Neuronal input triggers Ca2+ influx through AMPA receptors and voltage-gated Ca2+ channels in oligodendrocytes

Communication between neurons and developing oligodendrocytes (OLs) leading to OL Ca²⁺ rise is critical for axon myelination and OL development. Here, we investigate signaling factors and sources of Ca²⁺ rise in OLs in the mouse brainstem. Glutamate puff or axon fiber stimulation induces a Ca²⁺ rise in pre-myelinating OLs, which is primarily mediated by Ca²⁺-permeable AMPA receptors. During glutamate application, inward currents via AMPA receptors and elevated extracellular K⁺ caused by increased neuronal activity collectively lead to OL depolarization, triggering Ca²⁺ influx via P/Q- and L-type voltage-gated Ca²⁺ (Ca_v) channels. Thus, glutamate is a key signaling factor in dynamic communication between neurons and OLs that triggers Ca²⁺ transients via AMPARs and Ca_v channels in developing OLs. The results provide a mechanism for OL Ca²⁺ dynamics in response to neuronal input, which has implications for OL development and myelination.     

Neuronal ageing in long-term cultures: alterations of Ca2+ homeostasis

Deficiencies of Ca2+ homeostasis are proposed to play an important role in neuronal ageing and/or neurodegeneration. The aim of this study was to investigate, in a defined neuronal population, primary cerebellar granule neuron culture, the time-dependent changes in Ca2+ homeostasis and compare them with data obtained in cerebellar brain slices from aged rats. In neurons aged in culture (DIV 23), a small decrease in the resting [Ca2+]i was associated with a decrease in the maximal rate of [Ca2+]i increase upon KCl-induced depolarization and in the amplitude of the [Ca2+]i response, when compared with mature neurons (DIV 9). The most significant change of [Ca2+]i signal parameters was a 50% decrease in the rate of [Ca2+]i recovery after the stimulation. These results were similar to those obtained in aged brain slices, and indicate that primary neuronal cultures could serve as a model for studying the age-related changes in Ca2+ homeostasis.     

Neuronal Ca2+-sensor proteins: multitalented regulators of neuronal function

Many aspects of neuronal activity are regulated by Ca2+ signals. The transduction of temporally and spatially distinct Ca2+ signals requires the action of Ca2+-sensor proteins including various EF-hand-containing Ca2+-binding proteins. The neuronal Ca2+ sensor (NCS) protein family and the related Ca2+-binding proteins (CaBPs) have begun to emerge as key players in neuronal function. Many of these proteins are expressed predominantly or only in neurons, sometimes with cell-specific patterns of expression. Their ability to associate with membranes either constitutively or in response to elevated Ca2+ concentration allows the NCS proteins to discriminate between different spatial and temporal patterns of Ca2+ signals. Recent work has established several physiological roles of these proteins, including diverse actions on gene expression, ion channel function, membrane traffic of ion channels and receptors, and the control of apoptosis.     

Neuronal differentiation of Ca2+ channel by nerve growth factor

The inhibitory effect of nicardipine, a potent Ca2+ channel blocker in muscular cells, on the Ca2+ channel of clonal rat pheochromocytoma cells (PC12h) and cultured rat adrenal medullary cells was studied during the neuronal differentiation mediated by nerve growth factor (NGF). Nicardipine at nM-order concentrations suppressed the high-K+-evoked, Ca2+-dependent release of preloaded [3H]norepinephrine from PC12h cells and adrenal medullary cells, whereas it scarcely inhibited the release from the cultured rat brainstem cells. The inhibitory actions of nicardipine on both PC12h and newborn rat adrenal medullary cells were significantly decreased after these cells were cultured in the presence of NGF. These results suggest that the changes in Ca2+ channel are accompanied by the neuronal differentiation mediated by NGF.     

Tuberous Sclerosis Complex (TSC) Inactivation Increases Neuronal Network Activity by Enhancing Ca2+ Influx via L-Type Ca2+ Channels

