Action potential-dependent calcium transients in myenteric S neurons of the guinea-pig ileum.

Simultaneous intracellular microelectrode recording and Fura-2 imaging was used to investigate the relationship between intracellular calcium ion concentration ([Ca2+]i) and excitability of tonic S neurons in intact myenteric plexus of the guinea-pig ileum. S neurons were impaled in myenteric ganglia, at locations near connections with internodal strands. The calcium indicator Fura-2 was loaded via the recording microelectrode. The estimated [Ca2+]i of these neurons was approximately 95 nM (n = 25). Intracellular current injection (200 ms pulses, 0.2 nA, delivered at 0.05 Hz) resulted in action potential firing throughout the stimulus pulse, accompanied by transient increases in [Ca2+]i (to approximately 240 nM, n = 12). Increasing the number of evoked action potentials by increasing stimulus duration (100-500 ms) or intensity (0.05-0.3 nA) produced correspondingly larger [Ca2+]i transients. Single action potentials rarely produced resolvable [Ca2+]i events, while short bursts of action potentials (three to five events) invariably produced resolvable [Ca2+]i increases. Some neurons demonstrated spontaneous action potential firing, which was accompanied by sustained [Ca2+]i increases. Action potential firing and [Ca2+]i increases were also observed by activation of slow synaptic input to these neurons, in cases where the slow depolarization initiated action potential firing. Action potentials (evoked or spontaneous) and associated [Ca2+]i transients were abolished by tetrodotoxin (1 microM). Omega-conotoxin GVIA (100 nM) reduced [Ca2+]i transients by approximately 67%, suggesting that calcium influx through N-type calcium channels contributes to evoked [Ca2+]i increases. The S neurons in this study showed prominent afterhyperpolarizations following bursts of action potential firing. The time-course of afterhyperpolarizations was correlated with the time-course of evoked [Ca2+]i transients. Afterhyperpolarizations were blocked by tetrodotoxin and reduced by omega-conotoxin GVIA, suggesting that calcium influx through N-type channels contributes to these events. The electrical properties of Fura-2-loaded neurons were not significantly different from properties of neurons recorded without Fura-2 injection, suggesting that Fura-2 injection alone does not significantly influence the electrical properties of these cells. These data indicate that myenteric S neurons in situ show prominent, activity-dependent increases in [Ca2+]i. These events can be generated spontaneously, or be evoked by intracellular current injection or synaptic activation. [Ca2+]i transients in these neurons appear to involve action potential-dependent opening of N-type calcium channels, and the elevation in [Ca2+]i increase may underlie afterhyperpolarizations and regulate excitability of these enteric neurons.     

E(f)-current contributes to whole-cell calcium current in low calcium in frog sympathetic neurons

Because Ca(2+) plays diverse roles in intracellular signaling in neurons, several types of calcium channels are employed to control Ca(2+) influx in these cells. Our experiments focus on resolving the paradox of why whole-cell current has not been observed under typical recording conditions for one type of calcium channel that is highly expressed in frog sympathetic neurons. These channels, referred to as E(f)-channels, are present in the membrane at a density greater than the channels that carry approximately 90% of whole-cell current in low Ba(2+); but, E(f)-current has not been detected in low Ba(2+). Using Ca(2+) instead of Ba(2+) as the charge carrier, we recorded a possible E-type current in frog sympathetic neurons. The current was resistant to specific blockers of N-, L-, and P/Q-type calcium channels but was more sensitive to Ni(2+) block than was N- or L-current. Current amplitude in Ca(2+) is slightly greater than that in Ba(2+). In 3 mM Ca(2+), the current contributed approximately 12% of total current at peak voltage and increased at voltages more hyperpolarized to the peak, reaching approximately 40% at -30 mV, where whole-cell current starts to activate. The presence of E(f)-current in 3 mM Ca(2+) suggests a potential role for E(f)-channels in regulating calcium influx into sympathetic neurons.     

Mechanisms of GABA and glycine depolarization-induced calcium transients in rat dorsal horn neurons.

