Blockade of calcium entry accelerates arsenite-mediated apoptosis in rat cerebellar granule cells

Arsenical exposure can cause defects in the central nervous system, yet the underlying cellular and molecular mechanisms are largely unknown. We have recently demonstrated that sodium arsenite induces apoptosis of cultured cortical and cerebellar neurons, suggesting that arsenite-induced neuronal apoptosis may contribute to at least some of its neurotoxic effects. Here we investigated the effect of Ca2+ on arsenite-mediated cerebellar granule neuron death. Sodium arsenite induced apoptosis in cerebellar neurons which were maintained in the presence of serum and depolarizing concentrations of potassium chloride (25 mM KCI). Under these conditions, inhibition of calcium entry by N-methyl-D-aspartate (NMDA) receptor blocker DL-aminophosphonovalerate (APV) or calcium channel antagonist nifedipine increased arsenite-induced apoptosis, while APV or nifedipine alone had little effect on cell viability. In cortical neurons or cerebellar neurons maintained at low potassium (5 mM), arsenite also induced apoptosis. However, the addition of APV or nifedipine did not alter levels of arsenite-induced apoptosis. These data suggest that arsenite-mediated apoptosis is regulated by intracellular calcium levels.     

Phosphorylation of IkappaB-beta is necessary for neuronal survival

Cerebellar granule neurons undergo apoptosis when switched from culture medium containing depolarizing levels of potassium (high potassium or HK) to nondepolarizing medium (low potassium or LK). We showed that in healthy neurons maintained in HK medium, IkappaB-beta is phosphorylated at a novel site, Tyr-161. LK-induced neuronal apoptosis is accompanied by a decrease in the extent of IkappaB-beta phosphorylation at this residue. Tyr-161 shares similarity to the consensus sequence for phosphorylation by the nonreceptor tyrosine kinases Abl and Arg. Arg phosphorylates Tyr-161 differentially in vitro, and LK treatment does cause a down-regulation of Arg activity. Moreover, treatment of neurons with two structurally distinct and highly selective Abl inhibitors, PD173955 and Gleevec, blocks HK-induced phosphorylation of IkappaB-beta at Tyr-161 and induces neuronal apoptosis. Overexpression of wild-type IkappaB-beta blocks LK-induced apoptosis, but this effect is abolished when Arg is pharmacologically inhibited. On the other hand, forced overexpression of IkappaB-beta in which Tyr-161 is mutated inhibits survival in HK demonstrating the importance of this residue to neuronal survival. Phosphorylation of IkappaB-beta enhances its association with p65/RelA causing an increase in NF-kappaB DNA binding activity. Our results identified IkappaB-beta phosphorylation as a key event in neuronal survival and provided a mechanism by which this is mediated.     

Calcium Transport by Primary Cultured Neuronal and Glial Cells from Chick Embryo Brain

The uptake of calcium was examined in primary cultures of pure neurons and of glial cells from dissociated hemispheres of chick embryo brain. Neuronal cultures took up calcium at a rate of 2.0 nmol per min per mg cell protein at medium concentrations of 1.2 mM-Ca²⁺ and 5.4 mM-K⁺. The rate of calcium entry into neurons was increased 2.7-fold by elevating medium potassium to 60 MM. The effect of high external potassium was to increase the V_max value for calcium transport from 5.5 to 13 nmol per min per mg; the Michaelis constant for calcium, 1.2 mM, was unchanged. The potassium-dependent component of calcium entry into the neuronal cultures was eliminated by addition of 0.1 mM-D-600 (a verapamil derivative) or by 1 mM-CoCl₂, but 0.5 μM-tet-rodotoxin had no significant effect. When choline replaced potassium in uptake medium no change in calcium transport was detected in neurons, nor was the entry of calcium increased when choline replaced sodium. Glial cultures took up calcium at 20% of the basal rate for neuronal cultures on a weight-of-protein basis. Uptake was not increased by potassium; during depolarization by potassium the calcium transport activity of glia was less than 10% that of neurons. It was concluded that cultured neurons contain a depolarization-sensitive, calcium-specific channel. A similar calcium transport activity was not detected in cultured glial cells.     

Calcineurin enhances MEF2 DNA binding activity in calcium-dependent survival of cerebellar granule neurons.

