Processing of sensory signals by a non-spiking neuron in the leech

The non-spiking neurons 151 are present as bilateral pairs in each midbody ganglion of the leech nervous system and they are electrically coupled to several motorneurons. Intracellular recordings were used to investigate how these neurons process input from the mechanosensory P neurons in isolated ganglia. Induction of spike trains (15 Hz) in single P cells evoked responses that combined depolarizing and hyperpolarizing phases in cells 151. The phasic depolarizations, transmitted through spiking interneurons, reversed at around -20 mV. The hyperpolarization had two components, both reversing at around -65 mV, and which were inhibited by strychnine (10 micromol l(-1)). The faster component was transmitted through spiking interneurons and the slower component through a direct P-151 interaction. Short trains (<400 ms) of P cell spikes (15 Hz) evoked the phasic depolarizations superimposed on the hyperpolarization, while long spike trains (>500 ms) produced a succession of depolarizations that masked the hyperpolarizing phase. The amplitude and duration of the hyperpolarization reached their maximum at the initial spikes in a train, while the depolarizations persisted throughout the duration of the stimulus train. Both phases of the response were relatively unaffected by the spike frequency (5-25 Hz). The non-spiking neurons 151 processed the sensory signals in the temporal rather than in the amplitude domain.     

The quantal source of area supralinearity of flash responses in Limulus photoreceptors

The time integrals of the responses of dark-adapted Limulus ventral photoreceptors to flashes exhibit a supralinear dependence on intensity at intermediate intensities. By decomposing the responses into their elementary single-photon components ("bumps"), we are able to calculate the overall quantum efficiency and to display the time courses of the bump amplitude and rate of appearance. Since the time course of the flash response is not slow compared with that of the bump, it was necessary, in order to carry out the decomposition, to develop a new technique for noise analysis of dynamic signals. This new technique should have wide applications. Our main finding is that the supralinearity of the flash responses corresponds to an increase in bump amplitude, with little change in bump duration or quantum efficiency. The time courses of the bump rate and of the change in bump amplitude are peaked and have widths similar to that of the response itself. The peaks of the time courses of the bump rate and amplitude displayed against the starting times of the bumps do not coincide and occur approximately 80 and approximately 40 ms, respectively, before the peak of the response. The time from the start of a bump to its centroid is approximately 70 ms, which means that the time at which the bump centroid reaches its maximum follows the response peak by 30 ms. These results impose constraints on possible mechanisms for the amplitude enhancement.     

Simultaneous capacitance and amperometric measurements of exocytosis: a comparison.

We measured the exocytotic response induced by flash photolysis of caged compounds in isolated mast cells and chromaffin cells. Vesicle fusion was measured by monitoring the cell membrane capacitance. The release of vesicular contents was followed by amperometry. In response to a GTP gamma S stimulus we found that the time integral of the amperometric current could be superimposed on the capacitance trace. This shows that the integrated amperometric signal provides an alternative method of measuring the extent and kinetics of the secretory response. Very different results were obtained when photolysis of caged Ca2+ (DM-nitrophen) was used to stimulate secretion. In mast cells, there was an immediate, graded increase in membrane capacitance that was followed by step increases (indicative of granule fusion). During the initial phase of the capacitance increases, no release of oxidizable secretory products was detected. In chromaffin cells we also observed a considerable delay between increases in capacitance, triggered by uncaging Ca2+, and the release of oxidizable secretory products. Here we demonstrate that there can be large increases in the membrane capacitance of a secretory cell, triggered by flash photolysis of DM-nitrophen, which indicate events that are not due to the fusion of granules containing oxidizable substances. These results show that increases in capacitance that are not resolved as steps cannot be readily interpreted as secretory events unless they are confirmed independently.     

Fast kinetics of calcium liberation induced in Xenopus oocytes by photoreleased inositol trisphosphate.

