Rat hippocampal neurons in culture: Ca2+ and Ca2+-dependent K+ conductances

Rat hippocampal neurons grown in dissociated cell culture were studied in a medium containing 1 microM tetrodotoxin (TTX) and 25 mM tetraethylammonium (TEA), which eliminated the Na+ and K+ conductances normally activated by depolarizing current injections. In this medium depolarizing current pulses evoked depolarizing regenerative potentials and afterhyperpolarizations in most cells. Both of these events were blocked by close application of Co2+ or Cd2+. These events resemble Ca2+ spikes reported previously in hippocampal pyramidal cells. The membrane potential at which these Ca2+ spikes could be triggered and the rheobase current necessary were dependent on the potential at which the cell was conditioned: the more depolarized the holding potential, the more negative the absolute potential at which a spike could be triggered and the less rheobase current required. The duration of these Ca2+ spikes was also sensitive to the holding potential: the more depolarized the holding level, the longer the duration of the triggered spikes. The amplitude and duration of the Ca2+ spikes were enhanced in a reversible manner by 0.5-1.0 mM 4-aminopyridine (4-AP) delivered in the vicinity of the cell. Two-electrode voltage-clamp analysis of cells studied in TTX, TEA-containing medium revealed an inward current response that peaked in 25-50 ms during depolarizing commands. This response first became detectable during commands to -30 mV. It peaked in amplitude during commands to -10 mV and was enhanced in medium containing elevated [Ca2+]0. It was blocked by either 20 mM Mg2+, 0.2 mM Cd2+, 5 mM Co2+, or 5 mM Mn2+. These results have led us to identify this inward current response as ICa2+. 4-AP enhanced the magnitude and duration of ICa2+ independent of the drug's depressant effects on a transient K+ current also observed under these same experimental conditions. In many but not all cells the Ca2+ spike was followed by a long-lasting hyperpolarization associated with an increase in membrane conductance. This was blocked by Co2+. Under voltage clamp ICa2+ was followed by a slowly developing outward current response that was attenuated by Co2+ or Cd2+. These properties observed under current- and voltage-clamp recording conditions are superficially similar to those previously reported for Ca2+-dependent K+ conductance mechanisms (IC) recorded in these and other membranes. Long-lasting tail currents following activation of IC inverted in the membrane potential range for the K+ equilibrium potential found in these cells.     

Ca2+ influx modulation of temporal and spatial patterns of inositol trisphosphate-mediated Ca2+ liberation in Xenopus oocytes.

Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger by liberating intracellular Ca2+ and by promoting influx of extracellular Ca2+. We examined the effects of Ca2+ influx on the temporal and spatial patterns of intracellular Ca2+ liberation in Xenopus oocytes by fluorescence imaging of cytosolic free Ca2+ together with voltage clamp recording of Ca(2+)-activated Cl- currents. Oocytes were injected with a poorly metabolized InsP3 analogue (3-F-InsP3; see Introduction) to induce sustained activation of InsP3 signalling, and Ca2+ influx was controlled by applying voltage steps to change the driving force for Ca2+ entry. Positive- and negative-going potential steps (corresponding, respectively, to decreases and increases in Ca2+ influx) evoked damped oscillatory Cl- currents, accompanied by cyclical changes in cytosolic free Ca2+. The source of this Ca2+ was intracellular, since oscillations persisted when Ca2+ entry was suppressed by removing extracellular Ca2+ or by polarization close to the Ca2+ equilibrium potential. Fluorescence recordings from localized (ca 5 microns) spots on the oocyte showed repetitive Ca2+ spikes. Their frequency increased at more negative potentials, but they became smaller and superimposed on a sustained 'pedestal' of Ca2+. Spike periods ranged from about 50 s at +20 mV to 4s at potentials between -60 and -120 mV. Ca2+ spike frequency decreased after removing extracellular Ca2+, but the spike amplitude was not reduced and low frequency spikes continued for at least 30 min in the absence of extracellular Ca2+. Membrane current oscillations decayed in amplitude following voltage steps, while locally recorded Ca2+ spikes did not. This probably arose because Ca2+ release was initially synchronous across the cell, leading to large Ca(2+)-activated Cl- currents, but the currents then diminished as different areas of the cell began to release Ca2+ asynchronously. Fluorescence imaging revealed that Ca2+ liberation in 3-F-InsP3-loaded oocytes occurred as transient localized puffs and as propagating waves. Polarization to more negative potentials increased the frequency of puffs and the number of sites at which they were seen, and enhanced their ability to initiate waves. The frequency and velocity of Ca2+ waves increased at more negative potentials. When the potential was returned to more positive levels, repetitive Ca2+ spikes at first occurred synchronously across the recording area, but this synchronization was gradually lost and Ca2+ waves began at several foci. We conclude that influx of extracellular Ca2+ regulates the temporal and spatial patterns of Ca2+ liberation from InsP3-sensitive intracellular stores, probably as a result of dual excitatory and inhibitory actions of cytosolic Ca2+ on the InsP3 receptor.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of a nonspecific cation conductance by intracellular Ca2+ elevation in bursting pacemaker neurons of Helix pomatia

