Single-electrode Voltage-clamp Technique Research Articles

Selective actions of insecticides on desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach (Periplaneta americana) neurons.

Insect desensitizing nicotinic acetylcholine (nAChD) receptors are desensitized by low concentrations of agonists, including neonicotinoid insecticides, but are essentially insensitive to spinosyns, while non-desensitizing nicotinic acetylcholine (nAChN) receptors are selectively activated by spinosyns and relatively insensitive to neonicotinoids. The single-electrode voltage-clamp technique was used to measure the actions of newer nicotinic insecticides dinotefuran, sulfoxaflor, triflumezopyrim, spinetoram and GS-ω/k-hexatoxin-Hv1a on cockroach neuronal nAChD and nAChN currents. Like imidacloprid and clothianidin, newer orthosteric nicotinic agonist insecticides dinotefuran and sulfoxaflor act by desensitizing nAChD receptors. The mesoionic insecticide triflumezopyrim selectively inhibited nAChD current with an half maximal inhibitory concentration (IC50 ) of 1.2nmol L-1 , with no activation. Unlike other Group 4 insecticides, it did not activate nAChN current, but inhibited it with an IC50 of 3.8μmol L-1 , indicating that the compound is a true antagonist. Spinosad and the spinosyn-derived insecticide spinetoram potently and selectively activated nAChN receptors. GS-ω/k-hexatoxin-Hv1a had no effect on nAChN currents and it had a complex action on nAChD currents, inhibiting at sub-nanomolar concentrations and causing some activation and enhancement of ACh-evoked currents at 30 nmol L-1 and above. Some cells express GS-ω/k-hexatoxin-Hv1a-resistant nAChD receptors. Nicotinic acetylcholine receptor competitive modulators (IRAC Group 4) and nicotinic acetylcholine receptor allosteric modulators, site II (hexatoxins, IRAC Group 32) are selective for nAChD receptors, while nicotinic acetylcholine receptor allosteric modulators, site I (spinosyns, IRAC Group 5) are selective for nAChN receptors. It is proposed that IRAC Groups 5 and 32 be re-named non-desensitizing nicotinic acetylcholine receptor allosteric modulators and desensitizing nicotinic acetylcholine receptor allosteric modulators, respectively. © 2021 Society of Chemical Industry.

Antagonist pharmacology of desensitizing and non-desensitizing nicotinic acetylcholine receptors in cockroach neurons

Two α-bungarotoxin-sensitive nicotinic acetylcholine (ACh) receptor subtypes in neurons of the American cockroach have been identified as desensitizing (nAChD) and selectively inhibitable with 100nM imidacloprid, and non-desensitizing (nAChN) and selectively inhibitable with 100pM methyllycaconitine. In this paper, the single-electrode voltage-clamp technique was used to measure concentration-response relations for the action of ACh and five antagonists on pharmacologically separated nAChD and nAChN receptors of acutely dissociated neurons from thoracic ganglia of the American cockroach. A dual bath and U-tube perfusion system was used to achieve rapid application of ACh in the continued presence of antagonists, which was essential to accurately measure inhibition by rapidly-reversible antagonists. ACh activated both receptors with an EC50 of 7μM and the antagonist potencies were (nAChD/nAChN in nM): dihydro-β-erythroidine: 1.0/5.6, d-tubocurarine: 1000/34, condelphine: 0.39/0.65, phencyclidine: 74/980 and mecamylamine 47/1150. While each of these antagonists displayed some subtype selectivity, none are selective enough to be used as subtype-selective tools. These results bring to a total of 16 the number of nicotinic compounds that have been measured on nAChD and nAChN currents. Characterization of these receptors is important for understanding the role of nAChRs in the insect nervous system and the mechanism of action of insecticides.