Tuberous sclerosis complex (TSC) is a multisystem developmental disorder characterized by hamartomas in various organs, such as the brain, lungs, and kidneys. Epilepsy, along with autism and intellectual disability, is one of the neurologic impairments associated with TSC that has an intimate relationship with developmental outcomes and quality of life. Sustained activation of the mammalian target of rapamycin (mTOR) via TSC1 or TSC2 mutations is known to be involved in the onset of epilepsy in TSC. However, the mechanism by which mTOR causes seizures remains unknown. In this study, we showed that, human induced pluripotent stem cell-derived TSC2-deficient (TSC2^−/−) neurons exhibited elevated neuronal activity with highly synchronized Ca²⁺ spikes. Notably, TSC2^−/− neurons presented enhanced Ca²⁺ influx via L-type Ca²⁺ channels (LTCCs), which contributed to the abnormal neurite extension and sustained activation of cAMP response element binding protein (CREB), a critical mediator of synaptic plasticity. Expression of Cav1.3, a subtype of LTCCs, was increased in TSC2^−/− neurons, but long-term rapamycin treatment suppressed this increase and reversed the altered neuronal activity and neurite extensions. Thus, we identified Cav1.3 LTCC as a critical downstream component of TSC-mTOR signaling that would trigger enhanced neuronal network activity of TSC2^−/− neurons. We suggest that LTCCs could be potential novel targets for the treatment of epilepsy in TSC.SIGNIFICANCE STATEMENT There is a close relationship between elevated mammalian target of rapamycin (mTOR) activity and epilepsy in tuberous sclerosis complex (TSC). However, the underlying mechanism by which mTOR causes epilepsy remains unknown. In this study, using human TSC2^−/− neurons, we identified elevated Ca²⁺ influx via L-type Ca²⁺ channels as a critical downstream component of TSC-mTOR signaling and a potential cause of both elevated neuronal activity and neurite extension in TSC2^−/− neurons. Our findings demonstrate a previously unrecognized connection between sustained mTOR activation and elevated Ca²⁺ signaling via L-type Ca²⁺ channels in human TSC neurons, which could cause epilepsy in TSC.     

Orexin receptors couple to Ca2+ channels different from store-operated Ca2+ channels.

We have investigated Ca2+ release and receptor- and store-operated Ca2+ influxes in Chinese hamster ovary-K1 cells expressing human OX1 orexin receptor. Receptor-operated Ca2+ influx-response to 3 nM orexin-A was not affected by Gd3+ or 2-APB (2-aminoethoxydiphenyl borate), but was inhibited by Ni2+. Store-operated Ca2+ influx was blocked by Ni2+, Gd3+ and 2-APB, whereas the thapsigargin-induced release was unaffected. 2-APB did not block inositol-1,4,5- trisphosphate-dependent Ca2+ release in these cells. Thus, low concentrations of orexin-A cause activation of two Ca2+ influxes in the cells: primarily, a receptor-operated Ca2+ influx, and secondarily, a store-depletion activated Ca2+ influx, which is subsequent to receptor-activated Ca2+ influx and the therewith-caused IP3 production. The results show that these two rely on different molecular entities.     

Membrane-bound enzymes of erythrocytes in human muscular dystrophy: (Na+ + K+-ATPase, Ca2+-ATPase, K+- and Ca2+-p-nitrophenylphosphatase.

Four enzyme activities were studied in erythrocyte membranes from patients with Duchenne and congenital myotonic muscular dystrophy. (Na⁺ + K⁺)-stimulated, Mg²⁺-dependent adenosinetriphosphatase, measured in two different media, showed normal activity and ouabain inhibition, as did K⁺-stimulated p-nitrophenylphosphatase. The specific activity of Ca²⁺-stimulated p-nitrophenylphosphatase was twice normal in Duchenne membranes. Ca²⁺-stimulated, Mg²⁺-dependent adenosine-triphosphatase was augmented in membranes from both Duchenne and congenital myotonic muscular dystrophic patients. The cause of the increased activities may be the necessity for compensating an alteration in the calcium metabolism in the dystrophic erythrocytes.Several kinetic parameters of the two Ca²⁺-stimulated enzyme activities were studied in Duchenne and control membranes. Most were not changed, with the exception of the Na⁺-stimulation of Ca²⁺-ATPase. In Duchenne membranes two affinity sites were present with half maximal activating concentrations of 58 ± 4 and 4 ± 1 mM Na⁺. In control membranes only one affinity site was found with K_a = 26 ± 9 mM Na⁺.     

Alpha-synuclein potentiates Ca2+ influx through voltage-dependent Ca2+ channels

Alpha-synuclein localized in synaptic terminals plays an important role in the pathogenesis of neurodegenerative diseases. The central domain of the protein, the nonamyloid component, is probably responsible for alpha-synuclein toxicity. Here, we report that alpha-synuclein and its nonamyloid component induced Ca2+ influx in rat synaptoneurosomes. The effect of alpha-synuclein was eliminated by the N-type specific Ca2+ channel blocker, omega-conotoxin GVIA. The antioxidant, resveratrol, and the nitric oxide synthase inhibitor, Nomega-nitro-L-arginine, did not prevent alpha-synuclein-induced Ca2+ influx. Our findings indicate that alpha-synuclein stimulated Ca2+ influx through N-type voltage-dependent Ca2+ channels by a mechanism other than free radicals. A direct interaction between alpha-synuclein and N-type Ca2+ channels could be responsible for their effects on Ca2+ influx through voltage-dependent Ca2+ channels.     