Inositol 1,4,5-trisphosphate (InsP3) acts on intracellular receptors to cause liberation of Ca2+ ions into the cytosol as repetitive spikes and propagating waves. We studied the processes underlying this regenerative release of Ca2+ by monitoring with high resolution the kinetics of Ca2+ flux evoked in Xenopus oocytes by flash photolysis of caged InsP3. Confocal microfluorimetry was used to monitor intracellular free [Ca2+] from femtoliter volumes within the cell, and the underlying Ca2+ flux was then derived from the rate of increase of the fluorescence signals. A threshold amount of InsP3 had to be photoreleased to evoke any appreciable Ca2+ signal, and the amount of liberated Ca2+ then increased only approximately fourfold with maximal stimulation, whereas the peak rate of increase of Ca2+ varied over a range of nearly 20-fold, reaching a maximum of approximately 150 microMs-1. Ca2+ flux increased as a first-order function of [InsP3]. Indicating a lack of cooperativity in channel opening, and was half-maximal with stimuli approximately 10 times threshold. After a brief photolysis flash, Ca2+ efflux began after a quiescent latent period that shortened from several hundred milliseconds with near-threshold stimuli to 25 ms with maximal flashes. This delay could not be explained by an initial "foot" of Ca2+ increasing toward a threshold at which regenerative release was triggered, and the onset of release seemed too abrupt to be accounted for by multiple sequential steps involved in channel opening. Ca2+ efflux increased to a maximum after the latent period in a time that reduced from > 100 ms to approximately 8 ms with increasing [InsP3] and subsequently declined along a two-exponential time course: a rapid fall with a time constant shortening from > 100 ms to approximately 25 ms with increasing [InsP3], followed by a much smaller fail persisting for several seconds. The results are discussed in terms of a model in which InsP3 receptors must undergo a slow transition after binding InsP3 before they can be activated by cytosolic Ca2+ acting as a co-agonist. Positive feedback by liberated Ca2+ ions then leads to a rapid increase in efflux to a maximal rate set by the proportion of receptors binding InsP3. Subsequently, Ca2+ efflux terminates because of a slower inhibitory action of cytosolic Ca2+ on gating of InsP3 receptor-channels.     

Ca2+-dependent depolarization and burst firing of rat CA1 pyramidal neurones induced by N-methyl-D-aspartic acid and quinolinic acid: antagonism by 2-amino-5-phosphonovaleric and kynurenic acids

The excitatory effects of microiontophoretically applied quisqualic (QUIS), N-methyl-D-aspartic (NMDA), and quinolinic (QUIN) acids were investigated using intracellular recording from CAl pyramidal neurones in slices of rat hippocampus. QUIS evoked only simple action potentials superimposed upon a depolarization which attained a clear plateau. When this level had been reached, increased ejecting currents did not produce further depolarization. By contrast, with low currents NMDA and QUIN elicited small membrane depolarizations which triggered bursts of action potentials superimposed upon rhythmically occurring depolarizing shifts. Larger currents caused depolarization which if sufficiently large completely blocked spike activity. Tetrodotoxin (TTX) prevented the spikes evoked by QUIS and the bursts of action potentials seen with NMDA and QUIN, and the rhythmic depolarizing shifts then appeared as broad spikes of up to 50 mV in amplitude. These and the underlying membrane depolarization were blocked by Co2+, by the NMDA antagonist D(-)-2-amino-5-phosphonovaleric acid (DAPV), and by kynurenic acid (KYNU). It thus appears that the depolarization and burst firing of rat CAl pyramidal neurones elicited by NMDA and QUIN are Ca2+ dependent while the actions of QUIS are not.     

Charybdotoxin block of Ca2+-activated K+ channels in colonic muscle depends on membrane potential dynamics

Charybdotoxin(ChTX) is a specific blocker ofCa²⁺-activatedK⁺ channels. The voltage- andtime-dependent dynamics of ChTX block were investigated using caninecolonic myocytes and the whole cell patch-clamp technique with step andramp depolarization protocols. During prolonged step depolarizations,K⁺ current slowly increased in thecontinued presence of ChTX (100 nM). The rate of increase depended onmembrane potential with an e-foldchange for every 60 mV. During ramp depolarizations, the effectivenessof ChTX block depended significantly on the rate of the ramp (50% at0.01 V/s to 80% at 0.5 V/s). Results are consistent with a mechanismin which ChTX slowly "unbinds" in a voltage-dependent manner. Asimple kinetic model was developed in which ChTX binds to both open andclosed states. Slow unbinding is consistent with ChTX having littleeffect on electrical slow waves recorded from circular muscle whilecausing depolarization and contraction of longitudinal muscle, whichdisplays more rapid "spikes." Resting membrane potential andmembrane potential dynamics are important determinants of ChTX action.