A Fast Na+/Ca2+-Based Action Potential in a Marine Diatom

BackgroundElectrical impulses in animals play essential roles in co-ordinating an array of physiological functions including movement, secretion, environmental sensing and development. Underpinning many of these electrical signals is a fast Na+-based action potential that has been fully characterised only in cells associated with the neuromuscular systems of multicellular animals. Such rapid action potentials are thought to have evolved with the first metazoans, with cnidarians being the earliest representatives. The present study demonstrates that a unicellular protist, the marine diatom Odontella sinensis, can also generate a fast Na+/Ca2+ based action potential that has remarkably similar biophysical and pharmacological properties to invertebrates and vertebrate cardiac and skeletal muscle cells.Methodology/Principal FindingsThe kinetic, ionic and pharmacological properties of the rapid diatom action potential were examined using single electrode current and voltage clamp techniques. Overall, the characteristics of the fast diatom currents most closely resemble those of vertebrate and invertebrate muscle Na+/Ca2+ currents.Conclusions/SignificanceThis is the first demonstration of voltage-activated Na+ channels and the capacity to generate fast Na+-based action potentials in a unicellular photosynthetic organism. The biophysical and pharmacological characteristics together with the presence of a voltage activated Na+/Ca2+ channel homologue in the recently sequenced genome of the diatom Thalassiosira pseudonana, provides direct evidence supporting the hypothesis that this rapid signalling mechanism arose in ancestral unicellular eukaryotes and has been retained in at least two phylogenetically distant lineages of eukaryotes; opisthokonts and the stramenopiles. The functional role of the fast animal-like action potential in diatoms remains to be elucidated but is likely involved in rapid environmental sensing of these widespread and successful marine protists.

Cryptogein-Induced Anion Effluxes

Anion effluxes are amongst the earliest reactions of plant cells to elicitors of defence responses. However, their properties and their role in disease resistance remain almost unknown. We previously demonstrated that cryptogein, an elicitor of tobacco defence responses, induces a nitrate (NO3−) efflux. This efflux is an early prerequisite to the cryptogein-triggered hypersensitive response (HR). Here, we analyzed the electrophysiological properties of the elicitor-mediated NO3−− efflux and clarified the mechanisms through which it contributes to cell death. Application of the discontinuous single electrode voltage-clamp technique in tobacco cells elicited with cryptogein enabled us to record the activation of slow-type deactivating anion channel currents. Cryptogein-induced plasma membrane depolarization and Ca2+ influx, an essential component of elicitor signalling for HR cell death, were prevented by inhibiting the NO3−− efflux. Similarly, pharmacological blocking of the anion efflux suppressed vacuolar collapse, a hallmark of cell death. The role of NO3−− efflux in mediating proteases activation was further assessed. It is shown that cryptogein induced the activation of three proteases with apparent molecular masses of 95, 190 and 240 kDa. Their activation occurred independently on the anion efflux and, together with cell death, was strongly reduced by cycloheximide and the protease inhibitor PMSF. In contrast, the NO3− efflux was shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during the HR. Collectively, our data indicate that anion efflux is an early prerequisite to morphological and biochemical events participating to cell death.

Rapid modulation of inhibitory synaptic currents in cerebellar Purkinje cells by BDNF

The present study examined the acute effects of exogenous BDNF on inhibitory synaptic currents in Purkinje cells in cerebellar cultures. Miniature inhibitory postsynaptic currents (mIPSCs) were recorded in cultures (20-30 days in vitro), using discontinuous single electrode voltage clamp (dSEVC) technique. The effects of BDNF were studied in untreated control cultures and in cultures in which the endogenous levels of BDNF were decreased by chronic block of neural activity with tetrodotoxin (TTX). Chronic activity deprivation did not alter the amplitude of mIPSCs in Purkinje cells, and acute application of BDNF (50 ng/ml) to Purkinje cells in TTX-treated cultures significantly potentiated the amplitude and frequency of mIPSCs. By contrast, acute application of BDNF (50 ng/ml) produced no significant changes on mIPSC activity in control neurons. At higher concentrations of BDNF (100 ng/ml), comparable effects on mIPSC activity were also observed in control neurons. Preincubation of cerebellar cultures with K252a, an inhibitor of tyrosine kinases, effectively blocked the effects of BDNF on mIPSCs. These results indicate that functional inhibitory synapses develop in the absence of neural activity, and that activation of TrkB receptors by BDNF modulates inhibitory neurotransmission in Purkinje cells at both pre- and postsynaptic sites.