1. The mechanisms and effects of GABA- and glycine-evoked depolarization were studied in cultured rat dorsal horn neurons using indo-1 recordings of [Ca2+]i and patch clamp recordings in conventional whole-cell or perforated-patch mode. 2. Application of GABA to unclamped neurons caused [Ca2+]i increases that were dose dependent and exhibited GABAA receptor pharmacology. Calcium entered the neurons via high-threshold voltage-gated calcium channels (conotoxin and nimodipine sensitive). 3. In perforated-patch recordings employing cation-selective ionophores, GABAA receptor activation depolarized 123 of 132 cells to membrane potentials as depolarized as -33 mV (mean -50 mV in all 132 cells, +12 mV above resting potential). The ionic basis of the depolarization was determined by extracellular ion substitution; increased anionic conductance could account fully for the results. 4. Glycine, acting at a strychnine-sensitive receptor, also caused Ca2+ entry into these neurons through voltage-gated Ca2+ channels. Glycine and GABA both evoked [Ca2+]i responses in the same cells and the responses were highly correlated in amplitude. Glycine also depolarized all five cells tested with perforated recording. Each of the five cells was also depolarized by muscimol to a value similar to that obtained for glycine. 5. Both the depolarization and the increases in [Ca2+]i caused by GABA and glycine could potentially play a role in processes of development and differentiation and sensory transmission in the spinal cord dorsal horn.     

Local control of excitation-contraction coupling in rat heart cells.

1. Cytosolic free calcium ion concentration ([Ca2+]i) and whole-cell L-type Ca2+ channel currents were measured during excitation-contraction (E-C) coupling in single voltage-clamped rat cardiac ventricular cells. The measurements were used to compute the total cellular efflux of calcium ions through sarcoplasmic reticulum (SR) Ca2+ release channels (FSR,rel) and the influx of Ca2+ via L-type Ca2+ channels (FICa). 2. FSR,rel was elicited by depolarizing voltage-clamp pulses 200 ms in duration to membrane potentials from -30 to +80 mV. Over this range, peak FSR,rel had a bell-shaped dependence on clamp pulse potential. In all cells, the 'gain' of the system, measured as the ratio, FSR,rel(max)/FICa(max), declined from about 16, at 0 mV, to much lower values as clamp pulse voltage was made progressively more positive. We named this phenomenon of change in gain as a function of membrane potential, 'variable gain'. At clamp pulse potentials in the range -30 to 0 mV, the gain differed from cell to cell, being constant at about 16 in some cells, but decreasing from high values (approximately 65) at -20 mV in others. 3. At clamp pulse potentials at which Ca2+ influx (FICa) was maintained, FSR,rel also had a small maintained component. When macroscopic Ca2+ influx was brief (1-2 ms, during 'tails' of FICa), FSR,rel rose rapidly to a peak after repolarization and then declined with a half-time of about 9 ms (typically). 4. The rising phase of [Ca2+]i transients could be interrupted by stopping Ca2+ influx rapidly (by voltage clamp). We therefore termed this phenomenon 'interrupted SR Ca2+ release'.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effect of GIRK2(wv) on neurite growth, protein expression, and viability in the CNS-derived neuronal cell line, CATH.A-differentiated

Death occurs in the homozygous mutant mouse weaver among several classes of neuron in cerebellum and ventral midbrain, because these neurons carry a mutation in the G protein-gated inwardly rectifying potassium channel, Girk2. GIRK2 is expressed in all neuronal types killed by wv in cerebellum and midbrain as well as in neurons elsewhere that suffer lesser consequences. GIRK2(wv) affects neurons postnatally, after proliferation, at the time of final differentiation. To assess the impact of GIRK2(wv) on neuronal development and viability, we introduced cDNA encoding wild-type and mutant channels into a variant of a CNS derived catecholamine cell line (Cath.a) known as Cath.a-differentiated. When cultured in serum-free medium, Cath.a-differentiated cells cease proliferation and undergo morphological differentiation, growing long neurites. Cath.a-differentiated cells do not express endogenous Girk channels. Transfection of GIRK2(wv) resulted in the death of Cath.a-differentiated cells, in a cDNA-concentration dependent manner. The highest concentration of Girk2(wv) cDNA caused loss of about half the cells, the next highest concentration one-third, and the least had no effect on viability. However, even the lowest concentration resulted in disruption of neurite outgrowth and reduced the protein products of co-transfected genes. High concentrations of MK801, which prevent Na(+) influx through the mutant channel, prevented death induced by GIRK2(wv). Cell death and disruption of neurite outgrowth were counteracted in GIRK2(wv)-expressing cells by the presence of an unrelated inwardly rectifying potassium channel, Kir2.3. These results are consistent with wv being a gain-of-function mutation, causing disruption of cellular homeostasis by mechanisms such as increased Na(+) influx and chronic depolarization which may in turn result in an excessive metabolic burden on the cell.     