Myocyte enhancer factor 2 (MEF2) has been shown recently to be necessary for mediating activity-dependent neuronal survival. In this study, we show that calcium signals regulate MEF2 activity through a serine/threonine phosphatase calcineurin. In cultured primary cerebellar granule neurons, the electrophoretic mobility of MEF2A protein was sensitive to the level of extracellular potassium chloride (KCl) and depolarizing concentrations of KCl led to hypophosphorylation of the protein. The specific inhibitors of calcineurin cyclosporin A (CsA) and FK506 could overcome KCl-dependent MEF2A hypophosphorylation. The effects of CsA and FK506 were KCl specific as they had little effect on MEF2A phosphorylation when granule neurons were cultured in the presence of full media. Hyperphosphorylation of MEF2A led to the loss of its DNA binding activity as determined by DNA mobility shift assay. Consistent with this, CsA/FK506 also inhibited MEF2-dependent reporter gene expression. These findings demonstrate that regulation of MEF2A by calcium signals requires the action of protein phosphatase calcineurin. By maintaining MEF2A in a hypophosphorylated state, calcineurin enhances the DNA binding activity of MEF2A and therefore maximizes its transactivation capability. The identification of MEF2 as a novel target of calcineurin may provide in part a biochemical explanation for the therapeutic and toxic effects of immunosuppressants CsA and FK506.     

State-dependent inactivation of the Kv3 potassium channel.

Inactivation of Kv3 (Kv1.3) delayed rectifier potassium channels was studied in the Xenopus oocyte expression system. These channels inactivate slowly during a long depolarizing pulse. In addition, inactivation accumulates in response to a series of short depolarizing pulses (cumulative inactivation), although no significant inactivation occurs within each short pulse. The extent of cumulative inactivation does not depend on the voltage during the depolarizing pulse, but it does vary in a biphasic manner as a function of the interpulse duration. Furthermore, the rate of cumulative inactivation is influenced by changing the rate of deactivation. These data are consistent with a model in which Kv3 channel inactivation is a state-dependent and voltage-independent process. Macroscopic and single channel experiments indicate that inactivation can occur from a closed (silent) state before channel opening. That is, channels need not open to inactivate. The transition that leads to the inactivated state from the silent state is, in fact, severalfold faster then the observed inactivation of current during long depolarizing pulses. Long pulse-induced inactivation appears to be slow, because its rate is limited by the probability that channels are in the open state, rather than in the silent state from which they can inactivate. External potassium and external calcium ions alter the rates of cumulative and long pulse-induced inactivation, suggesting that antagonistic potassium and calcium binding steps are involved in the normal gating of the channel.     

A method for detection and characterization of GABA(A) receptor ligands using calcium-sensitive fluorescent probes

A method for detecting and characterizing possible ligands of neuronal GABA(A) receptors has been developed, which is based on measuring the calcium response to GABA by the fluorescence of a two-wavelength Ca-sensitive probe Fura-2. In a young (2–4 days) rat hippocampal cell culture, GABA induced depolarization and a transient increase in Ca²⁺ concentration in the cytosol of neurons due to activation of voltage-dependent calcium channels. A brief application of GABA could attenuate the calcium response to a subsequent addition of depolarizing agents (GABA or KCl). However, at modest amplitudes of calcium response to GABA, the reduction of the subsequent effect of KCl was insignificant, and the amplitudes of responses to KCl and to GABA proved to be linearly correlated, with a slope of ∼3.4. Therefore, the GABA calcium signals could be normalized in order to compare experiments performed on different days and different cultures. With such normalization, we estimated the EC₅₀ for GABA in neurons at ∼2.23 μM and the Hill coefficient at ∼1.9. A blocker of voltage-dependent calcium channels nifedipine suppressed the calcium responses both to KCl and to GABA, so that the linear relationship between their amplitudes was retained. To further validate the method, the IC50 and the type of inhibition were verified for known noncompetitive and competitive antagonists of GABA(A) receptors.     

NGF and bFGF protect rat hippocampal and human cortical neurons against hypoglycemic damage by stabilizing calcium homeostasis.

NGF and bFGF have recently been shown to have biological activity in central neurons, but their normal functions and mechanisms of action are unknown. Since central neurons are particularly vulnerable to hypoglycemia that occurs with ischemia or insulin overdose, we tested the hypothesis that growth factors can protect neurons against hypoglycemic damage. NGF and bFGF each prevented glucose deprivation-induced neuronal damage in human cerebral cortical and rat hippocampal cell cultures (EGF was ineffective). Protection was afforded when the growth factors were administered before (NGF and bFGF) or up to 12 hr following (NGF) the onset of hypoglycemia. Direct measurements of intracellular calcium levels and manipulations of calcium influx demonstrated that sustained elevations in intracellular calcium levels mediated the hypoglycemic damage. NGF and bFGF each prevented the hypoglycemia-induced elevations of intracellular calcium. These findings indicate that growth factors can stabilize neuronal calcium homeostasis in central neurons and thereby protect them against environmental insults.     