NAADP activates a Ca2+ current that is dependent on F-actin cytoskeleton.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is involved in the Ca2+ response observed at fertilization in several species, including starfish. In this study, we have employed Ca2+ imaging and the single-electrode voltage-clamp technique to investigate whether the NAADP-mediated Ca2+ entry discovered in our laboratory in starfish oocytes was underlain by a membrane current and whether the response to NAADP required an intact cytoskeleton. Uncaging of preinjected NAADP evoked a cortical Ca2+ flash that was followed by the spreading of the wave to the remainder of the cell. No Ca2+ increase was detected in Ca2+-free sea water. Under voltage-clamp conditions, the photoliberation of NAADP activated an inward rectifying membrane current, which reversed at potentials more positive than +50 mV and was abolished by removal of Ca2+ but not of Na+. The current was affected by preincubation with verapamil, SKF 96356, and thapsigargin but not by preinjection of heparin, 8-NH2- cyclic ADP-ribose, or both antagonists. The membrane current and the Ca2+ wave were inhibited by latrunculin-A and jasplakinolide, which depolymerize and stabilize actin cytoskeleton, respectively. These data offer the first demonstration that NAADP initiates a Ca2+ sweep by activating a Ca2+-permeable membrane current that requires an intact F-actin cytoskeleton as other Ca2+-permeable currents, such as ICRAC and IARC.

Ion currents involved in early Nod factor response in Medicago sativa root hairs: a discontinuous single-electrode voltage-clamp study.

Nod factor [NodRm-IV(Ac,S)], isolated from the bacterium Rhizobium meliloti, induces a well-known depolarization in Medicago sativa (cv Sitel) root hairs. Analysis of this membrane response using the discontinuous single-electrode voltage-clamp technique (dSEVC) shows that anion channel, K+ channel and H+-ATPase pump currents are involved in young growing root hairs. The early Nod-factor-induced depolarization is due to increase of the inward ion current and inhibition of the H+ pump. It involved an instantaneous inward anion current (IIAC) and/or a time-dependent inward K+ current (IRKC). These two ion currents are then down-regulated while the H+ pump is stimulated, allowing long-term rectification of the membrane potential (Em). Our results support the idea that the regulation of inward current plays a primary role in the Nod-factor-induced electrical response, the nature of the ions carried by these currents depending on the activated anion and/or K+ channels at the plasma membrane.

Electrophysiological and theoretical analysis of depolarization-dependent outward currents in the dendritic membrane of an identified nonspiking interneuron in crayfish.

Depolarization-dependent outward currents were analyzed using the single-electrode voltage clamp technique in the dendritic membrane of an identified nonspiking interneuron (LDS interneuron) in situ in the terminal abdominal ganglion of crayfish. When the membrane was depolarized by more than 20 mV from the resting potential (65.0 +/- 5.7 mV), a transient outward current was observed to be followed by a sustained outward current. Pharmacological experiments revealed that these outward currents were composed of 3 distinct components. A sustained component (I(s)) was activated slowly (half rise time > 5 msec) and blocked by 20 mM TEA. A transient component (I(t1)) that was activated and inactivated very rapidly (peak time < 2.5 msec, half decay time < 1.2 msec) was also blocked by 20 mM TEA. Another transient component (I(t2)) was blocked by 100 microM 4AP, activated rapidly (peak time < 10.0 msec) and inactivated slowly (half decay time > 131.8 msec). Two-step pulse experiments have revealed that both sustained and transient components are not inactivated at the resting potential: the half-maximal inactivation was attained at -21.0 mV in I(t1), and -38.0 mV in I(t2). I(s) showed no noticeable inactivation. When the membrane was initially held at the resting potential level and clamped to varying potential levels, the half-maximal activation was attained at -36.0 mV in I(s), -31.0 mV in I(t1) and -40.0 mV in I(t2). The activation and inactivation time constants were both voltage dependent. A mathematical model of the LDS interneuron was constructed based on the present electrophysiological records to simulate the dynamic interaction of outward currents during membrane depolarization. The results suggest that those membrane conductances found in this study underlie the outward rectification of the interneuron membrane as well as depolarization-dependent shaping of the excitatory synaptic potential observed in current-clamp experiments.