In vitro evidence that the reduction in mesencepalic dopaminergic neurons in the weaver heterozygote is not due to a failure in target cell interaction

 The murine weaver (wv) mutation is characterized by a genetically determined loss of several neuronal populations, which include the nigrostriatal dopaminergic neurons. Animals homozygous for the wv gene exhibit marked deficits in dopaminergic morphological and neurochemical parameters. The wv gene shows incomplete dominance in that heterozygous (wv/+) mice exhibit moderate reductions in midbrain dopaminergic neuron number. It is unclear whether the dopaminergic neuronal loss in homozygous and heterozygous animals results from an effect of the wv gene solely on the dopaminergic neurons or is due to a failure of interaction of dopaminergic neurons with target cells of the striatum. This issue has been addressed utilizing three-dimensional reaggregate tissue cultures to determine whether the wv gene acts directly on the mesencephalic dopaminergic neurons. Embryonic mesencephalon and striatum from wv/+ and wild-type (+/+) brains were dissociated and the cells recombined into four mesencephalic-striatal aggregate combinations: (1) mesencephalic_(+/+)-striatal_(+/+)aggregates; (2) mesencephalic _(wv/+) -striatal _(wv/+) aggregates; (3) mesencephalic _(wv/+) -striatal_(+/+)aggregates; and (4) mesencephalic_(+/+)-striatal _(wv/+) aggregates. At 29 days and 57 days of culture, the number of dopaminergic neurons and dopamine content from mesencephalic-striatal aggregates consisting of mixed genotype or from only wv/+ tissue were quantitated and compared with that from mesencephalic-striatal cultures prepared from +/+ tissue alone. At both culture time points, aggregates containing wv/+ mesencephalon coaggregated with either wv/+ or +/+ striatum contained fewer dopaminergic neurons than mesencephalic-striatal cultures composed of only +/+ cells. Coaggregation of +/+ mesencephalon with wv/+ striatum did not have a detrimental effect on dopaminergic cell number. The findings demonstrate that the difference in the number of mesencephalic dopaminergic neurons between wv/+ and +/+ animals seen in vivo can be reproduced in three-dimensional reaggregate culture. Since the coculture of +/+ striatum with wv/+ mesencephalon did not appear to rescue wv/+ dopaminergic neurons in the aggregates as compared to wv/+ striatum and, wv/+ striatum proved to be a perfectly adequate target for +/+ mesencephalic dopaminergic neurons, it appears that the effect of the wv gene is on the dopaminergic neurons themselves. Received: 7 August 1996 / Accepted: 20 December 1996     

Temperature sensitivity of dopaminergic neurons of the substantia nigra pars compacta: involvement of transient receptor potential channels

