Discontinuous Single-electrode Voltage-clamp Research Articles

Abstract 809: Chronic Testosterone Deficiency Increases Late Inward Sodium Current in Ventricular Myocytes from Aging Male C57BL/6 Mice

Clinical studies suggest that testosterone modulates the electrical activity of the heart and that low testosterone promotes cardiac arrhythmias. Here we investigated cellular mechanisms involved in the electrophysiological effects of chronic testosterone deficiency on the heart in aging male mice. Male C57BL/6 mice were subjected to either a gonadectomy (GDX) or a sham surgery at 1 month of age and then aged to 16-18 months. Ventricular myocytes were isolated and transmembrane voltage plus ionic currents were recorded with microelectrodes (20-25 MΩ; current clamp & discontinuous single electrode voltage clamp; 37 o C). Cells were paced at 2 and 4 Hz. Action potential duration at 90% repolarization (APD 90 ) was prolonged by GDX (57.9 ± 1.7 vs 108.2 ± 14.1 msec; p<0.05), while APD 50 and resting membrane potential were unchanged. We next determined whether an increase in late inward sodium current (I Na-L ) contributed to the increase in APD 90 in GDX cells. Voltage clamp studies showed that I Na-L was significantly larger in GDX cells compared to sham cells (-0.70 ± 0.16 vs -1.53 ± 0.27 pA/pF; p<0.05). When myocytes were superfused with the I Na-L antagonist ranolazine (10 μM), the increase in APD 90 was blocked in cells from GDX mice, so that APD 90 no longer differed between the two groups. By contrast, ranolazine had no effect on either APD 50 or resting membrane potential. Ranolazine also blocked the increase in I Na-L in cells from GDX mice. Prolongation of the action potential in GDX myocytes was associated with a significant increase in the incidence of early afterdepolarizations (EADs) when compared to sham controls. EADs were also inhibited by ranolazine. These data demonstrate that long-term GDX prolongs APD 90 at least in part by increasing the magnitude of I Na-L . These findings also suggest that an increase in I Na-L may promote arrhythmias in older men with low circulating testosterone levels.

Role of ATP Sensitive Potassium Channel in Exercise-Training Mediated Adaptations in Ventricular Repolarization

Regular exercise-training is known to reduce the incidences and severity of ischemic heart disease, but the mechanistic nature is unknown. We showed that regular exercise shortened the left ventricular action potential duration (APD) at high heart rate (HR), which would prevent Ca2+ overload and/or inadequate filling under conditions of metabolic stress. Here we test the hypothesis that exercise-training induced APD shortening at high HR is due to the up-regulation of ATP sensitive potassium channel (KATP). Female and male Sprague-Dawley rats were randomly assigned to voluntary wheel running or control groups. After 6-8 weeks training, cardiac myocytes were isolated from the apex and base regions of the left ventricle with collagenase–protease dispersion technique. APD was measured with glass micro-electrode, current-voltage relationship was recorded with discontinuous single electrode voltage clamp, and the expression level of KATP channel pore-forming subunit Kir6.2 and regulatory subunits SUR2A were determined by Western blots. At 1Hz, KATP activator pinacidil (100 µM) shortened APD in myocytes from both sexes and, the shortening was significantly greater in exercise-trained compared to sedentary groups. The KATP inhibitor glibenclamide (2 µM) prolonged the 10Hz APD more in exercise-trained than control rats. Regular exercise enhanced KATP outward current density in apex myocytes from both sexes; in base, trained females exhibited larger KATP inward current than controls. In both sexes, Kir6.2 expression was elevated by wheel-running in apex but not base region. Exercise-training increased SUR2A density in base region of both sexes, and eliminated its reginal difference in females. In conclusion, exercise-training induced APD shortening under energy demanding conditions (e.g. high HR) is caused at least in part by KATP up-regulation, an adaptation that reduces energy requirements for ion homeostasis and maintains a diastolic interval adequate for myocardial relaxation.