Apamin-sensitive conductance mediates the K(+) current response during chemical ischemia in CA3 pyramidal cells.

Pyramidal cells typically respond to ischemia with initial transient hyperpolarization, which may represent a neuroprotective response. To identify the conductance underlying this hyperpolarization in CA3 pyramidal neurons of rat hippocampal organotypic slice cultures, recordings were obtained using the single-electrode voltage-clamp technique. Brief chemical ischemia (2 mM 2-deoxyglucose and 3 mM NaN(3), for 4 min) induced a response mediated by an increase in K(+) conductance. This current was blocked by intracellular application of the Ca(2+) chelator, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid (BAPTA), reduced with low external [Ca(2+)], and inhibited by a selective L-type Ca(2+) channel inhibitor, isradipine, consistent with the activation of a Ca(2+)-dependent K(+) conductance. Experiments with charybdotoxin (10 nM) and tetraethylammonium (TEA; 1 mM), or with the protein kinase C activator, phorbol 12,13-diacetate (PDAc; 3 microM), ruled out an involvement of a large conductance-type or an apamin-insensitive small conductance, respectively. In the presence of apamin (1 microM), however, the outward current was significantly reduced. These results demonstrate that in rat hippocampal CA3 pyramidal neurons an apamin-sensitive Ca(2+)-dependent K(+) conductance is activated in response to brief ischemia generating a pronounced outward current.

LVA and HVA Ca(2+) currents in ventricular muscle cells of the Lymnaea heart.

The single-electrode voltage-clamp technique was used to characterize voltage-gated Ca(2+) currents in dissociated Lymnaea heart ventricular cells. In the presence of 30 mM tetraethylammonium (TEA), two distinct Ca(2+) currents could be identified. The first current activated between -70 and -60 mV. It was fully available for activation at potentials more negative than -80 mV. The current was fast to activate and inactivate. The inactivation of the current was voltage dependent. The current was larger when it was carried by Ca(2+) compared with Ba(2+), although changing the permeant ion had no observable effect on the kinetics of the evoked currents. The current was blocked by Co(2+) and La(3+) (1 mM) but was particularly sensitive to Ni(2+) ions ( approximately 50% block with 100 microM Ni(2+)) and insensitive to low doses of the dihydropyridine Ca(2+) channel antagonist, nifedipine. All these properties classify this current as a member of the low-voltage-activated (LVA) T-type family of Ca(2+) currents. The activation threshold of the current (-70 mV) suggests that it has a role in pacemaking and action potential generation. Muscle contractions were first seen at -50 mV, indicating that this current might supply some of the Ca(2+) necessary for excitation-contraction coupling. The second, a high-voltage-activated (HVA) current, activated at potentials between -40 and -30 mV and was fully available for activation at potentials more negative than -60 mV. This current was also fast to activate and with Ca(2+) as the permeant ion, inactivated completely during the 200-ms voltage step. Substitution of Ba(2+) for Ca(2+) increased the amplitude of the current and significantly slowed the rate of inactivation. The inactivation of this current appeared to be current rather than voltage dependent. This current was blocked by Co(2+) and La(3+) ions (1 mM) but was sensitive to micromolar concentrations of nifedipine ( approximately 50% block 10 microM nifedipine) that were ineffective at blocking the LVA current. These properties characterize this current as a L-type Ca(2+) current. The voltage sensitivity of this current suggests that it is also important in generating the spontaneous action potentials, and in providing some of the Ca(2+) necessary for excitation-contraction coupling. These data provide the first detailed description of the voltage-dependent Ca(2+) currents present in the heart muscle cells of an invertebrate and indicate that pacemaking in the molluscan heart has some similarities with that of the mammalian heart.

Paradoxical effect of QX-314 on persistent inward currents and bistable behavior in spinal motoneurons in vivo.