Changes in temperature of up to several degrees have been reported in different brain regions during various behaviors or in response to environmental stimuli. We investigated temperature sensitivity of dopaminergic neurons of the rat substantia nigra pars compacta (SNc), an area important for motor and emotional control, using a combination of electrophysiological techniques, microfluorometry, and RT-PCR in brain slices. Spontaneous neuron firing, cell membrane potential/currents, and intracellular Ca2+ level ([Ca2+]i) were measured during cooling by < or =10 degrees and warming by < or =5 degrees from 34 degrees C. Cooling evoked slowing of firing, cell membrane hyperpolarization, increase in cell input resistance, an outward current under voltage clamp, and a decrease of [Ca2+]i. Warming induced an increase in firing frequency, a decrease in input resistance, an inward current, and a rise in [Ca2+]i. The cooling-induced current, which reversed in polarity between -5 and -17 mV, was dependent on extracellular Na+. Cooling-induced whole cell currents and changes in [Ca2+]i were attenuated by 79% in the presence of 2-aminoethoxydiphenylborane (2-APB; 200 microM), and the outward current was reduced by 20% with ruthenium red (100 microM). RT-PCR conducted with tissue punches containing the SNc revealed mRNA expression for TRPV3 and TRPV4 channels, known to be activated in expression systems by temperature changes within the physiological range. 2-APB, a TRPV3 modulator, increased baseline [Ca2+]i, whereas 4alphaPDD, a TRPV4 agonist, increased spontaneous firing in 7 of 14 neurons tested. We conclude that temperature-gated TRPV3 and TRPV4 cationic channels are expressed in nigral dopaminergic neurons and are constitutively active in brain slices at near physiological temperatures, where they affect the excitability and calcium homeostasis of these neurons.     

Calcium homeostasis in dissociated embryonic neurons: a flow cytometric analysis.

1. Ca2+ homeostasis in freshly dissociated neurons from embryonic rat hypothalamus, cortex, and brain stem was investigated with flow cytometry. Cells were dissociated from embryonic brain by enzymatic and mechanical means and were incubated with the acetoxymethylester derivative of the Ca(2+)-sensitive dye indo-1. Neurons hydrolyzed and retained the dye as determined by the intensity of fluorescence emission, whereas similarly treated cultured astrocytes gave very low-level fluorescence. 2. The fluorescence of the indo-1 dye was measured at two wavelengths (405 and 485 nm) for each cell. Data were collected only from those cells (presumptive neurons) with high levels of fluorescence. Methods were developed to calibrate the level of intracellular free calcium ([Ca2+]i) as the ratio of fluorescence at 410 and 485 nm. The level of intracellular free Ca2+ was then calculated for each neuron. 3. A wide distribution of resting [Ca2+]i was found, with a median of approximately 90 nM. After addition of ionomycin to cells in Ca(2+)-free medium, there was a transient increase in [Ca2+]i, suggesting that all embryonic neurons had internal Ca2+ stores. The presence of active calcium extrusion mechanisms was demonstrated with the use of ionomycin in Ca(2+)-containing medium and with metabolic inhibitors. Furthermore, incubation in sodium-free medium resulted in a transient increase in [Ca2+]i and a reduced ability to eliminate elevated [Ca2+]i from the cytoplasm, suggesting that calcium homeostasis was dependent on the activity of the Na(+)-Ca2+ exchange mechanism. 4. Depolarization with K+ or veratrine increased [Ca2+]i in approximately 20% of the cells. This increase was blocked by eliminating extracellular free Ca2+ or adding Co2+, nifedipine, or verapamil, suggesting mediation by voltage-sensitive calcium channels. 5. Neurons were sorted on the basis of high [Ca2+]i and placed into dissociated culture. After 24 h, neurons in culture retained indo-1 fluorescence, suggesting that populations of neurons can be collected on the basis of their levels of [Ca2+]i. 6. These results demonstrate that flow cytometric analysis allows the characterization of a variety of Ca(2+)-regulatory mechanisms in populations of freshly dissociated embryonic neurons. Although only a proportion of embryonic day 17 neurons exhibit voltage-sensitive calcium channels, all neurons have developed the ability to sequester and extrude Ca2+.     

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation.

