Examination of the role of calcium in neuronal death.
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Animals↗
Biomedical subjects
J M Dubinsky
Publications and source records attributed to J M Dubinsky.
Effects of calcium chelators on intracellular calcium and excitotoxicity.
In an attempt to probe the relationship between excitotoxicity and increases in intracellular calcium ([Ca2+]i), BAPTA-AM and its analogs were applied to cultured hippocampal neurons. Chelation of [Ca2+]i depressed and prolonged transient responses to glutamate and did not effect elevation of [Ca2+]i by prolonged exposure. This explains the inability of the chelators to prevent glutamate-induced toxicity.
Animals↗
Aurintricarboxylic acid protects hippocampal neurons from glutamate excitotoxicity in vitro.
Aurintricarboxylic acid (ATA), an endonuclease inhibitor, has been shown to protect several cell types from an apoptotic form of cell death. We tested ATA for protective effects against glutamate excitotoxicity in 2-week-old cultured hippocampal neurons. Cell viability was determined 24 h after glutamate exposure either by trypan blue exclusion or by measurement of lactate dehydrogenase release. When ATA was added during exposure to glutamate, there was a dramatic increase in the number of viable neurons compared with cultures that did not receive ATA. If ATA was added after glutamate exposure, the rate of survival approached 100%. Several cellular processes may be the targets for ATA action. If the mechanisms of ATA protection are similar for excitotoxicity and apoptosis, then these distinct forms of cell death may share a common intracellular pathway.
Animals↗
Changes in intracellular pH associated with glutamate excitotoxicity.
Excitotoxic neuronal injury is known to be associated with increases in cytosolic calcium ion concentrations. However, it is not known if perturbations in other intracellular ions are also associated with glutamate (GLU)-induced neuronal death. Accordingly, intracellular hydrogen ion concentrations were measured in cultured hippocampal neurons with the fluorescent dye BCECF during and after toxic exposures. Five minute GLU applications produced an initial cytosolic acidification. During the hour after GLU removal, intracellular pH (pHi) recovered steadily, resulting in a rebound cytosolic alkalinization. Lowering extracellular calcium depressed the initial GLU-induced acidification, suggesting that the rapid acidification may result partly as a consequence of calcium entry. An acidification-induced rebound alkalinization appeared to be activated by GLU exposure. Inhibitors of intracellular pH regulation, harmaline, 4,4'-disothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and replacement of external Na+ with N-methyl-glucamine+ (NMG+), retarded the rate of recovery from GLU-induced acidification. The rapid acidification and rebound alkalinization could be mimicked by challenging neurons with elevated external K+ or replacement of external Na+ with NMG+. Two or more hours following toxic GLU exposure, hydrogen ion concentration did not stabilize at initial levels but progressively increased. High K+ or Na+ removal did not produce this long-term acidification and were not toxic. The cumulative increase in intracellular hydrogen ion may reflect the declining health of injured neurons and could contribute directly to neuronal death. Therefore, cytosolic acidification may act synergistically with increases in calcium concentration in mediating excitotoxicity.
4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
Intracellular calcium levels during the period of delayed excitotoxicity.
Intracellular calcium concentrations ([Ca2+]i) among cultured hippocampal neurons were monitored during and in the hours following an excitotoxic glutamate application to determine the time course of changes involved in delayed excitotoxicity. After a 5 min toxic insult, [Ca2+]i increased immediately and remained elevated for an hour. Subsequently, [Ca2+]i declined to normal resting levels and remained so up to 13 hr following insult. Only a few neurons displayed greatly elevated [Ca2+]i at these extended times. Survival experiments in sister cultures indicated that 85% of the neurons died after 24 hr. Therefore, intracellular calcium returned to baseline levels prior to neuronal death. Additionally, during this period when basal calcium levels had recovered, the majority of neurons responded to a second excitatory amino acid application with a second increase in [Ca2+]i.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Relationship of intracellular calcium to dependence on nerve growth factor in dorsal root ganglion neurons in cell culture.