The pacemaker current of a bursting neuron of Helix pomatia was investigated using voltage-clamp and pressure-injection techniques. In the steady state the net membrane current was zero near threshold of the action potential at -45 mV. Negative to this potential the membrane current was inward and steady. During burst activity a long-lasting inward current instantaneously appeared with voltage steps to membrane potentials below -20 mV. This inward current was already present when the clamp step fell into the rising phase of the first spike and became larger during the depolarizing phase of the spike. The repolarization phase and the interspike interval did not add much current. As the spike duration became longer in the course of the burst discharge the inward current grew in amplitude, but its increase was not proportional to that of the spike duration. This was observed with clamp steps to the potassium equilibrium potential (EK = -70 mV). The inward current decayed during a hyperpolarizing step with a half time of approximately 400 ms, which was invariant to voltage as measured between -40 and -100 mV. It decreased linearly from -100 to -40 mV with an extrapolated zero potential of about -20 mV. The inward current was not generated by spikes if the Ca2+ conductance was blocked by Ni2+. At membrane potentials positive to EK the development of an outward current, probably carried by K+, could be observed during the burst. It overlasted the inward current and decayed with time constants of 6-7 s. This current grew successively in amplitude in the course of the burst discharge and finally nullified the inward-current component at potentials around spike threshold, thus terminating the burst. An inward current with properties similar to the spike-induced inward current was produced by pressure injecting CaCl2 into the neurons. This current was unselectively carried by cations as shown by both ion-substitution experiments and measurements with ion-selective microelectrodes. Large cations such as choline, TEA, and Tris passed through the channels nearly as well as Na+. Changes in the H+ or Cl- concentration were not seen to affect the inward current. Spike as well as the injection-induced currents were largest in bursting pacemaker cells compared with other cells of similar size. Both currents were found to be small or absent in nonbursting but regularly firing pacemaker cells, albeit these cells reveal a larger Ca2+ current density than the bursting pacemaker cell.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca2+ enhances U-type inactivation of N-type (CaV2.2) calcium current in rat sympathetic neurons

Ca2+ -dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (tau approximately 150 ms) and slow (tau approximately 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5-30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.     