Ca2+ mobilization and capacitative Ca2+ entry regulate DNA synthesis in cultured chick retinal neuroepithelial cells.

Release of Ca2+ from intracellular Ca2+ stores (Ca2+ mobilization) and capacitative Ca2+ entry have been shown to be inducible in neuroepithelial cells of the early embryonic chick retina. Both types of Ca2+ responses decline parallel with retinal progenitor cell proliferation. To investigate their potential role in the regulation of neuroepithelial cell proliferation, we studied the effects of 2,5-di-tert-butylhydroquinone (DBHQ), an inhibitor of the Ca2+ pump of intracellular Ca2+ stores, and of SK&F 96365, an inhibitor of capacitative Ca2+ entry, on DNA synthesis in retinal organ cultures from embryonic day 3 (E3) chicks and in dissociated cultures from E7 and E9 chick retinae. We demonstrate that both antagonists inhibit [3H]-thymidine incorporation in a dose-dependent manner without affecting cell viability or morphology. The inhibition of [3H]-thymidine incorporation by SK&F 96365 occurred in the same concentration range (IC50: approximately 4 microM) as the blockade of capacitative Ca2+ entry in the E3 retinal organ culture. At a concentration of 5 microM SK&F 96365. DNA synthesis was reduced by 71, 40 and 32% in the E3, E7 and E9 cultures, respectively. Application of DBHQ at concentrations which led to depletion of intracellular Ca2+ stores also inhibited [3H]-thymidine incorporation with IC50 values of 20-30 microM in the different cultures. Our results suggest the involvement of Ca2+ mobilization and capacitative Ca2+ entry in the regulation of DNA synthesis in the developing neural retina.     

Ca2+-permeable and Ca2+-impermeable AMPA receptors coexist on horizontal cells

Fura-2 fluorescent calcium imaging was used for analyzing the subtype of AMPA receptors in freshly dissociated horizontal cells of carp retina. Exogenous application of AMPA induced an increase of intracellular concentration of free Ca2+ ([Ca2+]i) in horizontal cells, while the [Ca2+]i increase was partly inhibited by nifedipine. The residual [Ca2+]i increase was completely eliminated by joro spider toxin-3, a blocker of Ca2+-permeable AMPA receptors. On the other hand, the application of pentobarbital, which blocked Ca2+-impermeable AMPA receptors, could also partly inhibit the increase of [Ca2+]i, implying that the application of AMPA induced the activation of both Ca2+-permeable and Ca2+-impermeable AMPA receptors and the consequent activation of voltage-gated Ca2+ channels. Taken together, these results suggested that Ca2+-permeable and Ca2+-impermeable AMPA receptors were coexpressed on horizontal cells.     

Ca2+/calmodulin-dependent facilitation and inactivation of P/Q-type Ca2+ channels.

Trains of action potentials cause Ca(2+)-dependent facilitation and inactivation of presynaptic P/Q-type Ca(2+) channels that can alter synaptic efficacy. A potential mechanism for these effects involves calmodulin, which associates in a Ca(2+)-dependent manner with the pore-forming alpha(1A) subunit. Here, we report that Ca(2+) and calmodulin dramatically enhance inactivation and facilitation of P/Q-type Ca(2+) channels containing the auxiliary beta(2a) subunit compared with their relatively small effects on channels with beta(1b). Tetanic stimulation causes an initial enhancement followed by a gradual decline in P/Q-type Ca(2+) currents over time. Recovery of Ca(2+) currents from facilitation and inactivation is relatively slow (30 sec to 1 min). These effects are strongly inhibited by high intracellular BAPTA, replacement of extracellular Ca(2+) with Ba(2+), and a calmodulin inhibitor peptide. The Ca(2+)/calmodulin-dependent facilitation and inactivation of P/Q-type Ca(2+) channels observed here are consistent with the behavior of presynaptic Ca(2+) channels in neurons, revealing how dual feedback regulation of P/Q-type channels by Ca(2+) and calmodulin could contribute to activity-dependent synaptic plasticity.     