Application of active electrode compensation to perform continuous voltage-clamp recordings with sharp microelectrodes

Objective. Electrophysiological recordings of single neurons in brain tissues are very common in neuroscience. Glass microelectrodes filled with an electrolyte are used to impale the cell membrane in order to record the membrane potential or to inject current. Their high resistance induces a high voltage drop when passing current and it is essential to correct the voltage measurements. In particular, for voltage clamping, the traditional alternatives are two-electrode voltage-clamp technique or discontinuous single electrode voltage-clamp (dSEVC). Nevertheless, it is generally difficult to impale two electrodes in a same neuron and the switching frequency is limited to low frequencies in the case of dSEVC. We present a novel fully computer-implemented alternative to perform continuous voltage-clamp recordings with a single sharp-electrode. Approach. To reach such voltage-clamp recordings, we combine an active electrode compensation algorithm (AEC) with a digital controller (AECVC). Main results. We applied two types of control-systems: a linear controller (proportional plus integrative controller) and a model-based controller (optimal control). We compared the performance of the two methods to dSEVC using a dynamic model cell and experiments in brain slices. Significance. The AECVC method provides an entirely digital method to perform continuous recording and smooth switching between voltage-clamp, current clamp or dynamic-clamp configurations without introducing artifacts.

Electrophysiological Properties of the Microstome and Macrostome Morph of the Polymorphic Ciliate Tetrahymena vorax

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately -30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca(2+) spikes with a maximum rate of rise of 7.5 Vs(-1) in microstome and 4.7 Vs(-1) in macrostome cells. Depolarizing voltage steps from a holding potential of -40 mV induced an inward Ca(2+) -current (I(ca) ) peaking at -10 mV, reaching approximately the same value in microstome (-1.4 nA) and macrostome cells (-1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I(Ca) in these morphs supports the notion that the voltage-gated Ca(2+) channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na(+) -free solution and was only partially inhibited by extracellular Cs(+) . The inward rectification was completely blocked by replacing Ca(2+) with Co(2+) or Ba(2+) in the recording solution, and is probably due to Ca(2+) -activated inward K(+) current secondary to Ca(2+) influx through channels activated by hyperpolarization.

Accurate measurement of junctional conductance between electrically coupled cells with dual whole-cell voltage-clamp under conditions of high series resistance

Accurate measurement of the junctional conductance ( G j) between electrically coupled cells can provide important information about the functional properties of coupling. With the development of tight-seal, whole-cell recording, it became possible to use dual, single-electrode voltage-clamp recording from pairs of small cells to measure G j. Experiments that require reduced perturbation of the intracellular environment can be performed with high-resistance pipettes or the perforated-patch technique, but an accompanying increase in series resistance ( R s) compromises voltage-clamp control and reduces the accuracy of G j measurements. Here, we present a detailed analysis of methodologies available for accurate determination of steady-state G j and related parameters under conditions of high R s, using continuous or discontinuous single-electrode voltage-clamp (CSEVC or DSEVC) amplifiers to quantify the parameters of different equivalent electrical circuit model cells. Both types of amplifiers can provide accurate measurements of G j, with errors less than 5% for a wide range of R s and G j values. However, CSEVC amplifiers need to be combined with R s-compensation or mathematical correction for the effects of nonzero R s and finite membrane resistance ( R m). R s-compensation is difficult for higher values of R s and leads to instability that can damage the recorded cells. Mathematical correction for R s and R m yields highly accurate results, but depends on accurate estimates of R s throughout an experiment. DSEVC amplifiers display very accurate measurements over a larger range of R s values than CSEVC amplifiers and have the advantage that knowledge of R s is unnecessary, suggesting that they are preferable for long-duration experiments and/or recordings with high R s.

Repetitive Firing Triggers Clustering of Kv2.1 Potassium Channels in Aplysia Neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.

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.

The slow and the quick anion conductance in whole guard cells: their voltage-dependent alternation, and the modulation of their activities by abscisic acid and CO2.