During development, neural crest-derived sensory neurons require nerve growth factor (NGF) for survival, but lose this dependency postnatally. Similarly, dissociated embryonic sensory neurons lose their NGF dependence during the first 3 weeks in cell culture. It has been hypothesized that, in sympathetic neurons, intracellular levels of calcium are related to trophic factor dependence. In vitro during the period in which embryonic-day-15 sensory neurons become independent of NGF, intracellular calcium concentrations progressively increased in parallel to the decline in NGF dependence. This elevation of intracellular calcium was directly related to the absolute age of the neurons, not to the length of time in culture. Without NGF, immature sensory, i.e., dependent, neurons survived in the presence of high extracellular potassium, a condition that produces elevated intracellular calcium. In another paradigm, measurements of intracellular calcium were determined in NGF-dependent neurons "committed to die" after NGF withdrawal. These measurements were determined prior to the time that extensive morphological changes, consistent with cell death, were noted by phase-contrast microscopy. No elevation in intracellular calcium was found in these dying neurons, but rather, a small decrease was observed prior to the disintegration of the neurons. These findings support the hypothesis that trophic factor dependence of neurons may be inversely related to levels of intracellular calcium.
Animals↗
Intracellular calcium concentrations during "chemical hypoxia" and excitotoxic neuronal injury.
Because hypoxic/ischemic neurodegeneration appears to be in part linked to glutamate neurotoxicity, we measured intracellular calcium (Ca2+i) levels in cultured hippocampal neurons during exposure to toxic doses of glutamate (GLU) and to an anoxic environment simulated by sodium cyanide (NaCN). Changes in Ca2+i produced by cyanide greatly exceeded those induced by GLU. The NaCN response was mimicked when oxidative metabolism was also disrupted by sodium azide, oligomycin, or dinitrophenol. Noncompetitive NMDA receptor antagonists and enzymatic GLU degradation abolished the GLU-induced Ca2+i increases and attenuated those produced by NaCN. Only NaCN-induced increases were blocked when dantrolene and ruthenium red were applied to prevent release from intracellular pools. All responses were reduced proportionally in the absence of added external calcium. These results suggest that extracellular GLU accumulation and subsequent activation of GLU receptors were involved in the NaCN response. During such metabolic compromise, however, GLU-induced elevations of Ca2+i were enormously amplified. In parallel toxicity studies, NaCN was not neurotoxic despite the large elevations in Ca2+i, indicating that a general elevation in cytoplasmic calcium does not necessarily predict neurodegeneration.
Animals↗
A role for cAMP in the development of functional neuromuscular transmission.
We have found that the incidence of functionally connected neuron-myotube pairs in chick ciliary-myotube cultures increases from 58% to more than 90% when the cells are treated for several hours with 8-bromo-cyclic adenosine monophosphate (8-br-cAMP) or with agents known to increase intracellular cAMP. The increase in connectivity was not accompanied by a change in neuron survival, or in the length of neurite-myotube contact. Moreover, there was no change in the shape of the presynaptic action potential, in mean end plate potential (epp) amplitude or in the sensitivity of postsynaptic acetylcholine receptors (AChRs). One interpretation of these results in that a cAMP-dependent phosphorylation acts as a trigger to activate a previously "silent" synapse.
8-Bromo Cyclic Adenosine Monophosphate↗
Formation of acetylcholine receptor clusters in chick myotubes: migration or new insertion?
Experiments were performed to study the feasibility of two mechanisms of acetylcholine receptor (ACHR) accumulation in chick myotubes: diffusion and trapping of previously dispersed surface receptors and localized insertion of new receptors at accumulation sites. Fluorescence photobleaching recovery (FPR) measurements indicated that the majority of diffusely distributed ACHRs in chick myotube membranes were mobile whereas nearly all receptors within high density clusters were effectively immobile. Unlike previous reports, two rates of ACHR movement characterized the mobile population. Moreover, we found that the estimated diffusion coefficient depended critically on the objective (spot size) used to assay recovery from bleaching. Implications of this finding for mechanisms of receptor immobilization are discussed. Extracts of chick brain, known to increase the number of surface receptors, did not alter receptor mobility. Extracts of Torpedo electric organ that increase the number of receptor aggregates, decreased the mobile fraction of ACHRs. Simulations of the diffusion and trapping mechanism indicated that captured receptors should congregate around the periphery of a receptor patch during the first hour after they were inserted into the membrane. However, newly inserted ACHRs were found to be located centrally within receptor patches under neurites, and this was not consistent with an exclusive diffusion-trapping mechanism. We also studied the mobility of ACHRs near points of contact made by cholinergic growth cones. The rate of receptor movement was increased in the vicinity of growth cones, but the magnitude of this effect was small.