     

Prolonged Time Course of Glutamate Release from Nerve Terminals: Relationship Between Stimulus Duration and the Secretory Event

Abstract: The kinetics of synaptosomal [³H]glutamate release were measured on a subsecond time scale to study the relationship between the length of depolarization and the duration of the secretory event. The time course of release evoked by elevated K⁺ was complex, proceeding for several seconds after a 200-ms depolarization. We developed a protocol for depolarizing excitable membranes on a millisecond time scale to deliver brief depolarizations, termed the synthetic action potential, by using batrachotoxin to activate Na⁺ channels. Depolarization is achieved by superfusing with solutions containing elevated concentrations of Na⁺, and the duration of the depolarization is limited by including tetrodotoxin (TTX) in the superfusion solution to block Na⁺ entry. Direct measurements of the time courses of Na⁺ current and membrane depolarizations were made in batrachotoxin-treated sensory neurons using patch clamp recording methods. Rapid increases in Na⁺ and TTX concentrations produced transient increases in inward Na⁺ current that decayed with a time course proportional to TTX concentration. Current clamp measurements indicated that, with 10 µ M TTX, depolarizations last ∼30 ms. Nonetheless, synaptosomal release of [³H]glutamate triggered by the synthetic action potential remained prolonged. Brief neuronal action potentials at some synapses may trigger transmitter release that persists for several seconds.     

Activation of Nuclear Calcium Dynamics by Synaptic Stimulation in Cultured Cortical Neurons

L-type voltage-sensitive Ca2+ channels (VSCCs) are enriched on the neuronal soma and trigger gene expression during synaptic activity. To understand better how these channels regulate somatic and nuclear Ca2+ dynamics, we have investigated Ca2+ influx through L-type VSCCs following synaptic stimulation, using the long-wavelength Ca2+ indicator fluo-3 combined with laser scanning confocal microscopy. Single synaptic stimuli resulted in rapid Ca2+ transients in somatic cytoplasmic compartments (<5 ms rise time). Nuclear Ca2+ elevations lagged behind cytoplasmic levels by approximately 60 ms, consistent with a dependence on diffusion from a cytoplasmic source. Pharmacological experiments indicated that L-type VSCCs mediated approximately 50% of the nuclear and somatic (cytoplasmic) Ca2+ elevation in response to strong synaptic stimulation. In contrast, relatively weak excitatory postsynaptic potentials (EPSPs; approximately 15 mV) or single action potentials were much less effective at activating L-type VSCCs. Antagonist experiments indicated that activation of the NMDA-type glutamate receptor leads to a long-lasting somatic depolarization necessary to activate L-type VSCCs effectively during synaptic stimuli. Simulation of action potential and somatic EPSP depolarization using voltage-clamp pulses indicated that nuclear Ca2+ transients mediated by L-type VSCCs were produced by sustained depolarization positive to -25 mV. In the absence of synaptic stimulation, action potential stimulation alone led to elevations in nuclear Ca2+ mediated by predominantly non-L-type VSCCs. Our results suggest that action potentials, in combination with long-lived synaptic depolarizations, facilitate the activation of L-type VSCCs. This activity elevates somatic Ca2+ levels that spread to the nucleus.     

Gating of the squid sodium channel at positive potentials. I. Macroscopic ionic and gating currents.