Effects of temperature on calcium transients and Ca2+-dependent afterhyperpolarizations in neocortical pyramidal neurons

In neocortical pyramidal neurons, the medium (mAHP) and slow AHP (sAHP) have different relationships with intracellular [Ca2+]. To further explore these differences, we varied bath temperature and compared passive and active membrane properties and Ca2+ transients in response to a single action potential (AP) or trains of APs. We tested whether Ca(2+)-dependent events are more temperature sensitive than voltage-dependent ones, the slow rise time of the sAHP is limited by diffusion, and temperature sensitivity differs between the mAHP and sAHP. The onset and decay kinetics of the sAHP were very temperature sensitive (more so than diffusion). We found that the decay time course of Ca2+ transients was also very temperature sensitive. In contrast, the mAHP (amplitude, time to peak, and exponential decay) and sAHP peak amplitude were moderately sensitive to temperature. The amplitudes of intracellular Ca2+ transients evoked either by a single spike or a train of spikes showed modest temperature sensitivities. Pyramidal neuron input resistance was increased by cooling. With the exception of threshold, which remained unchanged between 22 and 35 degrees C, action potential parameters (amplitude, half-width, maximum rates of rise and fall) were modestly affected by temperature. Collectively, these data suggest that temperature sensitivity was higher for the Ca(2+)-dependent sAHP than for voltage-dependent AP parameters or for the mAHP, diffusion of Ca2+ over distance cannot explain the slow rise of the sAHP in these cells, and the kinetics of the sAHP and mAHP are affected differently by temperature.     

Supraoptic neurons sustain high frequency firing when extracellular Ca2+ is replaced with other divalent cations in rat brain slices

In slices of hypothalamus, maintained in vitro, the discharge of supraoptic neurons in standard artificial cerebrospinal fluid was compared with that produced when extracellular Ca2+ was replaced with Mg2+, Co2+ or Mn2+. Interspike interval histograms were constructed for periods before, during and after replacement of extracellular Ca2+. Of the 31 cells recorded in normal medium, 16 fired slowly and irregularly, 9 were phasic, 3 fired continuously at more than 3 spikes/s and 3 produced short, high frequency bursts of activity that were separated by slow irregular discharge. Interspike interval distributions were broad showing little preference for any one interval and intervals shorter than 30-50 ms were rare. The cell firing rate could be increased by the electrophoretic application of glutamate and under these conditions, the interval distributions became narrower as shorter intervals predominated. However, when cells discharged above 20 spikes/s, the spike amplitude declined rapidly and became indistinguishable from the noise. Replacement of extracellular Ca2+ with Mg2+, Co2+ or Mn2+ produced reversible changes in interspike interval distribution, although no consistent change in mean firing frequency was observed. Supraoptic neurons were now able to maintain relatively high frequency discharge (15-25 spikes/s) for longer periods; firing either continuously or periodically, and interspike intervals became grouped more closely at the shorter end of the normal distribution. However, no very short interspike were recorded. Less than 2% of all recorded intervals were shorter than 30 ms, even in cells exposed to test medium for 1-3 h and excited by application of glutamate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 microM). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 microM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50-100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by niflumic acid (50-100 microM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary conductance (10-17 pS) and open with a high probability (相似文献   

Ca2+ dynamics at the frog motor nerve terminal

Rises in free [Ca2+]i in response to various tetanic stimuli (Ca2+ transient) in frog motor nerve terminals were measured by recording fluorescence changes of Ca2+ indicators and analyzed in relation to short-term synaptic plasticity. Ca2+ transients reached a plateau after 10-20 impulses at 100 Hz and decayed in a three-exponential manner, in which the fast component was predominant. The plateau and fast component of the Ca2+ transient were elevated infralinearly with an increase in tetanus frequency. Computer simulation showed that the Ca2+ transients estimated from fluorescence changes faithfully reflect the true changes in [Ca2+]i except for the initial 20 ms. A slow Ca2+ chelator, EGTA, loaded into the nerve terminal, decreased the magnitude of both the fast and slow components of facilitation of transmitter release and the time constant of the former. A fast Ca2+ chelator, BAPTA, decreased the magnitude of fast facilitation but slightly increased its time constant. These results suggest that Ca2+ transients in the frog motor nerve terminals are primarily caused by Ca2+ entry and are dissipated by three components, in which the rate of the fast component is equivalent to that of free Ca2+ diffusion. The residual Ca2+ in the nerve terminals after stimulation accounts for the fast component of facilitation.     