Animals↗
Quantitative physiological characterization of a quinoxalinedione non-NMDA receptor antagonist.
The effects of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, or FG 9065) on excitatory amino acid responses in cultured neurons from rat hippocampus were studied using tight-seal whole-cell recording techniques. CNQX reduced the magnitude of peak inward currents produced by exogenously applied kainate, quisqualate, and N-methyl-D-aspartate (NMDA) with Ki's of 2.5, 3.5, and 96 microM, respectively. The antagonism was competitive against kainate and quisqualate, but noncompetitive against NMDA. Glycine markedly reduced CNQX antagonism of NMDA responses. The same recording technique using pairs of monosynaptically connected neurons demonstrated reversible diminution of excitatory postsynaptic potentials in 7 of 7 pairs, using CNQX at concentrations as low as 10 microM. CNQX applied alone did not evoke inward or outward currents at membrane potentials near the resting membrane potential and did not affect the current-voltage relationship at membrane potentials between -90 and -30 mV. These observations represent the first quantitative characterization of glutamate receptor antagonism by CNQX with respect to physiological rather than biochemical parameters and demonstrate that CNQX is far more potent and more selective than currently available non-NMDA antagonists. The results suggest that CNQX will be a useful pharmacologic tool for the study of synaptic transmission in a variety of systems in which glutamate or related excitatory amino acids are involved.
6-Cyano-7-nitroquinoxaline-2,3-dione↗
Development of inhibitory synapses among striatal neurons in vitro.
The development of excitatory and inhibitory synaptic connections has been studied in postnatal neurons from the caudate and putamen maintained in tissue culture. Excitatory postsynaptic potentials which were sensitive to the glutamate antagonist CNQX (6-cyano-7-nitroquinoxaline-2,3-dione) appeared between 4 and 8 d in vitro. This is the first indication that glutamatergic excitatory neurons may be intrinsic to the striatum. Spontaneous inhibitory postsynaptic currents appeared at approximately the same time, several days after process outgrowth. Antibodies to glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, labeled neurons which produce bicuculline-sensitive, inhibitory postsynaptic currents. GAD immunoreactivity and immunoreactivity to synapsin I, a synaptic vesicle-associated protein, became localized to discrete sites along neurites 4-8 d after plating. It is concluded that the punctate GAD immunoreactivity identified possible sites of presynaptic transmitter release.
Acetylcholinesterase↗
Variation among acetylcholine receptor clusters induced by ciliary ganglion neurons in vitro.
We have examined the variation in receptor density and area among neurite-associated acetylcholine receptor patches (NARPs) induced by chick ciliary ganglion neurons on nearby myotubes in vitro. Quantitative analysis of rhodamine-alpha-bungarotoxin (RBTX) NARPs revealed that about 15% of the NARPs were "outstanding" in terms of size (greater than 60 micron 2) and fluorescence intensity (greater than 100 units on a 0-255 scale). The total number of receptors at different NARPs ranged over 3 orders of magnitude. It is likely that variation in NARP size and intensity reflects regional variation in the ability of myotubes to respond to the neuronal influence because (1) no gradient in NARP size or intensity with distance from the soma was evident; (2) the intensities and areas of uninnervated receptor clusters (hot spots) were similar to those of NARPs; (3) acetylcholinesterase was present at the same proportion of hot spots and NARPs at all times examined. We found no physiological or morphological evidence that outstanding NARPs were more effective sites of transmitter release. Outstanding NARPs were restricted to the longest neurite of individual neurons, so they may signal trophic interactions of the sort that promote neurite outgrowth and survival.
Acetylcholinesterase↗
Dual modulation of K channels by thyrotropin-releasing hormone in clonal pituitary cells.