A compartmental model of an external urethral sphincter motoneuron of Onuf's nucleus

This article discusses a model of the electrical behavior of an external urethral sphincter motoneuron, based on morphological parameters like soma size, dendritic diameters and spatial dendritic configuration, and several electrical parameters. Because experimental data about the exact ion conductance mix of external urethral sphincter neurons is scarce, the gaps in knowledge about external urethral sphincter motoneurons were filled in with known data of alpha-motoneurons. The constructed compartmental model of motoneurons of Onuf's nucleus contains six voltage-dependent ionic conductances: a fast sodium and potassium conductance and an anomalous rectifier in the soma; a fast delayed rectifier type potassium conductance and a fast sodium conductance in the initial axon segment; an L-type calcium channel in the dendritic compartments. This paper considers the simulation of external urethral sphincter motoneuron responses to current injections that evoke bistable behavior. Simulations show self-sustained discharge following a depolarizing pulse through the microelectrode; the firing was subsequently terminated by a short hyperpolarizing pulse. This behavior is highly functional for neurons that have to exhibit prolonged activation during sphincter closure. In addition to these 'on' and 'off ' responses, we also observed a particular firing behavior in response to long-lasting triangular current pulses. When the depolarizing current was slowly increased and then decreased (triangular pulse) the firing frequency was higher during the descending phase than during the initial ascending phase.     

The dynamics of a neuronal culture of dissociated cortical neurons of neonatal rats

Neuronal networks of dissociated cortical neurons from neonatal rats were cultured over a multielectrode dish with 64 active sites, which were used both for recording the electrical activity and for stimulation. After about 4 weeks of culture, a dense network of neurons had developed and their electrical activity was studied. When a brief voltage pulse was applied to one extracellular electrode, a clear electrical response was evoked over almost the entire network. When a strong voltage pulse was used, the response was composed of an early phase, terminating within 25 ms, and a late phase which could last several hundreds of milliseconds. Action potentials evoked during the early phase occurred with a precise timing with a small jitter and the electrical activity initiated by a localized stimulation diffused significantly over the network. In contrast, the late phase was characterized by the occurrence of clusters of electrical activity with significant spatio-temporal fluctuations. The late phase was suppressed by adding small amounts of d(−)-2-amino-5-phosphonovaleric acid to the extracellular medium, or by increasing the amount of extracellular Mg²⁺. The electrical activity of the network was substantially increased by the addition of bicuculline to the extracellular medium. The results presented here show that the neuronal network may exist in two different dynamical states: one state in which the neuronal network behaves as a non-chaotic deterministic system and another state where the system exhibits large spatio-temporal fluctuations, characteristic of stochastic or chaotic systems. Received: 8 June 1999 / Accepted in revised form: 10 January 2000     

In vivo electrophysiological evidences for cortical neuron-glia interactions during slow (<1 Hz) and paroxysmal sleep oscillations.

The cortical activity results from complex interactions within networks of neurons and glial cells. The dialogue signals consist of neurotransmitters and various ions, which cross through the extracellular space. Slow (<1 Hz) sleep oscillations were first disclosed and investigated at the neuronal level where they consist of an alternation of the membrane potential between a depolarized and a hyperpolarized state. However, neuronal properties alone could not account for the mechanisms underlying the oscillatory nature of the sleeping cortex. Here I will show the behavior of glial cells during the slow sleep oscillation and its relationship with the variation of the neuronal membrane potential (pairs of neurons and glia recorded simultaneously and intracellularly) suggesting that, in contrast with previous assumptions, glial cells are not idle followers of neuronal activity. I will equally present measurements of the extracellular concentration of K(+) and Ca(2+), ions known to modulate the neuronal excitability. They are also part of the ionic flux that is spatially buffered by glial cells. The timing of the spatial buffering during the slow oscillation suggests that, during normal oscillatory activity, K(+) ions are cleared from active spots and released in the near vicinity, where they modulate the excitability of the neuronal membrane and contribute to maintain the depolarizing phase of the oscillation. Ca(2+) ions undergo a periodic variation of their extracellular concentration, which modulates the synaptic efficacy. The depolarizing phase of the slow oscillation is associated with a gradual depletion of the extracellular Ca(2+) promoting a progressive disfacilitation in the network. This functional synaptic neuronal disconnection is responsible for the ending of the depolarizing phase of the slow oscillation and the onset of a phasic hyperpolarization during which the neuronal network is silent and the intra- and extracellular ionic concentrations return to normal values. Spike-wave seizures often develop during sleep from the slow oscillation. Here I will show how the increased gap junction communication substantiates the facility of the glial syncytium to spatially buffer K(+) ions that were uptaken during the spike-wave seizures, and therefore contributing to the long-range recruitment of cortical territories. Similar mechanisms as those described during the slow oscillation promote the periodic (2-3 Hz) recurrence of spike-wave complexes.     