Spinal motoneurons can exhibit bistable behavior, which consists of stable self-sustained firing that is initiated by a brief excitatory input and terminated by brief inhibitory input. This bistable behavior is generated by a persistent inward current (I(PIC)). In cat motoneurons with low input conductances and slow axonal conduction velocities, I(PIC) exhibits little decay with time and thus self-sustained firing is long-lasting. In contrast, in cells that have high input conductances and fast conduction velocities, I(PIC) decays with time, and these cells cannot maintain long duration self-sustained firing. An alternative way to measure bistable behavior is to assess plateau potentials after the action potential has been blocked by intracellular injection of QX-314 to block sodium (Na(+)) currents. However, QX-314 also blocks calcium (Ca(2+)) currents and, because I(PIC) may be generated by a mixture of Ca(2+) and Na(+) currents, a reduction in amplitude of I(PIC) was expected. We therefore systematically compared the properties of I(PIC) in a sample of cells recorded with QX-314 to a control sample of cells without QX-314, which was obtained in a previous study. Single-electrode voltage-clamp techniques were applied in spinal motoneurons in the decerebrate cat preparation following administration of a standardized dose of the noradrenergic alpha1 agonist methoxamine. In the sample with QX-314, the average value of I(PIC) was only about half that in the control sample. However, the reduction of I(PIC) was much greater in cells with slow as compared with fast conduction velocities. Because a substantial portion of I(PIC) originates in dendritic regions and because conduction velocity covaries with the extent of the dendritic tree, this result suggests that QX-314 may fail to diffuse very far into the dendrites of the largest motoneurons. The analysis of the decay of I(PIC) and plateau potentials in cells with QX-314 also produced an unexpected result: QX-314 virtually eliminated time-dependent decay in both I(PIC) and plateau potentials. Consequently, I(PIC) became equally persistent in high and low input conductance cells. Therefore the decay in I(PIC) in high input conductance cells in the absence of QX-314 is not due to an intrinsic tendency of the underlying inward current to decay. Instead it is possible that the decay may result from activation of a slow outward current. Overall, these results show that QX-314 has a profound effect on I(PIC) and thus plateau potentials obtained using QX-314 do not accurately reflect the properties of I(PIC) in normal cells without QX-314.

Two Types of Voltage-Gated K+Currents in Dissociated Heart Ventricular Muscle Cells of the SnailLymnaea stagnalis

We have used a combination of current-clamp and voltage-clamp techniques to characterize the electrophysiological properties of enzymatically dissociated Lymnaea heart ventricle cells. Dissociated ventricular muscle cells had average resting membrane potentials of -55 +/- 5 mV. When hyperpolarized to potentials between -70 and -63 mV, ventricle cells were capable of firing repetitive action potentials (8.5 +/- 1.2 spikes/min) that failed to overshoot 0 mV. The action potentials were either simple spikes or more complex spike/plateau events. The latter were always accompanied by strong contractions of the muscle cell. The waveform of the action potentials were shown to be dependent on the presence of extracellular Ca(2+) and K(+) ions. With the use of the single-electrode voltage-clamp technique, two types of voltage-gated K(+) currents were identified that could be separated by differences in their voltage sensitivity and time-dependent kinetics. The first current activated between -50 and -40 mV. It was relatively fast to activate (time-to-peak; 13.7 +/- 0.7 ms at +40 mV) and inactivated by 53.3 +/- 4.9% during a maintained 200-ms depolarization. It was fully available for activation below -80 mV and was completely inactivated by holding potentials more positive than -40 mV. It was completely blocked by 5 mM 4-aminopyridine (4-AP) and by concentrations of tetraethylammonium chloride (TEA) >10 mM. These properties characterize this current as a member of the A-type family of voltage-dependent K(+) currents. The second voltage-gated K(+) current activated at more depolarized potentials (-30 to -20 mV). It activated slower than the A-type current (time-to-peak; 74.1 +/- 3.9 ms at +40 mV) and showed little inactivation (6.2 +/- 2.1%) during a maintained 200-ms depolarization. The current was fully available for activation below -80 mV with a proportion of the current still available for activation at potentials as positive as 0 mV. The current was completely blocked by 1-3 mM TEA. These properties characterize this current as a member of the delayed rectifier family of voltage-dependent K(+) currents. The slow activation rates and relatively depolarized activation thresholds of the two K(+) currents are suggestive that their main role is to contribute to the repolarization phase of the action potential.