We explored the functioning of the slowly activating anion conductance, S-type or SLAC, and of the quickly activating anion conductance, R-type or QUAC, in whole guard cells of Vicia faba L.; details of QUAC activity had not previously been demonstrated in guard cells possessing their walls. The discontinuous single-electrode voltage-clamp method was used to record current responses to voltage pulses and voltage ramps as well as the free-running membrane voltage. At all voltages tested between -200 and 60 mV, SLAC activated with two components, one had a time constant similar to 7 s, the other similar to 40 s. The current-voltage relationship resembled that obtained by patch-clamp experiments. In pulse experiments and 1-s ramps, QUAC activity appeared with half-maximum activation near -50 mV and full activation above -10 mV; it inactivated with a half-time of approximately 10 s. Inactivation of QUAC at -40 mV led to the appearance of SLAC. After deactivation of SLAC at -200 mV, QUAC could be activated again. We concluded that voltage-dependent interchanges between SLAC and QUAC had occurred. Frequently, SLAC and QUAC were active simultaneously in the same cell. Abscisic acid (ABA, 20 microM) activated SLAC as well as QUAC. External Ca2+ was not required, but enhanced the activation of QUAC. Rises in the partial pressure of CO2, in the range between 0 and 700 microbar, caused rapid and reversible increases in the activity of SLAC (and outward currents of K+). QUAC also responded to CO2, however in an unpredictable manner (either by increased or by decreased activity). Oscillations in the free-running membrane voltage arose either spontaneously or after changes in CO2. They were correlated with periodic activations and inactivations of QUAC and required the simultaneous activity of an electrogenic pump.

Alternation of the slow with the quick anion conductance in whole guard cells effected by external malate.

In previous investigations two anion conductances were discovered in guard-cell protoplasts: the quickly activating anion conductance (QUAC, R-type) and the slowly activating anion conductance (SLAC, S-type). In this investigation, effects of malate on the two anion conductances were tested in whole guard cells of Vicia faba L. by the use of the discontinuous single-electrode voltage-clamp method. Application of 1-s voltage ramps proved that QUAC displayed the malate shift of the activation threshold toward hyperpolarization also in complete guard cells. The sensitivity of SLAC to external malate was determined by responses to voltage pulses of 20 s duration at Cl- concentrations of 0.1, 3 or 50 mM. At no voltage were the currents measured at the end of the pulses in the presence and absence of malate significantly different from each other; the current-voltage relationship of SLAC appeared not to be affected by malate. However, in 32% of the cells exposed to malate, current activation in response to voltage steps occurred within 0.1 s, faster than was typical for SLAC, and activation was followed by inactivation with a half-time similar to 10 s: SLAC apparently had changed to QUAC. Simultaneously, the free-running membrane voltage depolarized at 0.1 mM Cl-, did not change at 3 mM Cl- and polarized at 50 mM Cl-, indicating that activation of QUAC increased the membrane conductance for anions and thereby drove the membrane voltage toward the equilibrium voltage of Cl-. The malate-induced changes were fully reversible at Cl- concentrations of 0.1 and 3 mM. These results reinforce the proposition that SLAC and QUAC represented two switching modes of the same anion channel (however, they do not suffice as proof); they also show that this interconvertibility can enable guard cells to control their membrane voltage rapidly.

Ionic currents in isolated and in situ squid Schwann cells.

Ionic currents from Schwann cells isolated enzymatically from the giant axons of the squids Loligo forbesi, Loligo vulgaris and Loligo bleekeri were compared with those obtained in situ. Macroscopic and single channel ionic currents were recorded using whole-cell voltage and patch clamp. In the whole-cell configuration, depolarisation from negative holding potentials evoked two voltage-dependent currents, an inward current and a delayed outward current. The outward current resembled an outwardly rectifying K+ current and was activated at -40 mV after a latent period of 5-20 ms following a step depolarisation. The current was reduced by externally applied nifedipine, Co2+ or quinine, was not blocked by addition of apamin or charibdotoxin and was insensitive to externally applied L-glutamate or acetylcholine. The voltage-gated inward current was activated at -40 mV and was identified as an L-type calcium current sensitive to externally applied nifedipine. Schwann cells were impaled in situ in split-open axons and voltage clamped using discontinuous single electrode voltage clamp. Voltage dependent outward currents were recorded that were kinetically identical to those seen in isolated cells and that had similar current-voltage relations. Single channel currents were recorded from excised inside-out patches. A single channel type was observed with a reversal potential close to the equilibrium potential for K+ (E(K)) and was therefore identified as a K+ channel. The channel conductance was 43.6 pS when both internal and external solutions contained 150 mM K+. Activity was weakly dependent on membrane voltage but sensitive to the internal Ca2+ concentration. Activity was insensitive to externally or internally applied L-glutamate or acetylcholine. The results suggest that calcium channels and calcium-activated K+ channels play an important role in the generation of the squid Schwann cell membrane potential, which may be controlled by the resting intracellular Ca2+ level.