Electrophysiology of dentate gyrus granule cells

The orthodromic synaptic responses, membrane properties, and responses of dentate gyrus granule cells (DGCs) to several convulsant agents were studied in the in vitro hippocampal slice preparation. Orthodromic stimulation via the perforant pathway (PP) evoked excitatory-inhibitory postsynaptic potentials (EPSP-IPSP) sequences in 27 of 34 DGCs studied. In the majority, only one action potential could be evoked by supramaximal orthodromic stimulation. In recordings from DGC somata, overshooting spikes could be evoked either orthodromically or by current injections. Small-amplitude, fast transients were seen in 5 of 34 DGCs. The current/voltage (I-V) characteristic of most DGCs was linear throughout a range of membrane potentials between 15 and 20 mV negative and 5 and 15 mV positive to the resting potential. At the extremes of this range nonohmic behavior was noted. Exposure of slices to agents that block IPSPs, such as penicillin, bicuculline, picrotoxin, and media containing low Cl- concentrations, eliminated PP-evoked hyperpolarizations in DGCs and prolonged the repolarizing phase of the PP EPSP. In contrast to findings in hippocampal pyramidal cells and neocortical neurons, blockade of IPSPs did not lead to the development of orthodromically evoked slow depolarizations and burst discharges. After slices were exposed to 5 mM tetraethylammonium, current pulses evoked slow spikes, which were resistant to tetrodotoxin and presumably mediated by Ca2+. Spontaneous burst discharges or bursts evoked by brief depolarizing pulses did not occur under these conditions. Substitution of Ba2+ for Ca2+ in the perfusion solution resulted in development of spontaneous slow membrane depolarizations and burst discharges in DGCs. Burst discharges could be directly evoked and spikes were prolonged and resistant to tetrodotoxin (TTX). After hyperpolarizations lasting 200-1,000 ms, associated with a conductance increase and presumably due to a Ca2+-activated K+ conductance, followed directly evoked spike trains in 5 of 20 DGCs. These data suggest that Ca2+ conductances may be evoked in DGCs under certain circumstances but are not prominent during activation of DGCs under standard in vitro recording conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blockade of SK-type Ca2+-activated K+ channels uncovers a Ca2+-dependent slow afterdepolarization in nigral dopamine neurons

Sharp electrode current-clamp recording techniques were used to characterize the response of nigral dopamine (DA)-containing neurons in rat brain slices to injected current pulses applied in the presence of TTX (2 microM) and under conditions in which apamin-sensitive Ca2+-activated K+ channels were blocked. Addition of apamin (100-300 nM) to perfusion solutions containing TTX blocked the pacemaker oscillation in membrane voltage evoked by depolarizing current pulses and revealed an afterdepolarization (ADP) that appeared as a shoulder on the falling phase of the voltage response. ADP were preceded by a ramp-shaped slow depolarization and followed by an apamin-insensitive hyperpolarizing afterpotential (HAP). Although ADPs were observed in all apamin-treated cells, the duration of the response varied considerably between individual neurons and was strongly potentiated by the addition of TEA (2-3 mM). In the presence of TTX, TEA, and apamin, optimal stimulus parameters (0.1 nA, 200-ms duration at -55 to -68 mV) evoked ADP ranging from 80 to 1,020 ms in duration (355.3 +/- 56.5 ms, n = 16). Both the ramp-shaped slow depolarization and the ensuing ADP were markedly voltage dependent but appeared to be mediated by separate conductance mechanisms. Thus, although bath application of nifedipine (10-30 microM) or low Ca2+, high Mg2+ Ringer blocked the ADP without affecting the ramp potential, equimolar substitution of Co2+ for Ca2+ blocked both components of the voltage response. Nominal Ca2+ Ringer containing Co2+ also blocked the HAP evoked between -55 and -68 mV. We conclude that the ADP elicited in DA neurons after blockade of apamin-sensitive Ca2+-activated K+ channels is mediated by a voltage-dependent, L-type Ca2+ channel and represents a transient form of the regenerative plateau oscillation in membrane potential previously shown to underlie apamin-induced bursting activity. These data provide further support for the notion that modulation of apamin-sensitive Ca2+-activated K+ channels in DA neurons exerts a permissive effect on the conductances that are involved in the expression of phasic activity.     