Temperature sensitivity of transduction and action potential conduction in a spider mechanoreceptor.

Previous work has suggested that the activation energy of mechanotransduction is higher than expected from the simple electrochemistry of ion channels, but the temperature sensitivity of mechanically activated receptor current has not been measured directly before. We used the single-electrode voltage-clamp technique to measure receptor currents in sensory neurons of the VS-3 slit-sense organ in the spider, Cupiennius salei. Receptor currents were generated by deforming the cuticular slits. Conduction velocity in afferent axons from the same organ was also measured by recording action potentials at two locations in the leg during mechanical stimulation of the slits. Activation energies of mechanotransduction and conduction velocity were estimated by making the measurements at a range of temperatures. The mean activation energy for receptor current was 23.1 kcal/mol (96.6 kJ/mol), corresponding to a Q10 value of 3.2. Conduction velocity in the afferent axons was approximately equal to 5 m/s at room temperature and it was much less temperature sensitive, with an activation energy of 6.3 kcal/mol (26.3 kJ/mol), corresponding to a Q10 value of 1.5. These results provide the first direct measurements of the activation energy of mechanically activated currents and support previous suggestions that a high thermal energy barrier is involved in mechanotransduction.

Estimated single-channel conductance of mechanically-activated channels in a spider mechanoreceptor

Noise analysis was used to estimate the single-channel conductance and number of channels responsible for the mechanically-activated current in the sensory neurons of a spider mechanoreceptor organ. External slits of the VS-3 slit-sense organ in the patellar cuticle of Cupiennius salei were moved with a piezoelectric stimulator while glass microelectrodes penetrated the adjacent cell bodies. Receptor currents were measured by the switching single-electrode voltage clamp technique during both step and ramp displacements of the slits. Current records were segmented in time, and the variance and amplitude of the current were obtained from each segment, to allow fitting of the variance vs. amplitude relationship by a standard equation based on a two-state channel. Mean values of 7.5 pS and 253 were obtained for the conductance and number of channels from 75 separate recordings. These values are in good agreement with the small number of other estimates of these parameters from different mechanoreceptor preparations.

NMDA-dependent currents in granule cells of the dentate gyrus contribute to induction but not permanence of kindling.

Single-electrode voltage-clamp techniques and bath application of the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (APV) were used to study the time course of seizure-induced alterations in NMDA-dependent synaptic currents in granule cells of the dentate gyrus in hippocampal slices from kindled and normal rats. In agreement with previous studies, granule cells from kindled rats examined within 1 wk after the last of 3 or 30-35 generalized tonic-clonic (class V) seizures demonstrated an increase in the NMDA receptor-dependent component of the perforant path-evoked synaptic current. Within 1 wk of the last kindled seizure, NMDA-dependent charge transfer underlying the perforant path-evoked current was increased by 63-111% at a holding potential of -30 mV. In contrast, the NMDA-dependent component of the perforant-evoked current in granule cells examined at 2.5-3 mo after the last of 3 or 90-120 class V seizures did not differ from age-matched controls. Because the seizure-induced increases in NMDA-dependent synaptic currents declined toward control values during a time course of 2.5-3 mo, increases in NMDA-dependent synaptic transmission cannot account for the permanent susceptibility to evoked and spontaneous seizures induced by kindling. The increase in NMDA receptor-dependent transmission was associated with the induction of kindling but was not responsible for the maintenance of the kindled state. The time course of alterations in NMDA-dependent synaptic current and the dependence of the progression of kindling and kindling-induced mossy fiber sprouting on repeated NMDA receptor activation are consistent with the possibility that the NMDA receptor is part of a transmembrane signaling pathway that induces long-term cellular alterations and circuit remodeling in response to repeated seizures, but is not required for permanent seizure susceptibility in circuitry altered by kindling.