Improved hybrid clamp: resolution of tail currents following single action potentials

Hybrid clamp protocols, in which a discontinuous single electrode voltage clamp (dSEVC) amplifier is switched from current to voltage clamp during the recording, are frequently used to investigate conductance changes after high frequency trains of action potentials. This technique is advantageous because it combines physiological stimulation of the cell with the possibility of analyzing the consecutive conductance changes quantitatively. In this study an improved hybrid clamp protocol, called dynamic hybrid clamp, is developed that enables the experimenter to study tail currents after single action potentials. The protocol employs real time action potential detection to assure precise timing of the mode switch and utilizes an external sample and hold amplifier to avoid voltage steps during the switch to voltage clamp. With the use of whole-cell patch clamp recordings and high switching frequencies (≥25 kHz), dSEVC can easily be started with a minimal delay (<1.5 ms) after single action potentials and tail currents underlying afterhyperpolarisations (AHPs) and afterdepolarisations ensuing single spikes are clearly resolved. The dynamic hybrid clamp should also be useful for analysis of spontaneously occurring events such as intrinsic or population bursts.

Enhancement of bistability in spinal motoneurons in vivo by the noradrenergic alpha1 agonist methoxamine.

Enhancement of bistability in spinal motoneurons in vivo by the noradrenergic alpha1 agonist methoxamine. Like many types of motoneurons, spinal motoneurons in the adult mammal can exhibit bistable behavior. This means that short periods of excitatory input can initiate long periods of self-sustained firing and that equally short periods of inhibition can return the cell to the quiescent state. Usually, the presence of one of the monoamines (either serotonin or norepinephrine) is required for spinal motoneurons to express bistable behaviors. Because the decerebrate cat preparation has tonic activity in monoaminergic fibers that originate in the brain stem and project to spinal motoneurons, these cells sometimes exhibit bistable behavior. However, exogenous application of the noradrenergic alpha1 agonist methoxamine greatly enhances bistable behavior in the decerebrate. The goal of this study was to identify the mechanisms of this action of methoxamine. The total persistent inward current (IPIC) in spinal motoneurons in the decerebrate cat was measured from I-V functions generated by triangular voltage commands applied using discontinuous single electrode voltage clamp. The effect of methoxamine on IPIC was assessed by comparing its properties in a control cell sample without methoxamine to its properties in a sample of cells obtained after application of methoxamine. In most experiments, at least one cell was obtained from each sample. Our results showed that methoxamine approximately doubled the amplitude of IPIC without changing its onset voltage, its offset voltage, or its persistence. The reduced amplitude was a consistent finding within experiments and so was unlikely to be caused by interanimal variability. In addition, methoxamine depolarized motoneurons without altering their input conductances, so that a smaller amount of current was required to reach the onset voltage of IPIC. These effects of methoxamine were approximately equal in all cells. As a result of these changes, methoxamine greatly enhanced the tendency for motoneurons to become bistable. It is proposed that the methoxamine-induced increase in the amplitude of IPIC is effective in enhancing the duration of bistable firing because this increase makes IPIC more resistant to the deactivating effects of the afterhyperpolarizations between spikes.

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.