Effects of Na+ and Ca2+ gradients on intracellular free Ca2+ in voltage-clamped Aplysia neurons

Selected neurons of the abdominal ganglion of Aplysia californica were voltage-clamped and intracellular free Ca [( Ca2+]i) and Na [( Na+]i) concentrations were monitored with ion selective microelectrodes. Reducing [Na+]o from 500 mM (normal seawater, NSW) to 5 mM resulted in a decrease of the potential measured by the Ca electrode (VCa). Increasing [Ca2+]o from 10 to 50 mM increased [Ca2+]i two-fold, keeping [Ca2+]o at 50 mM and decreasing [Na+]o to 5 mM still led to a decrease in VCa. With 100 mM [Ca2+]o, which also increased [Ca2+]i, decreasing [Na+]o increased VCa in two of the eight cells tested. This indicates that in normal or moderately high resting [Ca2+]i, Ca2+ extrusion by Na/Ca exchange (forward mode) is not essential for [Ca2+]i buffering. [Na+]i was 12.9 +/- 3.6 mM (S.E.M., n = 7) in NSW; reducing [Na+]o to 5 mM decreased [Na+]i to 2.0 +/- 1.1 mM (S.E.M.). Keeping [Na+]o at 5 mM and increasing [Ca2+]o from 10 to 20 mM further decreased [Na+]i to about 1.0 mM, evidence of Na/Ca exchange operating in the reverse mode. Attempts to increase [Ca2+]i by bath application of the Ca ionophores A23187, X537A, ionomycin or ETH 1001 resulted in no measurable change of the resting [Ca2+]i. Application of Ouabain caused an apparent increase in [Ca2+]i in two of the six cells tested. In cells injected with the metallochromic indicator arsenazo III (AIII), the rate of the falling phase of the AIII absorbance increase, following a voltage-clamp pulse, was significantly slower in 5 mM [Na+]o. This indicates that in its forward mode Na-Ca exchange is active in clearing large submembrane increases in [Ca2+]i.     

Cytosolic Ca2+ changes during in vitro ischemia in rat hippocampal slices: major roles for glutamate and Na+-dependent Ca2+ release from mitochondria.

This work determined Ca2+ transport processes that contribute to the rise in cytosolic Ca2+ during in vitro ischemia (deprivation of oxygen and glucose) in the hippocampus. The CA1 striatum radiatum of rat hippocampal slices was monitored by confocal microscopy of calcium green-1. There was a 50-60% increase in fluorescence during 10 min of ischemia after a 3 min lag period. During the first 5 min of ischemia the major contribution was from Ca2+ entering via NMDA receptors; most of the fluorescence increase was blocked by MK-801. Approximately one-half of the sustained increase in fluorescence during 10 min of ischemia was caused by activation of Ca2+ release from mitochondria via the mitochondrial 2Na+-Ca2+ exchanger. Inhibition of Na+ influx across the plasmalemma using lidocaine, low extracellular Na+, or the AMPA/kainate receptor blocker CNQX reduced the fluorescence increase by 50%. The 2Na+-Ca2+ exchange blocker CGP37157 also blocked the increase, and this effect was not additive with the effects of blocking Na+ influx. When added together, CNQX and lidocaine inhibited the fluorescence increase more than CGP37157 did. Thus, during ischemia, Ca2+ entry via NMDA receptors accounts for the earliest rise in cytosolic Ca2+. Approximately 50% of the sustained rise is attributable to Na+ entry and subsequent Ca2+ release from the mitochondria via the 2Na+-Ca2+ exchanger. Sodium entry is also hypothesized to compromise clearance of cytosolic Ca2+ by routes other than mitochondrial uptake, probably by enhancing ATP depletion, accounting for the large inhibition of the Ca2+ increase by the combination of CNQX and lidocaine.     

The effects of trimethyltin on the Ca+2, Mg+2 and Ca+2 + Mg+2-dependent ATPases of human neuroblastoma GM 3320

Data presented here indicate neuroblastoma GM 3320 tissue homogenates exhibit ouabain insensitive Ca+2-dependent, Mg+2-independent, Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities. Inclusion of trimethyltin in homogenate preparations of these cells appears to discriminate between these various ATPase activities. At low concentrations (25 microM), trimethyltin preferentially stimulated the Ca+2-dependent, Mg+2-independent ATPase activity while inhibiting the Ca+2 + Mg+2-ATPase activity approximately 70%. At 75 microM trimethyltin, the Ca+2 + Mg+2-dependent ATPase activity is inhibited greater than 95% while the Ca+2-dependent, Mg+2-independent activity is essentially unchanged from control activity and the Mg+2-dependent, Ca+2-independent activity is inhibited approximately 50%. At concentrations greater than 75 microM, trimethyltin significantly inhibits the Ca+2-dependent, Mg+2-independent ATPase activity. Thus, at trimethyltin concentrations of 50-75 microM, preferential inhibition of the Mg+2-dependent, Ca+2-independent and Ca+2 + Mg+2-dependent ATPase activities of neuroblastoma GM 3320 is achieved.     

