Ouabain (10(-5) M) depolarized cells and decreased input resistance from 0.487 +/- 0.060 to 0.292 +/- 0.040 G omega. The decrease in resistance was attributed to an increase in K+ conductance. Studies were also performed to measure the ouabain-dependent current. At 37 degrees C, in cells dialyzed with 19 mM intracellular Na+ concentration [( Na+]i), ouabain caused an inward current averaging 71.06 +/- 7.49 pA, which was attributed to blockade of pump current. At 24 degrees C or in cells dialyzed with low [Na+]i (11 mM), ouabain caused little change in holding current. With the input resistance of colonic cells, pump current appears capable of generating at least 35 mV. Thus an electrogenic Na+ pump could contribute significantly to membrane potential.

Noise in Integrate-and-Fire Neurons: From Stochastic Input to Escape Rates

2000 ◽  
Vol 12 (2) ◽  
pp. 367-384 ◽  
Author(s):  
Hans E. Plesser ◽  
Wulfram Gerstner
Keyword(s):  
Membrane Potential ◽  
Diffusion Approximation ◽  
Hazard Function ◽  
Time Dependent ◽  
Integrate And Fire ◽  
Probability Current ◽  
Periodic Input ◽  
Stochastic Input ◽  
Free Membrane ◽  
Noise Models

We analyze the effect of noise in integrate-and-fire neurons driven by time-dependent input and compare the diffusion approximation for the membrane potential to escape noise. It is shown that for time-dependent subthreshold input, diffusive noise can be replaced by escape noise with a hazard function that has a gaussian dependence on the distance between the (noise-free) membrane voltage and threshold. The approximation is improved if we add to the hazard function a probability current proportional to the derivative of the voltage. Stochastic resonance in response to periodic input occurs in both noise models and exhibits similar characteristics.

scholarly journals Extracellular Hco3− Dependence of Electrogenic Na/Hco3 Cotransporters Cloned from Salamander and Rat Kidney

2000 ◽  
Vol 115 (5) ◽  
pp. 533-546 ◽  
Author(s):  
Irina I. Grichtchenko ◽  
Michael F. Romero ◽  
Walter F. Boron
Keyword(s):  
Membrane Potential ◽  
Voltage Clamp ◽  
Xenopus Oocytes ◽  
Rat Kidney ◽  
Outward Current ◽  
Membrane Vesicles ◽  
Disulfonic Acid ◽  
Ambystoma Tigrinum ◽  
Ph Titration ◽  
Electrode Voltage

We studied the extracellular [HCOabstract 3 −] dependence of two renal clones of the electrogenic Na/HCO3 cotransporter (NBC) heterologously expressed in Xenopus oocytes. We used microelectrodes to measure the change in membrane potential (ΔVm) elicited by the NBC cloned from the kidney of the salamander Ambystoma tigrinum (akNBC) and by the NBC cloned from the kidney of rat (rkNBC). We used a two-electrode voltage clamp to measure the change in current (ΔI) elicited by rkNBC. Briefly exposing an NBC-expressing oocyte to HCOabstract 3 −/CO2 (0.33–99 mM HCOabstract 3−, pHo 7.5) elicited an immediate, DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid)-sensitive and Na+-dependent hyperpolarization (or outward current). In ΔVm experiments, the apparent Km for HCOabstract 3− of akNBC (10.6 mM) and rkNBC (10.8 mM) were similar. However, under voltage-clamp conditions, the apparent Km for HCOabstract 3− of rkNBC was less (6.5 mM). Because it has been reported that SOabstract 3=/HSO abstract 3− stimulates Na/HCO3 cotransport in renal membrane vesicles (a result that supports the existence of a COabstract 3= binding site with which SOabstract 3= interacts), we examined the effect of SOabstract 3=/HSO abstract 3− on rkNBC. In voltage-clamp studies, we found that neither 33 mM SOabstract 4= nor 33 mM SOabstract 3 =/HSOabstract 3− substantially affects the apparent Km for HCO abstract 3−. We also used microelectrodes to monitor intracellular pH (pHi) while exposing rkNBC-expressing oocytes to 3.3 mM HCOabstract 3 −/0.5% CO2. We found that SO abstract 3=/HSOabstract 3 − did not significantly affect the DIDS-sensitive component of the pHi recovery from the initial CO2 -induced acidification. We also monitored the rkNBC current while simultaneously varying [CO2]o, pHo, and [COabstract 3=]o at a fixed [HCOabstract 3−]o of 33 mM. A Michaelis-Menten equation poorly fitted the data expressed as current versus [COabstract 3=]o . However, a pH titration curve nicely fitted the data expressed as current versus pHo. Thus, rkNBC expressed in Xenopus oocytes does not appear to interact with SOabstract 3 =, HSOabstract 3−, or COabstract 3=.

Sign in / Sign up

Export Citation Format

Share Document