Imperfect Space Clamp Permits Electrotonic Interactions between Inhibitory and Excitatory Synaptic Conductances, Distorting Voltage Clamp Recordings

1. The magnitude and kinetics of inhibitory postsynaptic currents (IPSCs) evoked in the goldfish Mauthner (M-) cell by intracellular stimulation of identified presynaptic interneurons (unitary responses) and by activation of the recurrent collateral network were determined with single-and double electrode voltage-clamp techniques. 2. The peak magnitude of the inhibitory conductance changes were 5610 +/- 4800 nS (mean +/- SD; n = 13) for the collateral response, and 144 +/- 44 nS (n = 7) for the unitary IPSCs. These synaptic conductances, which are due to the opening of Cl- channels, were independent of the degree of Cl- -loading of the M-cell. 3. The peak amplitude of the collateral inhibitory postsynaptic potential (IPSP) was a constant fraction (0.52 +/- 0.06) of the driving force, which was determined from current-voltage plots for both types of IPSCs and ranged from 10 to 37 mV. These findings confirm indirect measurements from previous current-clamp studies and validate the normalization procedure used to previously calculate synaptic conductances from IPSP amplitudes, a method that therefore may be applicable to other central neurons. 4. At the resting membrane potential, the rise time of the unitary IPSCs was 0.34 +/- 0.07 ms (n = 18), whereas their decay was exponential, with a time constant of 5.7 +/- 1.1 ms (n = 16). 5. Iontophoretic and intramuscular applications of the glycine antagonist strychnine reduced or blocked M-cell inhibitory responses, without altering the excitability of the presynaptic neurons, or the driving force. 6. Amplitude fluctuations of unitary IPSPs recorded during partial blockade by strychnine were analyzed according to a binomial model of quantal transmitter release. In one experimental series, comparison of the binomial parameters before and after applying the antagonist indicated that only quantal size, q, was reduced, whereas n, the number of available release units, and p, the probability of release, were unaffected by strychnine. In a second series, the individual presynaptic cells were injected with horseradish peroxidase (HRP), and it was found that the correlation between n and the number of stained presynaptic boutons and, therefore, of active zones, was maintained in the presence of the drug. No evidence was found for silent synapses in these conditions. 7. The quantal conductance, gq, was estimated from the binomially derived quantal size, in millivolts, and the voltage-clamp measurements of the IPSP driving force and M-cell input conductance. gq averaged 21.5 nS in control conditions and 12.3 nS in the presence of strychnine.(ABSTRACT TRUNCATED AT 400 WORDS)

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Determination of Effective Synaptic Conductances Using Somatic Voltage Clamp v1 (protocols.io.wm4fc8w)

protocols.io ◽

10.17504/protocols.io.wm4fc8w ◽

2018 ◽

Author(s):

Songting Li ◽

Nan Liu ◽

Xiaohui Zhang ◽

Douglas Zhou ◽

David Cai

Keyword(s):

Voltage Clamp ◽

Synaptic Conductances

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Faculty Opinions recommendation of Whole-cell voltage clamp measurements of anthrax toxin pore current.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1029941.348418 ◽

2005 ◽

Author(s):

Sjur Olsnes

Keyword(s):

Voltage Clamp ◽

Cell Voltage ◽

Anthrax Toxin ◽

Whole Cell ◽

Whole Cell Voltage Clamp

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Faculty Opinions recommendation of Dendritic spines prevent synaptic voltage clamp.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732320538.793540955 ◽

2017 ◽

Author(s):

Jeffry Isaacson

Keyword(s):

Voltage Clamp ◽

Dendritic Spines

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Characterization of Transport Activity of SLC11 Transporters in Xenopus laevis Oocytes by Fluorophore Quenching

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/24725552211004123 ◽

2021 ◽

pp. 247255522110041

Author(s):

Raffaella Cinquetti ◽

Francesca Guia Imperiali ◽

Salvatore Bozzaro ◽

Daniele Zanella ◽

Francesca Vacca ◽

...