Macroscopic ionic sodium currents and gating currents were studied in voltage-clamped, dialyzed giant axons of the squid Loligo pealei under conditions of regular and inverse sodium gradients. Sodium currents showed regular kinetics but inactivation was incomplete, showing a maintained current for depolarizations lasting 18 ms. The ratio of the maintained current to the peak current increased with depolarization and it did not depend on the direction of the current flow or the sodium gradient. The time constant of inactivation was not affected by the sodium gradient. Double-pulse experiments allowed the separation of a normal inactivating component and a noninactivating component of the sodium currents. In gating current experiments, the results from double-pulse protocols showed that the charge was decreased by the prepulse and that the slow component of the 'on' gating current was preferentially depressed. As expected, charge immobilization was established faster at higher depolarizations than at low depolarizations, however, the amount of immobilized charge was unaffected by the pulse amplitude. This indicates that the incomplete sodium inactivation observed at high depolarizations is not the result of decreased charge immobilization; the maintained current must be due to a conductance that appears after normal charge immobilization and fast inactivation.     

Effects of acoustic stimuli on neuronal activity in the auditory cortex of the rat

Spontaneous activity of cortical neurons exhibits alternative fluctuations of membrane potential consisting of phased depolarization called "up-state" and persistent hyperpolarization called "down-state" during slow wave sleep and anesthesia. Here, we examined the effects of sound stimuli (noise bursts) on neuronal activity by intracellular recording in vivo from the rat auditory cortex (AC). Noise bursts increased the average time in the up-state by 0.81+/-0.65 s (range, 0.27-1.74 s) related to a 10 s recording duration. The rise times of the spontaneous up-events averaged 69.41+/-18.04 ms (range, 40.10-119.21 ms), while those of the sound-evoked up-events were significantly shorter (p<0.001) averaging only 22.54+/-8.81 ms (range, 9.31-45.74 ms). Sound stimulation did not influence ongoing spontaneous up-events. Our data suggest that a sound stimulus does not interfere with ongoing spontaneous neuronal activity in auditory cortex but can evoke new depolarizations in addition to the spontaneous ones.     

Membrane capacity of squid giant axon during hyper- and depolarizations

Summary The change in membrane capacitance and conductance of squid giant axons during hyper- and depolarizations was investigated. The measurements of capacitance and conductance were performed using an admittance bridge with resting, hyperpolarized and depolarized membranes. The duration of DC pulses is 20–40 msec and is long enough to permit the admittance measurements between 1 and 50 kHz. The amplitudes of DC pulses were varied between 0 and 40mV for both depolarization and hyperpolarization. Within these limited experimental conditions, we found a substantial increase in membrane capacitance with depolarization and a decrease with hyperpolarization. Our results indicate that the change in membrane capacitance will increase further if low frequencies are used with larger depolarizing pulses. The change in membrane capacitance is frequency dependent and it increases with decreasing frequencies. The analyses based on an equivalent circuit (vide infra) gives rise to a time constant of active membrane capacitance close to that of sodium currents. This result indicates that the observed capacitance changes may arise from sodium channels. A brief discussion is given on the nature of frequency-dependent membrane capacitance of nerve axons.     

Calcium-dependent potassium current in barnacle photoreceptor

When barnacle lateral eye photoreceptors are depolarized to membrane potentials of 0 to +50 mV in the dark, the plot of outward current through the cell membrane against time has two distinct maxima. The first maximum occurs 5-10 ms after the depolarization began. The current then decays to a minimum at approximately 500 ms after the onset of depolarization, and then increases to a second maximum 4-6 s after the depolarization began. If depolarization is maintained, the current again decays to reach a steady value approximately 1 min after depolarization began. The increase in current to the maximum at 4-6s from the minimum at approximately 500 ms is termed the "late current." It is maximum for depolarizations to around +25 mV and is reduced in amplitude at more positive potentials. It is not observed when the membrane is depolarized to potentials more positive than +60 mV. The late current is inhibited by external cobaltous ion and external tetraethylammonium ion, and shows a requirement for external calcium ion. When the calcium-sequestering agent EGTA is injected, the late current is abolished. Illumination of a cell under voltage clamp reduces the amplitude of the late current recorded subsequently in the dark. On the basis of the voltage dependence and pharmacology of the late current, it is proposed that the current is a calcium-dependent potassium current.     