Electrophysiological recordings and calcium measurements in striatal large aspiny interneurons in response to combined O2/glucose deprivation. The effects of combined O2/glucose deprivation were investigated on large aspiny (LA) interneurons recorded from a striatal slice preparation by means of simultaneous electrophysiological and optical recordings. LA interneurons were visually identified and impaled with sharp microelectrodes loaded with the calcium (Ca2+)-sensitive dye bis-fura-2. These cells showed the morphological, electrophysiological, and pharmacological features of large striatal cholinergic interneurons. O2/glucose deprivation induced a membrane hyperpolarization coupled to a concomitant increase in intracellular Ca2+ concentration ([Ca2+]i). Interestingly, this [Ca2+]i elevation was more pronounced in dendritic branches rather than in the somatic region. The O2/glucose-deprivation-induced membrane hyperpolarization reversed its polarity at the potassium (K+) equilibrium potential. Both membrane hyperpolarization and [Ca2+]i rise were unaffected by TTX or by a combination of ionotropic glutamate receptors antagonists, D-2-amino-5-phosphonovaleric acid and 6cyano-7-nitroquinoxaline-2, 3-dione. Sulfonylurea glibenclamide, a blocker of ATP-sensitive K+ channels, markedly reduced the O2/glucose-deprivation-induced membrane hyperpolarization but failed to prevent the rise in [Ca2+]i. Likewise, charybdotoxin, a large K+-channel (BK) inhibitor, abolished the membrane hyperpolarization but did not produce detectable changes of [Ca2+]i elevation. A combination of high-voltage-activated Ca2+ channel blockers significantly reduced both the membrane hyperpolarization and the rise in [Ca2+]i. In a set of experiments performed without dye in the recording electrode, either intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid or external barium abolished the membrane hyperpolarization induced by O2/glucose deprivation. The hyperpolarizing effect on membrane potential was mimicked by oxotremorine, an M2-like muscarinic receptor agonist, and by baclofen, a GABAB receptor agonist. However, this membrane hyperpolarization was not coupled to an increase but rather to a decrease of the basal [Ca2+]i. Furthermore glibenclamide did not reduce the oxotremorine- and baclofen-induced membrane hyperpolarization. In conclusion, the present results suggest that in striatal LA cells, O2/glucose deprivation activates a membrane hyperpolarization that does not involve ligand-gated K+ conductances but is sensitive to barium, glibenclamide, and charybdotoxin. The increase in [Ca2+]i is partially due to influx through voltage-gated high-voltage-activated Ca2+ channels.     

Estradiol inhibits atp-induced intracellular calcium concentration increase in dorsal root ganglia neurons

Estrogen has been implicated in modulation of pain processing. Although this modulation occurs within the CNS, estrogen may also act on primary afferent neurons whose cell bodies are located within the dorsal root ganglia (DRG). Primary cultures of rat DRG neurons were loaded with Fura-2 and tested for ATP-induced changes in intracellular calcium concentration ([Ca(2+)](i)) by fluorescent ratio imaging. ATP, an algesic agent, induces [Ca(2+)](i) changes via activation of purinergic 2X (P2X) type receptors and voltage-gated Ca(2+) channels (VGCC). ATP (10 microM) caused increased [Ca(2+)](i) transients (226.6+/-16.7 nM, n = 42) in 53% of small to medium DRG neurons. A 5-min incubation with 17 beta-estradiol (100 nM) inhibited ATP-induced [Ca(2+)](i) (164+/-14.6 nM, P<0.05) in 85% of the ATP-responsive DRG neurons, whereas the inactive isomer 17 alpha-estradiol had no effect. Both the mixed agonist/antagonist tamoxifen (1 microM) and specific estrogen receptor antagonist ICI 182780 (1 microM) blocked the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. Estradiol coupled to bovine serum albumin, which does not diffuse through the plasma membrane, blocked ATP-induced [Ca(2+)](i), suggesting that estradiol acts at a membrane-associated estrogen receptor. Attenuation of [Ca(2+)](i) transients was mediated by estrogen action on VGCC. Nifedipine (10 microM), an L-type VGCC antagonist mimicked the effect of estrogen and when co-administered did not increase the estradiol inhibition of ATP-induced [Ca(2+)](i) transients. N- and P-type VGCC antagonists omega-conotoxin GVIA (1 microM) and omega-agatoxin IVA (100 nM), attenuated the ATP-induced [Ca(2+)](i) transients. Co-administration of these blockers with estrogen induced a further decrease of the ATP-induced [Ca(2+)](i) flux. Together, these results suggest that although ATP stimulation of P2X receptors activates L-, N-, and P-type VGCC, estradiol primarily blocks L-type VGCC. The estradiol regulation of this ATP-induced [Ca(2+)](i) transients suggests a mechanism through which estradiol may modulate nociceptive signaling in the peripheral nervous system.     