Chapter 4 Synaptic Integration in Bistable Motoneurons

Several types of motoneurons exhibit bistable behavior in which brief periods of excitation and inhibition can toggle self-sustained firing on and off. In spinal motoneurons in the adult cat, bistable behavior is especially strong, but recent studies have shown that this bistable behavior varies systematically across the motoneuron pool. Fully bistable cells can maintain self-sustained firing for many seconds, whereas partially bistable cells can generate at most 1–2 seconds of self-sustained firing. It is important to realize that fully bistable motoneurons tend to have slow axonal conduction velocities and low input conductances. In contrast, partially bistable cells tend to have fast conduction velocities and high input conductances. Thus, fully bistable cells are likely to innervate fatigue-resistant muscle fibers and to be either of type slow contracting (S) or fast, fatigue resistant (FR) in the type classification scheme of Burke and colleagues. Using the single electrode voltage clamp technique, this chapter measures the total persistent inward current (IPT) underlying bistable behavior. Although IPT is of similar amplitude in fully and partially bistable cells, it tends to slowly decay with time in the partially bistable group while exhibiting little or no decay in fully bistable cells. The chapter briefly reviews the evidence that IPT and the plateau potential largely originate in dendritic regions and then considers the implications of a large and persistent inward current in dendrites for synaptic integration. Finally, it presents some guidelines for detecting the signs of plateau potentials in single motor unit firing patterns in human subjects.

Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes.

1. Ionic currents and unloaded cell shortening were recorded from guinea-pig ventricular myocytes with single electrode voltage clamp techniques and video edge detection at 37 C. Patch pipettes (1-3 MOmega) were used to provide intracellular dialysis with pipette solutions. 2. Na+ currents were blocked with 200 microM lidocaine. Contractions initiated by the voltage-sensitive release mechanism (VSRM) and Ca2+-induced Ca2+ release (CICR) in response to L-type Ca2+ current (ICa,L) were separated with voltage clamp protocols. 3. Without 8-bromo cyclic adenosine 3',5'-monophosphate (8-Br-cAMP) in the pipette, small VSRM-induced contractions occurred transiently in only 13% of myocytes. In contrast, large ICa,L-induced contractions were demonstrable in 100% of cells. 4. Addition of 10 or 50 microM 8-Br-cAMP to the pipette increased the percentage of cells exhibiting VSRM contractions to 68 and 93%, respectively. With 50 microM 8-Br-cAMP, contractions initiated by the VSRM and ICa,L were not significantly different in amplitude. 5. 8-Br-cAMP-supported VSRM contractions had characteristics of the VSRM shown previously in undialysed myocytes. Cd2+ (100 microM) blocked ICa,L and ICa,L contractions but not VSRM contractions. 8-Br-cAMP-supported contractions exhibited steady-state inactivation with parameters characteristic of the VSRM, as well as sigmoidal contraction-voltage relations. 6. Without 8-Br-cAMP in the pipette, contraction-voltage relations determined with steps from a post-conditioning potential (Vpc) of either -40 or -65 mV were bell shaped, with a threshold near -35 mV. With 50 microM 8-Br-cAMP in the pipette, contraction-voltage relations from a Vpc of -65 mV were sigmoidal and the threshold shifted to near -55 mV. Contraction-voltage relations remained bell shaped in the presence of 8-Br-cAMP when the Vpc was -40 mV. 7. H-89, which inhibits cAMP-dependent protein kinase A (PKA), significantly reduced the amplitudes of VSRM contractions by approximately 84% with 50 microM 8-Br-cAMP in the pipette. H-89 also significantly reduced the amplitudes of peak ICa, L and ICa,L contractions, although to a lesser extent. 8. We conclude that intracellular dialysis with patch pipettes disrupts the adenylyl cyclase-PKA phosphorylation cascade, and that the VSRM requires intracellular phosphorylation to be available for activation. Intracellular dialysis with solutions that do not maintain phosphorylation levels inhibits a major mechanism in cardiac excitation- contraction coupling.

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.

Bistability in spinal motoneurons in vivo: systematic variations in persistent inward currents.