Role of potassium lateral diffusion in non-synaptic epilepsy: a computational study

An increase of extracellular potassium ion concentration can result in neuronal hyperexcitability, and thus contribute to non-synaptic epileptiform activity. It has been shown that potassium lateral diffusion alone is sufficient for synchronization in the low-calcium epilepsy in-vitro model. However, it is not yet known whether the lateral diffusion can, by itself, induce seizure activity. We hypothesize that spontaneous sustained neuronal activity can be generated by potassium coupling between neurons. To test this hypothesis, neuronal simulations with 2-cell or 4-cell models were used. Each model neuron was embedded in a bath of K+ and surrounded by interstitial space. Interstitial potassium concentration was regulated by both K+-pump and glial buffer mechanisms. Simulations performed with two coupled neurons with parameter values within physiological range show that, without chemical and electrical synapses, potassium lateral diffusion alone can generate and synchronize zero-Ca2+ non-synaptic epileptiform activity. Simulations performed with a network of four zero-Ca2+ CA1 pyramidal neurons modeled in zero-calcium conditions also show that spontaneous sustained activity can propagate by potassium lateral diffusion alone with a velocity of approximately 0.93 mm/sec. This diffusion model used for the simulations is based on physiological parameters, is robust for various kinetics, and is able to reproduce both the spontaneous triplet bursting of non-synaptic activity and speed of propagation in low-Ca2+ non-synaptic epilepsy experiments. These simulations suggest that potassium lateral diffusion can play an important role in the synchronization and generation on non-synaptic epilepsy.     

Contribution of different ion currents to generation of repetitive action potentials: A study by numerical experiment

A model of electrical activity in the somatic membrane of dorsal root ganglion (DRG) neurons of the rat was constructed from the experimental data about magnitudes and kinetic parameters of individual ion currents. Numerical experiments indicated several types of response to a short depolarizing pulse of the membrane potential, depending on the densities of voltage-dependent ion currents, and also distinct role of different currents in generation of repetitive responses. It was shown that activation of low-threshold calcium current and inactivating potassium current are most significant for the generation of repetitive responses to a single pulse. The article is the continuation of the printed matters represented by the International Center for Molecular Physiology of the National Academy of Sciences of Ukraine (see No. 1, Vol. 29).     

Use-dependent decline in rat aorta sensitivity to contraction by potassium.

Aortic rings excised from rats at 12 weeks of age showed a decrease in responsiveness during repeated contraction by increasing potassium concentration. By comparison, aortic rings obtained from rats at 22 and 26 weeks exhibited less loss or an increase in responsiveness to high potassium concentration during repeated contraction. The decrease in responsiveness to potassium in aortae of young rats was not due to the extended interval of incubation of these tissue in vitro. Aortic rings incubated without stimulation for 4 h following a reference contraction showed no change in contractile response to potassium. However, the magnitude of loss in responsiveness to potassium did appear to be related to the potassium concentration and the length of time the tissues were exposed to the high potassium solutions. Contraction of the tissue at 60 versus 30 mM KCl or extending the interval in depolarizing solution from 15 to 60 min significantly enhanced the decline in tissue responsiveness to potassium. The interruption of a series of potassium-induced contractions by exposure of the tissue to contractile (serotonin, norepinephrine) or relaxant (acetylcholine, isoproterenol) stimuli had no effect on the loss in responsiveness to potassium. However, injection of the calcium channel agonist, Bay K 8644, into the incubation media restored responsiveness to potassium. Concentration-response curves indicated that both sensitivity and the maximal response to potassium were reduced in aortic rings repeatedly contracted with potassium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Brief Subthreshold Events Can Act as Hebbian Signals for Long-Term Plasticity

Background

Action potentials are thought to be determinant for the induction of long-term synaptic plasticity, the cellular basis of learning and memory. However, neuronal activity does not lead systematically to an action potential but also, in many cases, to synaptic depolarizing subthreshold events. This is particularly exemplified in corticostriatal information processing. Indeed, the striatum integrates information from the whole cerebral cortex and, due to the membrane properties of striatal medium spiny neurons, cortical inputs do not systematically trigger an action potential but a wide range of subthreshold postsynaptic depolarizations. Accordingly, we have addressed the following question: does a brief subthreshold event act as a Hebbian signal and induce long-term synaptic efficacy changes?