Inhibitory synaptic drive patterns motoneuronal activity in rhythmic preparations of isolated thoracic ganglia in the stick insect

During active leg movements of an insect leg, the activity of the motoneuron pools of each individual leg joint is generated by the interaction between signals from central rhythm generating sources, peripheral signals as well as coordinating signals from other leg joints and legs. The nature of the synaptic drive from the central rhythm generators onto the motoneuron pools of the individual leg joints during rhythmic motor activity of the stick insect ( Carausius morosus) middle leg has been investigated. In the isolated mesothoracic ganglion central rhythm generators were activated pharmacologically by topical application of the muscarinic agonist pilocarpine. Motoneurons supplying the femur–tibia (FT) joint were investigated in detail. Recordings from neuropil processes of these motoneurons revealed that patterning of their rhythmic activity is based on cyclic hyperpolarizing synaptic inputs. These inputs are in clear antiphase for extensor and flexor motoneurons. DCC (discontinuous current clamp) and dSEVC (discontinuous single electrode voltage clamp) recordings showed reversal potentials of the inhibitory inputs between −80 to −85 mV (FETi, N=7; Flex MN, N=3). After intracellular injection of TEA rhythmic inhibition in FETi was decreased by about 84% ( N=4). Both findings indicate that the cyclic inhibition is mediated by potassium ions. Thus, it appears that central rhythm generators pattern motor activity in antagonistic tibial motoneuron pools by cyclic alternating inhibition.

On the nature of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus in vitro.

1. Intracellular sharp electrode current clamp and discontinuous single electrode voltage clamp recordings were made from thalamocortical neurones (n = 57) of the cat ventrobasal thalamus in order to investigate the mechanism underlying anomalous rectification. 2. Under current clamp conditions, voltage-current (V-I) relationships in a potential range of -55 to -110 mV demonstrated anomalous rectification with two components: fast rectification, which controlled the peak of negative voltage deviations, and time-dependent rectification. Time-dependent rectification was apparent as a depolarizing sag generated during the course of negative voltage deviations, was first formed at potentials in the range -60 to -70 mV, and was sensitive to 3 mM Cs+ (n = 6). Similarly, under voltage clamp conditions, instantaneous and steady-state I-V relationships demonstrated anomalous rectification. A slowly activating inward current with an activation threshold in the range of -65 to -70 mV formed time-dependent rectification. This current was sensitive to Cs+ (3 mM) (n = 3) and had properties similar to the slow inward mixed cationic current (Ih). 3. 4-(N-Ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)-pyrimidinium++ + chloride (ZD 7288) (100-300 microM) irreversibly blocked time-dependent rectification mediated by Ih (n = 23 of 25 neurones), and led to a hyperpolarization of the resting membrane potential (6.8 +/- 0.5 mV). In the presence of ZD 7288, V-I and I-V relationships, exhibited fast anomalous rectification, first activated from potential more negative than -80 mV. 4. Ba2+ (100 microM) (n = 8), in the continuous presence of ZD 7288, reversibly linearized peak V-I and instantaneous I-V relationships over a potential range of -70 to -120 mV, and led to a membrane depolarization (13.3 +/- 4.2 mV) or tonic inward current (192 +/- 36 pA). 5. The co-application of ZD 7288 and Ba2+ revealed a depolarizing sag in negative voltage deviations under current clamp conditions, or a large inward current with kinetics two to three times slower than those of Ih under voltage clamp conditions. This novel form of time-dependent rectification was first apparent at potentials more negative than about -85 mV, was sensitive to 5 mM Cs+ (n = 4), and is termed Ih,slow. Ih,slow tail currents reversed between -65.3 and -56.6 mV (with potassium acetate electrodes, n = 3) or -57.6 and -50.3 mV (with KCl electrodes, n = 3). 6. Computer simulations confirmed that the pattern of anomalous rectification in thalamocortical neurones of the cat ventrobasal thalamus is mediated by the concerted action of Ih and a Ba(2+)-sensitive current with properties similar to an inwardly rectifying K+ current (IKIR).