Keyword(s):

Xenopus Laevis ◽

Voltage Clamp ◽

Expression System ◽

Peptide Transporter ◽

Xenopus Laevis Oocytes ◽

Substrate Uptake ◽

Transport Activity ◽

Experimental Conditions ◽

Metal Transporters ◽

Electrode Voltage

Membrane proteins are involved in different physiological functions and are the target of pharmaceutical and abuse drugs. Xenopus laevis oocytes provide a powerful heterologous expression system for functional studies of these proteins. Typical experiments investigate transport using electrophysiology and radiolabeled uptake. A two-electrode voltage clamp is suitable only for electrogenic proteins, and uptake measurements require the existence of radiolabeled substrates and adequate laboratory facilities. Recently, Dictyostelium discoideum Nramp1 and NrampB were characterized using multidisciplinary approaches. NrampB showed no measurable electrogenic activity, and it was investigated in Xenopus oocytes by acquiring confocal images of the quenching of injected fluorophore calcein. This method is adequate to measure the variation in emitted fluorescence, and thus transporter activity indirectly, but requires long experimental procedures to collect statistically consistent data. Considering that optimal expression of heterologous proteins lasts for 48–72 h, a slow acquiring process requires the use of more than one batch of oocytes to complete the experiments. Here, a novel approach to measure substrate uptake is reported. Upon injection of a fluorophore, oocytes were incubated with the substrate and the transport activity measured, evaluating fluorescence quenching in a microplate reader. The technique permits the testing of tens of oocytes in different experimental conditions simultaneously, and thus the collection of significant statistical data for each batch, saving time and animals. The method was tested with different metal transporters (SLC11), DMT1, DdNramp1, and DdNrampB, and verified with the peptide transporter PepT1 (SLC15). Comparison with traditional methods (uptake, two-electrode voltage clamp) and with quenching images acquired by fluorescence microscopy confirmed its efficacy.

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Epileptiform activity in the hippocampus produced by tetraethylammonium

Journal of Neurophysiology ◽

10.1152/jn.1990.64.4.1077 ◽

1990 ◽

Vol 64 (4) ◽

pp. 1077-1088 ◽

Cited By ~ 24

Author(s):

P. A. Rutecki ◽

F. J. Lebeda ◽

D. Johnston

Keyword(s):

Potassium Channel ◽

Voltage Clamp ◽

Channel Blocker ◽

Mossy Fiber ◽

Reversal Potential ◽

Evoked Response ◽

Epileptiform Discharge ◽

Extracellular Potassium ◽

Potassium Channel Blocker ◽

Epileptiform Discharges

1. The epileptiform discharges in the CA3 region of the rat hippocampal slice produced by bath application of the potassium channel blocker tetraethylammonium (TEA) were investigated. The effects of a convulsant (5 mM) and subconvulsant (0.5 mM) concentration of TEA on the mossy fiber-evoked synaptic currents were studied by the use of voltage-clamp techniques to determine whether TEA, like 4-aminopyridine (4-AP), another potassium channel blocker and convulsant, increased both inhibitory and excitatory components of the synaptic response. 2. At extracellular potassium concentrations of 2.5 mM, TEA (5 mM) was found to produce spontaneously occurring epileptiform discharges that could be recorded extracellularly. The intracellular correlate of the epileptiform discharge, the paroxysmal depolarizing shift (PDS), could be reversed in polarity by depolarizing the membrane and was associated with a large increase in membrane conductance. These results suggest that a synaptically mediated potential underlies the generation of the epileptiform discharge. 3. The reversal potential for the PDS was dependent on the time, relative to the extracellularly recorded field discharge, at which the measurement was made. In current clamp the mean reversal potential of the PDS measured at the midpoint of the extracellular discharge was -3.3 +/- 2.9 (SE) mV (n = 9). The reversal potential of the PDS was considerably more negative when measured either before or after the midpoint of the extracellular discharge, suggesting the presence of an inhibitory synaptic component. In voltage clamp similar results were obtained and a large conductance change was found to be associated with the PDS. These results suggest that the synaptic conductance associated with the PDS has both inhibitory and excitatory components. 4. TEA increased significantly the mossy fiber-evoked, early-inhibitory conductance. A convulsant concentration (5 mM) increased the conductance measured 15 ms after the stimulus from 39.7 +/- 8.7 to 87.2 +/- 8.0 nS (n = 6). The reversal potential associated with the conductance depolarized from -68.3 +/- 3.4 to -58.3 +/- 4.0 mV after 5 mM TEA. A subconvulsant concentration of TEA (0.5 mM) also increased the conductance of the mossy fiber-evoked response at 15 ms after the stimulus from 49.5 +/- 3.1 to 63.1 +/- 6.1 nS (n = 4) without an associated shift in reversal potential. 5. The late-inhibitory component of the mossy fiber-evoked response, when present, was increased by 5 mM TEA and unchanged by 0.5 mM TEA. 6. The excitatory mossy fiber-evoked synaptic current was studied in the presence of picrotoxin and was found to be increased and prolonged by 5 mM TEA.(ABSTRACT TRUNCATED AT 400 WORDS)

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