Methodology/Principal Findings

Here, using perforated patch-clamp recordings on rat brain corticostriatal slices, we demonstrate, that brief (30 ms) subthreshold depolarizing events in quasi-coincidence with presynaptic activity can act as Hebbian signals and are sufficient to induce long-term synaptic plasticity at corticostriatal synapses. This “subthreshold-depolarization dependent plasticity” (SDDP) induces strong, significant and bidirectional long-term synaptic efficacy changes at a very high occurrence (81%) for time intervals between pre- and postsynaptic stimulations (Δt) of −110<Δt<+110 ms. Such subthreshold depolarizations are able to induce robust long-term depression (cannabinoid type-1 receptor-activation dependent) as well as long-term potentiation (NMDA receptor-activation dependent).

Conclusion/Significance

Our data show the existence of a robust, reliable and timing-dependent bidirectional long-term plasticity induced by brief subthreshold events paired with presynaptic activity. The existence of a subthreshold-depolarization dependent plasticity extends considerably, beyond the action potential, the neuron''s capabilities to express long-term synaptic efficacy changes.     

Brief exposure to neurotrophins produces a calcium-dependent increase in choline acetyltransferase activity in cultured rat septal neurons

We demonstrate that brief (30-min) exposure of cultured embryonic rat septal neurons to neurotrophins (NTs) increases choline acetyltransferase (ChAT) activity by 20-50% for all tested NTs (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, each at 100 ng/ml). The increase in ChAT activity was first detected 12 h after NT exposure, persisted at least 48 h, and was not mediated by increased neuronal survival or action-potential activity. Under some conditions, the response to brief NT exposure was as great as that produced by continuous exposure. NT stimulation of ChAT activity was inhibited by inhibitors of p75- and Trk kinase-mediated signaling, by removal of extracellular Ca2+ during the period of NT exposure, and by buffering intracellular Ca2+ with BAPTA. Application of nerve growth factor and brain-derived neurotrophic factor transiently increased [Ca2+] within a subpopulation of neurons. NT stimulation of ChAT activity was not affected significantly by cyclic AMP agonists or antagonists. These findings suggest that brief exposure to NTs can have a long-lasting effect on cholinergic transmission, and that this effect requires Ca2+, but not cyclic AMP.     

Adaptation in the Visual Cortex: Influence of Membrane Trajectory and Neuronal Firing Pattern on Slow Afterpotentials

The input/output relationship in primary visual cortex neurons is influenced by the history of the preceding activity. To understand the impact that membrane potential trajectory and firing pattern has on the activation of slow conductances in cortical neurons we compared the afterpotentials that followed responses to different stimuli evoking similar numbers of action potentials. In particular, we compared afterpotentials following the intracellular injection of either square or sinusoidal currents lasting 20 seconds. Both stimuli were intracellular surrogates of different neuronal responses to prolonged visual stimulation. Recordings from 99 neurons in slices of visual cortex revealed that for stimuli evoking an equivalent number of spikes, sinusoidal current injection activated a slow afterhyperpolarization of significantly larger amplitude (8.5±3.3 mV) and duration (33±17 s) than that evoked by a square pulse (6.4±3.7 mV, 28±17 s; p<0.05). Spike frequency adaptation had a faster time course and was larger during plateau (square pulse) than during intermittent (sinusoidal) depolarizations. Similar results were obtained in 17 neurons intracellularly recorded from the visual cortex in vivo. The differences in the afterpotentials evoked with both protocols were abolished by removing calcium from the extracellular medium or by application of the L-type calcium channel blocker nifedipine, suggesting that the activation of a calcium-dependent current is at the base of this afterpotential difference. These findings suggest that not only the spikes, but the membrane potential values and firing patterns evoked by a particular stimulation protocol determine the responses to any subsequent incoming input in a time window that spans for tens of seconds to even minutes.