Shared calcium signaling pathways in the induction of long-term potentiation and synaptic disinhibition in CA1 pyramidal cell dendrites

1. Calcium signaling pathways were examined in the induction of long-term synaptic disinhibition following tetanization. Effects of tetanization on gamma-aminobutyric acid-A (GABAA receptor-mediated inhibitory responses were measured and compared with excitatory responses under experimental conditions previously used for examining induction mechanisms of N-methyl-D-aspartate (NMDA)-dependent long-term potentiation (LTP). Intracellular recordings were performed in current-clamp and discontinuous single-electrode voltage-clamp (dSEVC) modes in CA1 pyramidal cell apical dendrites in hippocampal slices of adult guinea pigs with the use of sharp electrodes. Test pulses and tetanic stimuli were applied to the Schaffer collateral fibers in stratum radiatum. 2. Under standard control conditions [3 M K Ac in the recording pipette and artificial cerebrospinal fluid as extracellular solution], tetanization-induced sustained increases of excitatory responses were accompanied by marked decreases of parameters of GABAA-mediated synaptic inhibition: at 40 min after tetanization [posttetanus 40 (PT 40)], orthodromically evoked excitatory postsynaptic potential (EPSP) peak amplitudes were on average 195 +/- 15% (mean +/- SE) and excitatory postsynaptic currents (IPSPs) were 166 +/- 10% of pretetanus controls. Peak amplitudes of orthodromically evoked inhibitory postsynaptic potentials (IPSPs) were 30 +/- 5% and inhibitory postsynaptic currents (IPSCs) were 21 +/- 4% at PT 40. Synaptic GABAA conductances (measured as chord conductances) were reduced to 22 +/- 4% at PT 40. Iontophoretic GABAA responses measured as conductance changes were 28 +/- 4% of pretetanus controls at PT 40. 3. A role of NMDA receptors in induction of long-term synaptic disinhibition was tested by preventing NMDA receptor activation 1) by pharmacological means and 2) by holding the membrane clamped at -80 mV (in dSEVC) during tetanization. In the presence of the NMDA-receptor antagonist D-2-amino5-phosphonopentanoic acid (D-AP5) 10-40 microM), orthodromically evoked EPSP amplitudes were 107 +/- 9%, EPSCs were 104 +/- 6%, GABAA-mediated IPSPs were 88 +/- 8%, IPSCs were 97 +/- 8%, synaptic GABAA conductances were 84 +/- 9%, and iontophoretic GABAA conductances were 102 +/- 13% at PT 40. In recordings in which the dendritic membrane potential was clamped at -80 mV during tetanization, orthodromically evoked peak amplitudes of EPSPs were 105 +/- 11%, EPSCs were 102 +/- 8, IPSPs were 103 +/- 4%, IPSCs were 102 +/- 5%, GABAA chord conductances were 101 +/- 9%, and iontophoretically evoked GABAA conductances were 105 +/- 5% at PT 40. 4. In recordings in which the intracellular pipette was preloaded with the Ca2+ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N'N"-tetraacetic acid (BAPTA) (5mM), long-term changes of synaptic transmission (increases of excitation, decreases of synaptic inhibition) were prevented. At PT 40, EPSP peak amplitudes were 93 +/- 7%, EPSCs were 115 +/- 6%, IPSPs were 115 +/- 9%, IPSCs were 117 +/- 8%, and synaptic GABAA conductances were 108 +/- 17%. Iontophoretic conductances at PT 40 were 109 +/- 9% over pretetanus controls when recorded with BAPTA-containing electrodes. 5. In recordings in which the intracellular pipette was preloaded with cypermethrin, a potent and selective inhibitor of phosphatase 2B, respective long-term changes of synaptic transmission (increases of excitation, decreases of synaptic inhibition) were prevented. At PT 40, EPSP peak amplitudes were 98 +/- 6%, EPSCs were 105 +/- 10%, IPSPs were 99 +/- 5%, IPSCs were 104 +/- 7%, synaptic GABAA conductances were 97 +/- 6% and iontophoretic GABAA conductances were 113 +/- 18% over pretetanus controls in cypermethrin-containing recordings. 6. In conclusion, the data presented demonstrate shared cellular pathways in the induction of both LTP and long-term synaptic disinhibition in apical dendrites of CA1 pyramidal cells after tetanization of the Schaffer collaterals.

Calcium currents in ventricular myocytes of prehypertrophic cardiomyopathic hamsters.