Bistable behavior in spinal motoneurons consists of self-sustained firing evoked by a brief period of input. However, not all motoneurons possess an equal capacity for bistable behavior. In the companion paper, we found that self-sustained firing was persistent for long periods only in motoneurons with low rheobases and slow axonal conduction velocities. High rheobase, fast conduction velocity motoneurons tend to be only partially bistable in that self-sustained firing lasts at most 1-2 s. The mechanisms underlying these differences between fully and partially bistable motoneurons were investigated by measuring their current voltage (I-V) relationships in the decerebrate cat preparation after administration of the noradrenergic alpha1 agonist methoxamine. Slow (8 mV/s) triangular voltage commands were applied using the discontinuous single-electrode voltage-clamp technique. Both fully and partially bistable cells exhibited a region of negative I-V slope due to activation of a strong, persistent inward current. The peak amplitude of the total persistent inward current (IPIC) was equally large in fully and partially bistable cells, but there were substantial differences in how IPIC was activated and deactivated. In fully bistable cells, the offset of IPIC on the descending phase of the triangular voltage command occurred at a substantially more hyperpolarized voltage then its onset on the rising phase. Thus the I-V function of fully bistable cells exhibited marked hysteresis. Partially bistable cells had significantly less hysteresis. The lack of hysteresis in partially bistable cells was due to a greater decay of IPIC with time than that seen in fully bistable cells. Furthermore, the range over which activation and deactivation of IPIC occurred was more depolarized in partially than in fully bistable cells. The I-V functions were compared with frequency-current (F-I) functions from the same cells, the characteristics of which were presented in the companion paper. The strong onset-offset difference in IPIC in fully bistable cells corresponded to a similarly large hysteresis for the thresholds of their F-I functions. The reduced onset-offset difference for IPIC in partially bistable cells corresponded to a lack of hysteresis in F-I thresholds. Thus the properties of IPIC accounted for the main differences in the F-I behavior seen between fully and partially bistable cells.

Effects of transient oxygen-glucose deprivation on G-proteins and G-protein-coupled receptors in rat CA3 pyramidal cells in vitro.

The role of guanosine triphosphate-binding proteins (G-proteins) in the generation of the outward current during transient oxygen-glucose deprivation (OGD) was investigated in CA3 pyramidal cells in rat hippocampal organotypic slice cultures using the single-electrode voltage-clamp technique with KMeSO4-filled microelectrodes. To simulate ischaemia, brief chemical OGD (2 mM 2-deoxyglucose and 3 mM NaN3 for 4-9 min) was used, which induced an outward K+ current associated with an increase in input conductance. OGD failed to induce the outward current under conditions where G-protein function was disrupted by loading cells with guanosine 5'-O-(2-thiodiphosphate) [GDPbetaS] or after prolonged injection of guanosine 5'-O(3-thiotdphosphate) [GTPgammaS]. However, in slices treated with pertussis toxin (PTX), OGD still elicited the outward current, indicating that PTX-insensitive G-proteins are involved. Consistent with this insensitivity to PTX, neither adenosine receptors nor GABA(B) (gamma-aminobutyric acid) receptors, which operate via PTX-sensitive G-proteins, mediate the OGD-induced outward current. When adenosine receptors or GABA(B) receptors were blocked with 1,3-dipropyl-8-psulphophenylxanthine (DPSPX, 5 microM) or CGP 52 432 (10 microM), respectively, the OGD-induced response was not modified. The response also persisted following pretreatment of slice cultures with tetanus toxin to prevent vesicular release of neurotransmitters and neuromodulators from presynaptic terminals. Both PTX-sensitive and PTX-insensitive G-protein-mediated responses were suppressed during OGD. The inward current induced by the metabotropic glutamate receptor agonist 1 S, 3R-1-aminocyclopentane-1,3-dicarboxylate (1S,3R-ACPD) and the outward current elicited by adenosine or baclofen were strongly or completely attenuated. In contrast, the ionotropic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) response was not affected. These findings suggest that during OGD there is a functional uncoupling of receptors from G-proteins, and a direct receptor-independent activation of PTX-insensitive G-proteins leading to an increase in membrane K+ conductance.