Calcium signal transmission in chick sensory neurones is diffusion based

In many cells, the cytosol is an excitable medium through which calcium waves propagate by calcium induced calcium release (CICR). Many labs. have reported CICR in neurones subsequent to calcium influx through voltage gated channels. However, these have used long depolarizations. We have imaged calcium within chick sensory neurones following 50 ms depolarizations. Calcium signals travelled rapidly throughout the cell, such that changes at the cell centre were delayed by 24 ms compared to regions 3 microm from the plasma membrane. The nuclear envelope imposed a delay of 9 ms. A simple diffusion model with few unknowns gave good fits to the measured data, indicating that passive diffusion is responsible for signal transmission in these neurones. Simulations run without indicator dye did not reveal markedly different spatiotemporal dynamics, although concentration changes were larger. Simulations of calcium changes during action potentials revealed that large calcium transients occurring in the cytosol close to the nucleus are significantly attenuated by the nuclear envelope. Our results indicate that for the brief depolarisations that neurones will experience during normal signal processing calcium signals are transmitted by passive diffusion only. Diffusion is perfectly capable of transmitting the calcium signal into the interior of nerve cell bodies, and into the nucleoplasm.     

Orientational exchange approach to fluorescence anisotropy decay.

Fluorescence depolarization is a powerful technique in resolving dynamics of molecular systems. Data obtained in fluorescence depolarization experiments are highly complex. Mathematical models for analyzing data from depolarization due to rotational motion have been largely based on the rotational diffusion equation. These results have been verified by Monte Carlo simulations. It has been implicitly stated that a 90 degrees jump model between predefined orientations such as presented by G. Weber (1971. J. Chem. Phys. 55:2399-2411) should, for the specific case of fluorescence depolarization, give the same answer as the diffusion equation. Since the highly symmetric cases considered by G. Weber gave the same result as the diffusion equation, it has been desirable to use this method in cases where depolarization arises from both discrete processes and rotational diffusion. We have derived, in a compartmental formalism, the general result for excitation and emission dipoles not necessarily coincident with any of the principal rotational axes of the fluorophore from this exchange model, and have found it to be different from that of the diffusion equation approach. We have also verified this difference with a Monte Carlo simulation of our exchange model. This derivation allows us to define the limits of validity of the 90 degrees exchanges to model rotational diffusion. Also, for systems where movements may be jumps between a few preferred orientations, the actual physical mechanism of depolarization may not be accurately represented by continuous diffusion. The compartmental formalism developed here can be used to easily combine rotational motions with discrete position jumps or other level kinetics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rabbit oocyte maturation: changes of membrane resistance, capacitance, and the frequency of spontaneous transient depolarizations

Immature oocytes from rabbits were examined with electrophysiological techniques to determine if their membrane properties change during maturation. The input resistance increased and input capacitance decreased during maturation, although no significant change in membrane potential was observed. The changes observed were consistent with a decrease of corona radiata-oocyte electrical coupling accompanying maturation. Spontaneous transient depolarizations were recorded from immature oocytes surrounded by corona radiata, but not from mature ova. Each event consisted of a rapid depolarization, sustained for 1-100 sec, and a slow repolarization to the resting potential. Spontaneous inward currents with a time course similar to the spontaneous transient depolarizations occurred when the oocyte's membrane potential was held constant by voltage clamp. The frequency with which spontaneous transient depolarizations occurred decreased during maturation. These findings are consistent with a model in which spontaneous depolarizations originate in corona radiata cells and are detected in the oocyte via electrical coupling.     

Dibucaine mitigates spreading depolarization in human neocortical slices and prevents acute dendritic injury in the ischemic rodent neocortex

Background

Spreading depolarizations that occur in patients with malignant stroke, subarachnoid/intracranial hemorrhage, and traumatic brain injury are known to facilitate neuronal damage in metabolically compromised brain tissue. The dramatic failure of brain ion homeostasis caused by propagating spreading depolarizations results in neuronal and astroglial swelling. In essence, swelling is the initial response and a sign of the acute neuronal injury that follows if energy deprivation is maintained. Choosing spreading depolarizations as a target for therapeutic intervention, we have used human brain slices and in vivo real-time two-photon laser scanning microscopy in the mouse neocortex to study potentially useful therapeutics against spreading depolarization-induced injury.