Transmembrane electrical activity in pituitary tumor cells can be altered by substances that either stimulate or inhibit their secretory activity. Using patch recording techniques, we have measured the resting membrane potentials, action potentials, transmembrane macroscopic ionic currents, and single Ca2+-activated K channel currents of GH3 and GH4/C1 rat pituitary tumor cells in response to thyrotropin-releasing hormone (TRH). TRH, which stimulates prolactin secretion, causes a transient hyperpolarization of the membrane potential followed by a period of elevated action potential frequency. In single cells voltage clamped and internally dialyzed with solutions containing K+, TRH application results in a transient increase in Ca2+-activated K currents and a more protracted decrease in voltage-dependent K currents. However, in cells internally dialyzed with K+-free solutions, TRH produces no changes in inward Ca2+ or Ba2+ currents through voltage-dependent Ca channels. The time courses of the effects on Ca2+-activated and voltage-dependent K currents correlate with the phases of hyperpolarization and hyperexcitability, respectively. During application of TRH to whole cells, single Ca2+-activated K channel activity increases in cell-attached patches not directly exposed to TRH. In contrast, TRH applied directly to excised membrane patches produces no change in single Ca2+-activated K channel behavior. We conclude that TRH (i) triggers intracellular Ca2+ release, which opens Ca2+-activated K channels, (ii) depresses voltage-dependent K channels during the hyperexcitable phase, which further elevated intracellular Ca2+, and (iii) does not directly modulate Ca channel activity.
Action Potentials↗
Ionic currents in two strains of rat anterior pituitary tumor cells.
The ionic conductance mechanisms underlying action potential behavior in GH3 and GH4/C1 rat pituitary tumor cell lines were identified and characterized using a patch electrode voltage-clamp technique. Voltage-dependent sodium, calcium, and potassium currents and calcium-activated potassium currents were present in the GH3 cells. GH4/C1 cells possess much less sodium current, less voltage-dependent potassium current, and comparable amounts of calcium current. Voltage-dependent inward sodium current activated and inactivated rapidly and was blocked by tetrodotoxin. A slower-activating voltage-dependent inward calcium current was blocked by cobalt, manganese, nickel, zinc, or cadmium. Barium was substituted for calcium as the inward current carrier. Calcium tail currents decay with two exponential components. The rate constant for the slower component is voltage dependent, while the faster rate constant is independent of voltage. An analysis of tail current envelopes under conditions of controlled ionic gradients suggests that much of the apparent decline of calcium currents arises from an opposing outward current of low cationic selectivity. Voltage-dependent outward potassium current activated rapidly and inactivated slowly. A second outward current, the calcium-activated potassium current, activated slowly and did not appear to reach steady state with 185-ms voltage pulses. This slowly activating outward current is sensitive to external cobalt and cadmium and to the internal concentration of calcium. Tetraethylammonium and 4-aminopyridine block the majority of these outward currents. Our studies reveal a variety of macroscopic ionic currents that could play a role in the initiation and short-term maintenance of hormone secretion, but suggest that sodium channels probably do not make a major contribution.
Action Potentials↗
The calcium current in inner segments of rods from the salamander (Ambystoma tigrinum) retina.
Solitary rod inner segments were isolated from salamander retinae. Their Ca current was studied with the 'whole-cell, gigaseal' technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The soluble constituents of the cytoplasm exchanged with the solution in the pipette. The external solution could be changed during continuous perfusion. Membrane voltage was controlled with a voltage clamp. After permeant ions other than Ca were replaced with impermeant ions (i.e. tetraethylammonium as a cation, and aspartate or methanesulphonate as an anion), an inward current remained. It activated at approximately -40 mV, reached a maximum at approximately 0 mV, and decreased as the membrane was further depolarized. The size of the current increased when Ba was substituted for external Ca. The current was blocked when Ca was replaced with Co. The voltage at which the current was half-maximum shifted from approximately -22 to -31 mV during the initial 3 min of an experiment. The maximum amplitude of the current continuously declined during the entire course of an experiment. The time course for activation of the Ca current following a step of depolarization could be described by the sum of two exponentials. The time constant of the slower exponential was voltage dependent. Deactivation following repolarization could also be described by the sum of two exponentials. Both time constants for deactivation were independent of voltage (between -30 and 0 mV) and faster than the slower time constant for activation. When the internal Ca concentration was buffered by 10 mM-EGTA, the Ca current did not inactivate during several seconds of maintained depolarization. When the concentration of EGTA was reduced to 0.1 mM, the Ca current declined and the membrane conductance decreased during several seconds of maintained depolarization. This inactivation was incomplete and only occurred after a substantial quantity of Ca entered. Following repolarization the Ca conductance recovered from inactivation. In contrast, the continuous decline observed during the course of an experiment (item 3) was not reversible. The difference suggests that inactivation and the decline are distinct processes.
Ambystoma↗