Simultaneous recordings of cytosolic Ca2+ level and membrane potential and current during the response to thyroliberin in clonal rat anterior pituitary cells

The response to thyroliberin in prolactin-producing rat GH4C1 clonal cells was studied using fura-2 to monitor the cytosolic Ca2+ level ([Ca2+]i) in single cells, combined with recordings of membrane potential and current. The average value of [Ca2+]i was 109 nM (mean +/- SD, n = 112), and evoked action potentials caused transient elevations of about 100 nM. At higher firing frequencies these transients merged to a sustained elevation. In 100% of the cells thyroliberin caused an instant rise in [Ca2+]i, peaking at 795 +/- 300 nM (n = 112). This first phase of the thyroliberin response was associated with hyperpolarization in current clamp and outward current in voltage clamp, caused by the opening of Ca2(+)-activated K+ channels. In 75% of the cells the initial peak in [Ca2+]i was followed by a prolonged plateau phase at 247 +/- 76 nM (n = 84). In current clamp the second-phase elevation of [Ca2+]i was linked to either a modest depolarization in combination with enhanced firing frequency or a more pronounced depolarization in silent cells. This elevation of [Ca2+]i was reversed by hyperpolarizing current injection. No second-phase elevation of [Ca2+]i was observed during voltage clamp at a holding potential of -50 mV. Short exposure to Ca2(+)-free conditions eliminated the second-phase elevation in [Ca2+]i, whereas the first phase remained intact. Our experiments show a direct relationship between electrical activity and [Ca2+]i in the GH4C1 cells. The second-phase elevation of [Ca2+]i caused by thyroliberin is the result of influx through voltage-sensitive Ca2+ channels, without involving agonist-gated channels.     

Systematic differences in time of dopaminergic neuron origin between normal mice and homozygous weaver mutants

Immunocytochemical labeling for tyrosine hydroxylase and [³H]thymidine autoradiography were combined in wild-type mice and in mice homozygous for the weaver mutant gene (wv) to see whether the neurogenetic patterns of midbrain dopaminergic neurons was normal in the mutants and whether the degeneration of dopaminergic neurons was linked to their time of origin. Dams of wild-type and homozygous weaver mice were injected with [³H]thymidine on embryonic days (E) 11–E12, E12–E13, E13–E14, and E14-E15 to label neurons in the retrorubral field, the substantia nigra pars compacta, the ventral tegmental area, and the interfascicular nucleus as they were being generated. The quantitatively determined time of origin profiles indicated that wv/wv mice have the same time span of neurogenesis as +/+ mice (E10 to E14), but have significant deficits in the proportion of late-generated neurons in each dopaminergic population. In the retrorubral field and substantia nigra, weaver homozygotes had substantial losses of dopaminergic neurons and had a greater deficit in the proportion of neurons generated late while, in the ventral tegmental area and interfascicular nucleus, there were slight losses of dopaminergic neurons and only slight deficits in the proportion of late-generated neurons. These findings lead to the conclusion that the weaver gene is specifically targeting dopaminergic neurons that are generated late, mainly on E13 and E14.     

Distinct intracellular calcium profiles following influx through N- versus L-type calcium channels: role of Ca2+-induced Ca2+ release