Methodology/Principal Findings

We have shown that anoxic or terminal depolarization, a spreading depolarization wave ignited in the ischemic core where neurons cannot repolarize, can be evoked in human slices from pediatric brains during simulated ischemia induced by oxygen/glucose deprivation or by exposure to ouabain. Changes in light transmittance (LT) tracked terminal depolarization in time and space. Though spreading depolarizations are notoriously difficult to block, terminal depolarization onset was delayed by dibucaine, a local amide anesthetic and sodium channel blocker. Remarkably, the occurrence of ouabain-induced terminal depolarization was delayed at a concentration of 1 µM that preserves synaptic function. Moreover, in vivo two-photon imaging in the penumbra revealed that, though spreading depolarizations did still occur, spreading depolarization-induced dendritic injury was inhibited by dibucaine administered intravenously at 2.5 mg/kg in a mouse stroke model.

Conclusions/Significance

Dibucaine mitigated the effects of spreading depolarization at a concentration that could be well-tolerated therapeutically. Hence, dibucaine is a promising candidate to protect the brain from ischemic injury with an approach that does not rely on the complete abolishment of spreading depolarizations.     

Effects of membrane polarization on sarcoplasmic calcium release in skeletal muscle

Calcium release from the sarcoplasmic reticulum was investigated in voltage-clamped, tetrodotoxin-treated frog skeletal muscle fibres injected with arsenazo III. Short (5 ms) depolarizing pulses (test pulses) produced a transient change in arsenazo III absorption, signalling an increase in intracellular calcium in concentration (calcium transient). Conditioning subthreshold depolarizations, which preceded the test pulse, potentiated the calcium transient triggered by the test pulse. Conditioning hyperpolarizations, applied either before or after the test pulse, inhibited the calcium transient. These effects of conditioning polarizations on the calcium transient may explain similar effects of subthreshold polarizations on muscle contraction that have previously been reported. The potentiating effect of subthreshold depolarizations was observed only when the test pulse was short (5 ms). The potentiating effect develops at -48 mV with a time constant of about 7 ms at 6.5 degrees C; this seems to be slower than that predicted by the potential spread from the surface along the tubular system. Thus, part of the effect could arise from the coupling process between tubular depolarization and calcium release.     

Slow oxygen release on the first two flashes in chemically stressed Photosystem II membrane fragments results from hydrogen peroxide oxidation

Flash-induced amperometric signals were measured with a Joliot-type O₂ rate electrode in spinach Photosystem II (PS II) membrane fragments exposed to very low concentrations of added hydroxylamine or hydrogen peroxide. In both cases anomalous O₂ signals were observed on the first two flashes, and oscillating four-flash patterns were observed on subsequent flashes. The anomalous signals were eliminated in the presence of catalase but not EDTA. The rise times of the O₂-release kinetics associated with the anomalous signals were slow (ca. 20 ms with NH₂OH and ca. 120 ms with H₂O₂) compared to the kinetics of O₂ release on subsequent flashes and in control membranes (3–6 ms). It is proposed that when the intact PS II O₂-evolving complex is perturbed with small concentrations of added reductant, H₂O₂ can gain access and bind to the complex. Bound H₂O₂ can then reduce lower S states in some centers leading to anomalous O₂ signals on the first two flashes. A model is presented to explain both types of anomalous O₂ production. Oxygen observed on the third and subsequent flashes is due to the normal photosynthetic O₂-evolution process arising from the S₃-state. Anomalous O₂ production could be a protective mechanism in PS II centers subjected to stress conditions.Abbreviations DCIP 2,6-dichlorophenolindophenol - EDTA ethylenediaminetetraacetic acid - MES 4-morpholine-ethanesulfonic acid - OEC oxygen-evolving complex - PS II Photosystem II - Y_i O₂ flash yield on the i^th flash - Y_ss steady-state O₂ flash yield level in algae, chloroplasts, or thylakoids after flash-driven S-state oscillations have been damped Formerly, the Solar Energy Research Institute and operated by the Midwest Research Institute for the US Department of Energy under Contract DE-AC-02-83CH10093. Government and MRI retain non-exclusive, royalty-free license to publish or reproduce published articles, or allow others to do so for Government purposes.     