Possible changes in Ca2+ currents (ICa), which might contribute to Ca2+ overload in young (70- to 100-day-old) cardiomyopathic (CM) hamster hearts, were determined in isolated ventricular myocytes with conventional recording and discontinuous single-electrode voltage-clamp techniques. Action potential duration at 90% repolarization (APD90) was significantly longer in CM myocytes compared with normal cells (APD90 = 120.3 +/- 4.5 vs. 98.2 +/- 5.9 ms, P < 0.01). Input resistance, membrane time constant, and membrane capacitance were similar in normal and CM myocytes. Current-voltage (I-V) relations for peak ICa were depressed in CM cells compared with normal cells; this difference was statistically significant at the peak of the I-V curve. Activation and inactivation relations for ICa and recovery from inactivation were similar in myocytes from normal and CM hearts. These changes occurred in myocytes from young CM animals before development of heart failure. Results indicate that increased Ca2+ influx through L-type Ca2+ channels does not account for the longer APD in cardiomyopathy and is not involved in the development of Ca2+ overload in cardiomyopathy.

Calcium-dependent chloride current induced by axotomy in rat sympathetic neurons.

1. Seven to ten days after sectioning their axons, rat sympathetic neurons were studied using intracellular recording techniques in an in vitro preparation of the superior cervical ganglion. 2. In 75% of axotomized cells, an after-depolarization (ADP) was observed following spike firing or depolarization with intracellular current pulses. Discontinuous single-electrode voltage-clamp techniques were employed to study the ADP. When the membrane potential was clamped at the resting level just after an action potential, a slow inward current was recorded in cells that showed an ADP. 3. In the presence of TTX and TEA, inward peaks and outward currents were recorded during depolarizing voltage jumps, followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. The inward peak and tail currents activated between -10 and -20 mV and reached maximum amplitudes around 0 mV. With depolarizing jumps to between +40 and +50 mV, net outward currents were recorded during the depolarizing jumps but inward tail currents were still activated. 4. In the presence of the Ca2+ channel blocker cadmium, or when Ca2+ was substituted by Mg2+, the ADP disappeared. In voltage-clamped cells, cadmium blocked the inward tail currents. The reversal potential for the inward tail current was approximately -15 mV. Substitution of the extracellular NaCl by sucrose or sodium isethionate increased the amplitude of the inward tail current, and displaced its equilibrium potential to more positive values. Changes in extracellular [K+] did not appreciably affect the inward tail current amplitude or equilibrium potential. Niflumic acid, a blocker of chloride channels activated by Ca2+, almost completely blocked the tail current. 5. No ADPs were observed in non-axotomized neurons, and when depolarizing pulses were applied while in voltage clamp no inward tail currents were evoked in these normal cells. 6. It is concluded that axotomy of sympathetic ganglion cells produces the appearance of a Ca(2+)-dependent chloride current responsible for the ADP observed following spike firing.

Interaction between thyrotropin-releasing hormone (TRH) and NMDA-receptor-mediated responses in hypoglossal motoneurones

The effect of thyrotropin-releasing hormone (TRH) on the responses to excitatory amino acids was investigated in hypoglossal motoneurones in an in vitro preparation of the brainstem from guinea pigs using current clamp and discontinuous single electrode voltage clamp (dSEVC). Bath application of 20–50 μM TRH markedly potentiated the response to iontophoretically applied NMDA, whereas no potentiation of the response to glutamate, aspartate or quisqualic acid was seen. Voltage clamp experiments showed that TRH did not increase the current flowing through NMDA channels, thus a direct modulatory role of TRH on NMDA channels was not a likely explanation of the potentiation. Voltage clamp studies of the current-voltage relationship showed that the potentiation of the response to NMDA and lack of potentiation of the response to quisqualic acid was a result of an interaction between the actions of TRH and the amino acids on the electroresponsive profile of the membrane. Endogenous NMDA receptor activation was produced by tetanic stimulation of the reticular formation dorsolaterally to the hypoglossal nucleus, evoking large APV sensitive EPSPs in the presence of CNQX, a non-NMDA blocker. The amplitude and duration of these potentials were increased at more positive membrane potentials in response to TRH. It is concluded that TRH can act as a neuromodulator-potentiating the response to NMDA receptor activation-simply by changing the electroresponsive properties of the membrane.