Selective activation of neuronal functions by Ca(2+) is determined by the kinetic profile of the intracellular calcium ([Ca(2+)](i)) signal in addition to its amplitude. Concurrent electrophysiology and ratiometric calcium imaging were used to measure transmembrane Ca(2+) current and the resulting rise and decay of [Ca(2+)](i) in differentiated pheochromocytoma (PC12) cells. We show that equal amounts of Ca(2+) entering through N-type and L-type voltage-gated Ca(2+) channels result in significantly different [Ca(2+)](i) temporal profiles. When the contribution of N-type channels was reduced by omega-conotoxin MVIIA treatment, a faster [Ca(2+)](i) decay was observed. Conversely, when the contribution of L-type channels was reduced by nifedipine treatment, [Ca(2+)](i) decay was slower. Potentiating L-type current with BayK8644, or inactivating N-type channels by shifting the holding potential to -40 mV, both resulted in a more rapid decay of [Ca(2+)](i). Channel-specific differences in [Ca(2+)](i) decay rates were abolished by depleting intracellular Ca(2+) stores with thapsigargin or by blocking ryanodine receptors with ryanodine, suggesting the involvement of Ca(2+)-induced Ca(2+) release (CICR). Further support for involvement of CICR is provided by the demonstration that caffeine slowed [Ca(2+)](i) decay while ryanodine at high concentrations increased the rate of [Ca(2+)](i) decay. We conclude that Ca(2+) entering through N-type channels is amplified by ryanodine receptor mediated CICR. Channel-specific activation of CICR provides a mechanism whereby the kinetics of intracellular Ca(2+) leaves a fingerprint of the route of entry, potentially encoding the selective activation of a subset of Ca(2+)-sensitive processes within the neuron.     

Mechanisms of unmodified CdSe quantum dot-induced elevation of cytoplasmic calcium levels in primary cultures of rat hippocampal neurons

Quantum dots (QDs) have shown great promise for applications in biology and medicine, which is being challenged by their potential nanotoxicity. Reactive oxygen species (ROS) produced by QDs are believed to be partially responsible for QD cytotoxicity. Cytoplasmic Ca(2+) plays an important role in the development of ROS injury. Here we found unmodified cadmium selenium (CdSe) QDs could elevate cytoplasmic calcium levels ([Ca(2+)](i)) in primary cultures of hippocampal neurons, involved both extracellular Ca(2+) influx and internal Ca(2+) release. More specifically, verapamil and mibefradil (L-type and T-type calcium channels antagonists, respectively) failed to prevent extracellular Ca(2+) influx under QD insult, while omega-conotoxin (N-type antagonist) could partially block this Ca(2+) influx. Surprisingly, this Ca(2+) influx could be well blocked by voltage-gated sodium channels (VGSCs) antagonist, tetrodotoxin (TTX). QD-induced internal Ca(2+) release could be avoided by clonazepam, a specific inhibitor of mitochondrial sodium-calcium exchangers (MNCX), and also by TTX. Furthermore, dantrolene, an antagonist of ryanodine (Ry) receptors in endoplasmic reticulum (ER), almost abolished internal Ca(2+) release, while 2-APB [inositol triphosphate (IP(3)) receptors antagonist] failed to block this Ca(2+) release, indicating that released Ca(2+) from mitochondria, which was induced by extracellular Na(+) influx, further triggered much more Ca(2+) release from ER. Our results imply that more research on the biocompatibility and biosafety of QD is both warranted and necessary.     

Voltage-gated calcium channels in autonomic neuroeffector transmission

Calcium influx through voltage-gated calcium channels (VGCCs) is required for neurotransmitter release. Recent research has characterised several pharmacologically and electrophysiologically distinct VGCC subtypes, some of which are involved in neurotransmitter release. Transmitter release from autonomic neurons can be coupled to calcium entry through N-, P/Q- and/or R-type VGCCs; the precise combination of VGCC subtypes appears to vary according to the neurotransmitter, tissue and species. L-type channels rarely appear to be important in autonomic neurotransmitter release. There does not appear to be a general rule regarding the nature of the VGCCs coupled to release of a particular transmitter in different tissues or species. Release of the same neurotransmitter from different populations of neurons often reveals a different pattern of involvement of VGCCs. Transmitters released from the same population of neurons are sometimes coupled to calcium influx through different VGCC subtypes. However, release of transmitters thought to be co-localised within vesicles is coupled to calcium influx through the same VGCCs. The role of VGCC subtypes in transmitter release can be altered by mode of nerve stimulation. Different VGCC subtypes may be coupled to transmitter release at low versus high electrical stimulation frequencies, or in response to potassium depolarization or chemical stimulation. In certain disease processes, voltage-gated calcium channels on autonomic neurons can be targeted; for example antibodies to P/Q-type VGCCs in Lambert-Eaton myasthenic syndrome downregulate VGCCs, thereby inhibiting autonomic neuroeffector transmission.     