Calcium-dependent potassium conductance in rat supraoptic nucleus neurosecretory neurons

Intracellular recordings of rat supraoptic nucleus neurons were obtained from perfused hypothalamic explants. Individual action potentials were followed by hyperpolarizing afterpotentials (HAPs) having a mean amplitude of -7.4 +/- 0.8 mV (SD). The decay of the HAP was approximated by a single exponential function having a mean time constant of 17.5 +/- 6.1 ms. This considerably exceeded the cell time constant of the same neurons (9.5 +/- 0.8 ms), thus indicating that the ionic conductance underlying the HAP persisted briefly after each spike. The HAP had a reversal potential of -85 mV and was unaffected by intracellular Cl- ionophoresis of during exposure to elevated extracellular concentrations of Mg2+. In contrast, the peak amplitude of the HAP was proportional to the extracellular Ca2+ concentration and could be reversibly eliminated by replacing Ca2+ with Co2+, Mn2+, or EGTA in the perfusion fluid. During depolarizing current pulses, evoked action potential trains demonstrated a progressive increase in interspike intervals associated with a potentiation of successive HAPs. This spike frequency adaptation was reversibly abolished by replacing Ca2+ with Co2+, Mn2+, or EGTA. Bursts of action potentials were followed by a more prolonged afterhyperpolarization (AHP) whose magnitude was proportional to the number of impulses elicited (greater than 20 Hz) during a burst. Current injection revealed that the AHP was associated with a 20-60% decrease in input resistance and showed little voltage dependence in the range of -70 to -120 mV. The reversal potential of the AHP shifted with the extracellular concentration of K+ [( K+]o) with a mean slope of -50 mV/log[K+]o.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of T-type calcium current in enkephalinergic neurones in guinea-pig hypothalamic slices

 The guinea-pig hypothalamic magnocellular dorsal nucleus (mdn) exclusively contains enkephalinergic neurones providing inputs to the septum. This nucleus is believed to play a role in hippocampo-septo-hypothalamic relationships. mdn neurones display prominent low-threshold Ca²⁺ spikes, which differ in their propensity to trigger either a burst of Na⁺ spikes or a single spike. In the present study, whole-cell voltage-clamp experiments were carried out on thick slices at 34°C to characterize the pharmacological and physical properties of the transient Ca²⁺ current (I _T) underlying the low-threshold spikes. Recorded cells were dye-labelled and identified as belonging to the mdn. In bursting and non-bursting neurones, I _T was reduced by amiloride (1 μM) and octanol (1 mM), and during replacement of Ca²⁺ by Ba²⁺. The Ca²⁺ channel blocker mibefradil (10 μM) had only a slight blocking action. Nifedipine (100 μM) and flunarizine (1 μM) had no effect. I _T activated between –80 mV and –50 mV and the mean peak current was 1050 pA. Steady-state activation and inactivation curves were fitted by a Boltzmann equation. The half-activation voltage was –70 mV, slope factor=3.6, and half-inactivation voltage was about –80 mV, slope factor=4.5. Time-to-peak and time constant of inactivation were voltage dependent. Recovery from activation occurred within 500 ms. When compared with results on other I _T, the present data show that the current possesses distinct pharmacological and physical properties. Nevertheless, all investigated cells displayed a homogenous profile of I _T, suggesting that the differences in spike pattern between mdn neurones are not due to different populations of Ca²⁺ channels. Received: 20 October 1998 / Received after revision and accepted: 5 January 1999     

Differential time-course of slow afterhyperpolarizations and associated Ca2+ transients in rat CA1 pyramidal neurons: further dissociation by Ca2+ buffer.