A slow component of intramembranous charge movement during sarcoplasmic reticulum calcium release in frog cut muscle fibers

Cut muscle fibers from Rana temporaria were mounted in a double Vaseline-gap chamber and equilibrated with an end-pool solution that contained 20 mM EGTA and 1.76 mM Ca (sarcomere length, 3.3-3.8 microns; temperature, 14-16 degrees C). Sarcoplasmic reticulum (SR) Ca release, delta[CaT], was estimated from changes in myoplasmic pH (Pape, P.C., D.- S. Jong, and W.K. Chandler. 1995. J. Gen. Physiol. 106:259-336). The maximal value of delta[CaT] obtained during a depleting depolarization was assumed to equal the SR Ca content before stimulation, [CaSR]R (expressed as myoplasmic concentration). After a depolarization to -55 to -40 mV in fibers with [CaSR]R = 1,000-3,000 microM, currents from intramembranous charge movement, Icm, showed an early I beta component. This was followed by an I gamma hump, which decayed within 50 ms to a small current that was maintained for as long as 500 ms. This slow current was probably a component of Icm because the amount of OFF charge, measured after depolarizations of different durations, increased according to the amount of ON charge. Icm was also measured after the SR had been depleted of most of its Ca, either by a depleting conditioning depolarization or by Ca removal from the end pools followed by a series of depleting depolarizations. The early I beta component was essentially unchanged by Ca depletion, the I gamma hump was increased (for [CaSR]R > 200 microM), the slow component was eliminated, and the total amount of OFF charge was essentially unchanged. These results suggest that the slow component of ON Icm is not movement of a new species of charge but is probably movement of Q gamma that is slowed by SR Ca release or some associated event such as the accompanying increase in myoplasmic free [Ca] that is expected to occur near the Ca release sites. The peak value of the apparent rate constant associated with this current, 2-4%/ms at pulse potentials between -48 and -40 mV, is decreased by half when [CaSR]R approximately equal to 500-1,000 microM, which gives a peak rate of SR Ca release of approximately 5-10 microM/ms.     

Inactivation of A currents and A channels on rat nodose neurons in culture

Cultured sensory neurons from nodose ganglia were investigated with whole-cell patch-clamp techniques and single-channel recordings to characterize the A current. Membrane depolarization from -40 mV holding potential activated the delayed rectifier current (IK) at potentials positive to -30 mV; this current had a sigmoidal time course and showed little or no inactivation. In most neurons, the A current was completely inactivated at the -40 mV holding potential and required hyperpolarization to remove the inactivation; the A current was isolated by subtracting the IK evoked by depolarizations from -40 mV from the total outward current evoked by depolarizations from -90 mV. The decay of the A current on several neurons had complex kinetics and was fit by the sum of three exponentials whose time constants were 10-40 ms, 100-350 ms, and 1-3 s. At the single-channel level we found that one class of channel underlies the A current. The conductance of A channels varied with the square root of the external K concentration: it was 22 pS when exposed to 5.4 mM K externally, the increased to 40 pS when exposed to 140 mM K externally. A channels activated rapidly upon depolarization and the latency to first opening decreased with depolarization. The open time distributions followed a single exponential and the mean open time increased with depolarization. A channels inactivate in three different modes: some A channels inactivated with little reopening and gave rise to ensemble averages that decayed in 10-40 ms; other A channels opened and closed three to four times before inactivating and gave rise to ensemble averages that decayed in 100-350 ms; still other A channels opened and closed several hundred times and required seconds to inactivate. Channels gating in all three modes contributed to the macroscopic A current from the whole cell, but their relative contribution differed among neurons. In addition, A channels could go directly from the closed, or resting, state to the inactivated state without opening, and the probability for channels inactivating in this way was greater at less depolarized voltages. In addition, a few A channels appeared to go reversibly from a mode where inactivation occurred rapidly to a slow mode of inactivation.