Intercellular Ca2+ waves induce temporally and spatially distinct intracellular Ca2+ oscillations in glia

Mechanically induced intercellular Ca²⁺ waves propagated for approximately 300 μm in primary glial cultures. Following the wave propagation, 34% of the cells displayed Ca²⁺ oscillations in a zone 60–120 μm from the stimulated cell. The initiation, frequency, and duration of these Ca²⁺ oscillations were dependent on the cells' distance from the wave origin but were not dependent on the cell type nor on the magnitude of the Ca²⁺ wave. When an individual cell propagated two sequential intercellular Ca²⁺ waves originating from different sites, the characteristics of the Ca²⁺ oscillations initiated by each wave were determined by the distance of the cell from the origin of each wave. Each Ca²⁺ oscillation commonly occurred as an intracellular Ca²⁺ wave that was initiated from a specific site within the cell. The position of the initiation site and the direction of the intracellular Ca²⁺ wave were independent of the orientation of the initial intercellular Ca²⁺ wave. Because initiation and frequency of Ca²⁺ oscillations are dependent on the intracellular inositol trisphosphate concentration ([IP₃]_i), we propose that the zone of cells displaying Ca²⁺ oscillations is determined by an intercellular gradient of [IP₃]_i, established by the diffusion of IP₃ through gap junctions during the propagation of the intercellular Ca²⁺ wave. Exposure to acetylcholine, a muscarinic agonist that initiates IP₃ production, shifted the zone of oscillating cells about 45 μm farther away from the origin of the mechanically induced wave. These findings indicate that a glial syncytium can resolve information provided by a local Ca²⁺ wave into a distinct spatial and temporal pattern of Ca²⁺ oscillations. GLIA 28:97–113, 1999. © 1999 Wiley‐Liss, Inc.     

Probable Ca2+-mediated inactivation of Ca2+ currents in mammalian brain neurons

In rat hippocampal neurons, current- and single-electrode voltage-clamp analyses revealed a pronounced inactivation of probable Ca2+ currents (ICa), which was dependent on the amount of Ca2+ influx. Studies were conducted in cesium-loaded, tetrodotoxin-treated brain slice neurons in which known contaminating currents were blocked. These results therefore provide the first clear evidence that apparent Ca2+-mediated inactivation of ICa is an important mechanism with which mammalian brain neurons limit Ca2+ influx.     

Platelet Ca2+ homeostasis: Na+-Ca2+ exchange in plasma membrane vesicles

A vesicular plasma membrane-enriched fraction obtained from human platelets exhibited 45Ca2+ uptake in exchange for intravesicular Na+. The rate of Ca2+ uptake was linear up to 4 sec. The apparent Km for Ca2+ was 22 microM and the Vmax 280 pmol/mg/sec. Ca2+ efflux from Ca2+ loaded vesicles was obtained upon dilution into a NaCl but not a KCl medium. The extent of Ca2+ uptake was monotonically increased as the pH increased from 6 to 9. Na+-Ca2+ exchange was shown to be electrogenic. Ca2+ uptake was distinguished from binding by the induction of Ca2+ release after A23187 addition. These findings support a role for Na+-Ca2+ exchange in human platelet Ca2+ transport.     

Ca(2+)- and voltage-dependent inactivation of Ca2+ currents in rat intermediate pituitary.

We used single electrode voltage-clamp methods to investigate the inactivation of Ca2+ currents in melanotrophs of the intermediate lobe of the pituitary. The low threshold transient current was inactivated by brief prepulses to potentials above -30 mV and inhibition remained complete as prepulse potential was increased from 0 to +70 mV. Both the high threshold transient and sustained currents, however, were inhibited to the greatest extent (60%) by prepulses to 0 mV. Prepulses to more positive potentials close to the Ca2+ reversal potential produced much less (15%) inactivation. Buffering intracellular Ca2+ by including BAPTA in the recording electrode or replacing extracellular Ca2+ with Ba2+ reduced the effect of prepulses. Slowing Ca2+ extrusion by reducing the Na+ gradient across the cell increased the duration of the effect of prepulses. We conclude that the low threshold, transient current is inactivated primarily by membrane voltage while both the high threshold currents are inhibited by elevation of intracellular Ca2+ although the two currents display different sensitivities to Ca2+ concentration. Inhibition of the high threshold transient current by the neurotransmitter dopamine, however, acts by a different mechanism not mediated by Ca(2+)-dependent inactivation.