Hippocampal neurons exhibit a slow afterhyperpolarization following membrane depolarization; this is thought to reflect an underlying Ca2+-dependent K+ current. This current is potentiated by intermediate concentrations (0.1-1.0 mM) of exogenous Ca2+ buffer [Schwindt P. C. et al. (1992) Neuroscience 47, 571-578; Zhang L. et al. (1995) J. Neurophysiol. 74, 2225-2241]. The relationship between the slow afterhyperpolarization and associated Ca2+ transients was investigated in the presence and absence of added exogenous Ca2+ buffer. Slow afterhyperpolarizations and underlying K+ currents were measured using whole-cell patch-clamp recordings from hippocampal CA1 neurons in acute rat brain slices. Inclusion of fluorescent Ca2+ indicators in the patch pipette solution allowed simultaneous measurement of the evoked subcellular Ca2+ transients using a confocal microscope. The peak Ca2+ signal exhibited an incremental increase with each action potential. This increase eventually reached a plateau with increasing numbers of action potentials, suggesting dye saturation with peak Ca2+ concentrations. As the K(D) for Ca2+ of the indicator dyes used was between 200 and 300 nM, it is predicted that saturation will occur when the peak Ca2+ signal exceeds 1 microM. This occurred with fewer action potentials in dendritic vs somatic compartments. Neither compartment exhibited averaged Ca2+ transients matching the slow afterhyperpolarization time-course, dendritic Ca2+ transients being most divergent. Intracellular accumulation of exogenous Ca2+ buffer, either by inclusion in the patch pipette or by incubation of the brain slice with its membrane-permeable form, caused a prolongation of the slow afterhyperpolarization but not of the somatic Ca2+ transient. The initial rate of decline of the dendritic Ca2+ transient was diminished, but remained faster than that of the slow afterhyperpolarization. We conclude that neither dendritic nor somatic Ca2+ signals match the slow afterhyperpolarization time-course, with this dissociation being further magnified by addition of exogenous Ca2+ buffer. The implications of this result are discussed.     

A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons

1. A long-lasting afterhyperpolarization (AHP) follows current-induced repetitive firing in hippocampal CA1 neurons studied in vitro. A 10-25% increase in membrane slope conductance occurs during the AHP, suggesting that it may be mediated by an increased conductance to either K+ or Cl-. 2. Intracellular Cl- iontophoresis does not alter the AHP but does attenuate the IPSP. In contrast Ba2+, a cation that can decrease K+ conductance, eliminates the AHP but not the IPSP. These findings suggest the AHP is produced by a long-lasting increased conductance to K+, and is distinct from the IPSP. 3. Mn2+, a Ca2+-channel blocker, eliminates the AHP. In comparison, the AHP persists in the presence of the Na+-channel blocker, tetrodotoxin (TTX), and appears to be temporally associated with TTX-resistant "Ca2+ spikes." It is concluded that AHP is probably activated by Ca2+ influx. 4. These observations indicate that the AHP may be produced by a Ca2+ activated K+ current. A balance between cellular depolarization produced by Ca2+ entry and repolarization generated by a Ca2+-activated K+ current appears to operate to control excitability in some mammalian cortical neurons as it does in molluscan neurons. Disruption of this balance by Ba2+ produces spontaneous membrane-potential oscillations and recurrent burst firing in hippocampal neurons. Increases in the magnitude and duration of Ca2+ depolarization and/or decreases in the Ca2+-activated, K+-mediated repolarization may be mechanisms that lead to spontaneous, epileptiform bursting in mammalian cortical neurons.     

Cysteine-protease activity elicited by Ca2+ stimulus in Plasmodium

Bloodstage malaria parasites require proteolytic activity for key processes as invasion, hemoglobin degradation and merozoite escape from red blood cells (RBCs). We investigated by confocal microscopy the presence of cysteine-protease activity elicited by calcium stimulus in Plasmodium chabaudi and Plasmodium falciparum in free trophozoites or for the later parasite within RBC using fluorescence resonance energy transfer (FRET) peptides. Peptide probes access, to either free or intraerythrocytic parasites, was also tested by selecting a range of fluorescent peptides (653-3146 Da molecular mass) labeled with Abz or FITC. In the present work we show that Ca2+ stimulus elicited by treatment with either melatonin, thapsigargin, ionomicin or nigericin, promotes an increase of substrate hydrolysis, which was blocked by the specific cysteine-protease inhibitor E-64 and the intracellular Ca2+ chelator, BAPTA. When parasites were treated with cytoplasmic Ca2+ releasing compounds, a cysteine-protease was labeled in the parasite cytoplasm by the fluorescent specific irreversible inhibitor, Ethyl-Eps-Leu-Tyr-Cap-Lys(Abz)-NH2, where Ethyl-Eps is Ethyl-(2S,3S)-oxirane-2,3-dicarboxylate. In summary, we demonstrate that P. chabaudi and P. falciparum have a cytoplasmic dependent cysteine-protease activity elicited by Ca2+.     

Electrogenic Ca2+ entry in the rat colonic epithelium

Capacitative Ca2+ entry in isolated rat colonic crypts was induced by dialysing the cells in the whole-cell patch-clamp mode with a pipette solution having a high Ca(2+)-buffering capacity. Under these conditions crypt cell resting potential was lower than normal. Flufe-namate, La3+ and Gd3+, blockers of non-selective cation channels, hyperpolarized the crypt cells and decreased membrane current. This current exhibited a cation selectivity of Na+>Ca2+. In contrast to Na+, Ca(2+) inhibited the current at concentrations exceeding 1 mmol/l. Indirect evidence suggests that the non-selective cation conductance is activated after stimulation of muscarinic receptors. Carbachol, a cholinergic agonist, evoked a transient hyperpolarization and an increase in membrane outwards current. The half-time of the decay of the carbachol response was shortened strongly in the presence of La3+. Fura-2 experiments with isolated crypts confirmed that La3+ inhibited the carbachol-induced increase in intracellular Ca2+. In parallel Ussing chamber experiments, La3+ suppressed the induction of Cl- secretion by carbachol. These results demonstrate that a non-selective cation conductance activated by store depletion may be involved in the regulation of electrolyte transport by agonists of the Ca2+ signalling pathway.     

Direct measurement of intracellular free Ca2+ in rat peritoneal macrophages: correlation with oxygen-radical production

A novel method has been developed, based on osmotic lysis of intracellular pinocytotic vesicles, to introduce the Ca2+-activated photoprotein obelin into the cytoplasm of rat peritoneal macrophages. The change in osmolarity of the incubating medium necessary to induce lysis of the pinocytotic vesicles did not significantly affect the viability or responsiveness of the cells. The method enabled on average 3 fl of external medium to be introduced into each cell. Macrophages loaded with photoprotein had a resting intracellular Ca2+ concentration of 0.24 +/- 0.02 microM, calculated from the obelin consumption rate. The calcium ionophore, A23187, induced a prolonged rise in intracellular Ca2+ and also stimulated oxygen-radical production, monitored by luminol-dependent chemiluminescence. The chemotactic peptide, N-formyl-methionyl-leucyl-phenylalanine, 1 microM, produced a transient increase in cytoplasmic Ca2+ which reached a plateau of 1.2 +/- 0.64 microM (n = 7) and declined with a half-time of approximately 40 sec. Unopsonized particles, latex beads (diameter = 1 micron), did not produce any detectable rise in intracellular Ca2+. Incorporation of a calcium chelator EGTA-ethylene-glycol-bis-(aminoethylether) tetra-acetate--into the cytoplasm abolished the transient intracellular Ca2+ rise induced by chemotactic peptide. Oxygen-radical production was also abolished. However, oxygen radical production induced by unopsonized particles was unaffected by intracellular EGTA. It was concluded that oxygen-radical production detected by chemiluminescence can be triggered by a rise in intracellular Ca2+. Chemotactic peptide induces oxygen-radical production by this mechanism. However, unopsonized particles induce oxygen-radical production by a mechanism independent of a rise in intracellular Ca2+.     

Accumulation of cytoplasmic calcium, but not apamin-sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells

The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s(-1), the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s(-1) the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They suggest that the calcium is present at the SK channel for a very short time after each action potential, and the current decays at a rate set by the deactivation kinetics of the SK channel. At high rates, repetitive firing was governed by a fast apamin-insensitive AHP current that did not accumulate, but rather showed depression with increases in activation frequency. A slowly accumulating AHP current, also insensitive to apamin, was extremely small at low rates but became significant with higher firing rates.     

Synaptic regulation of the slow Ca2+-activated K+ current in hippocampal CA1 pyramidal neurons: implication in epileptogenesis.

The slow Ca2+-activated K+ current (sI(AHP)) plays a critical role in regulating neuronal excitability, but its modulation during abnormal bursting activity, as in epilepsy, is unknown. Because synaptic transmission is enhanced during epilepsy, we investigated the synaptically mediated regulation of the sI(AHP) and its control of neuronal excitability during epileptiform activity induced by 4-aminopyridine (4AP) or 4AP+Mg2+-free treatment in rat hippocampal slices. We used electrophysiological and photometric Ca2+ techniques to analyze the sI(AHP) modifications that parallel epileptiform activity. Epileptiform activity was characterized by slow, repetitive, spontaneous depolarizations and action potential bursts and was associated with increased frequency and amplitude of spontaneous excitatory postsynaptic currents and a reduced sI(AHP.) The metabotropic glutamate receptor (mGluR) antagonist (S)-alpha-methyl-4-carboxyphenylglycine did not modify synaptic activity enhancement but did prevent sI(AHP) inhibition and epileptiform discharges. The mGluR-dependent regulation of the sI(AHP) was not caused by modulated intracellular Ca2+ signaling. Histamine, isoproterenol, and (+/-)-1-aminocyclopentane-trans-1,3-dicarboxylic acid reduced the sI(AHP) but did not increase synaptic activity and failed to evoke epileptiform activity. We conclude that 4AP or 4AP+Mg-free-induced enhancement of synaptic activity reduced the sI(AHP) via activation of postsynaptic group I/II mGluRs. The increased excitability caused by the lack of negative feedback provided by the sI(AHP) contributes to epileptiform activity, which requires the cooperative action of increased synaptic activity.     

Modulation of intracellular Na+ and Ca2+ induced by the cardiac Na+-Ca2+ exchanger in guinea pig ventricular myocytes

The Na+-Ca2+ exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca2+](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca2+ (approximately 66 microM Ca2+) thought low enough to inhibit exchanger turnover, the removal of external Na+ (by replacement with Li+) induced both an outward shift of the holding current and an increase in [Ca2+](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na+ removal were blocked either by the extracellular application of 2 mM Ni2+ or by chelating extracellular Ca2+ with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na+ substitution with Li(+) on both membrane current and [Ca2+](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni2+-sensitive components. The experimental values of the reversal potential and [Ca2+](i) were used to calculate cytosolic Na+ ([Na+](i)) by assuming an exchanger stoichiometry of 3Na+ : 1Ca2+. These calculations suggested that in the nominal absence of external Ca2+ ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na+ had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca2+ and high intracellular Ca2+ buffering, the Na+-Ca2+ exchanger can still generate sufficient Ca2+ influx on the removal of external Na+ to markedly increase cytosolic free Ca2+.