Differences in calcium signalling in rat peripheral sensory neurons

Calcium influx and the resulting increase in intracellular calcium concentration [Ca2+]i can induce enhanced sensitivity to temperature increases in nociceptive neurons. Using the patch-clamp technique and simultaneous calcium microfluorimetry we show that experimental elevation of [Ca2+]i using the calcium ionophore ionomycin resulted in a significant potentiation of heat-activated currents. This was not the case when rises in [Ca2+]i were elicited by depolarization of the cell membrane by current injection via the patch pipette. Our data provide first, however, indirect evidence that in sensory neurons calcium ions may be guided into different intracellular microdomains depending on the type of ion channel or pore through which they enter the cell. We conclude that the compartmentalization of sensory neurons for calcium ions may be decisive on further signalling cascades accounting, for example, for neuronal plasticity.     

Facilitation of calcium influx by propylene glycol through the voltage-dependent calcium channels in PC12 cells

Propylene glycol (PG) raises an intracellular calcium concentration ([Ca2+]i) in PC12 cells. The present study has been undertaken to examine whether or not the voltage-dependent Ca2+ channels are involved in the PG-induced rise in [Ca2+]i and, if so, to determine which types participate in it. CdCl2 (50 micro M) and the Ca2+ -free saline depressed the action of PG (0.5 - 10 %v/v)-induced [Ca2+]i rise. Although NiCl2 (50 micro M) at the same concentration as CdCl2, and omega-agatoxin (50 and 300 nM) had no effect on the PG-induced [Ca2+]i rise, each of omega-conotoxin (1 micro M), nifedipine (10 micro M), nicardipine (10 micro M), varapamil (10 micro M) and diltiazem (10 micro M) significantly decreased it. Electrical stimulation and Bay K 8644 (1 micro M) enhanced the PG-induced [Ca2+]i rise. The second phase of the [Ca2]i rise was fallen fast by nicardipine (10 micro M), but not by omega-conotoxin (1 micro M). The results obtained suggested that the Ca2+ influx through the L- and N-type Ca2+ channels are involved in the PG-induced [Ca2+]i rise.     

Effect of streptozotocin-induced diabetes on the activity of calcium channels in rat dorsal horn neurons

We have previously found that spinal dorsal horn neurons from streptozotocin-diabetic rats, an animal model for diabetes mellitus, show the prominent changes in the mechanisms responsible for [Ca2+]i regulation. The present study aimed to further characterize the effects of streptozotocin-induced diabetes on neuronal calcium homeostasis. The cytoplasmic Ca2+ concentration ([Ca2+]i) was measured in Fura-2AM-loaded dorsal horn neurons from acutely isolated spinal cord slices using fluorescence technique. We studied Ca2+ entry through plasmalemmal Ca2+ channels during potassium (50 mM KCl)-induced depolarization. The K+-induced [Ca2+]i elevation was inhibited to a different extent by nickel ions, nifedipine and omega-conotoxin suggesting the co-expression of different subtypes of plasmalemmal voltage-gated Ca2+ channels. The suppression of [Ca2+]i transients by Ni2+ (50 microM) was the same in control and diabetic neurons. On the other hand, inhibition of [Ca2+]i transients by nifedipine (50 microM) and omega-conotoxin (1 microM) was much greater in diabetic neurons compared with normal animals. These data suggest that under diabetic conditions the activity of N- and L- but not T-type voltage-gated Ca2+ channels substantially increased in dorsal horn neurons.     

Presynaptic calcium channels and the depletion of synaptic cleft calcium ions

The entry of calcium ions (Ca(2+)) through voltage-gated calcium channels is an essential step in the release of neurotransmitter at the presynaptic nerve terminal. Because the calcium channels are clustered at the release sites, the flux of Ca(2+) into the terminal inevitably removes the ion from the adjacent extracellular space, the synaptic cleft. We have used the large calyx-type synapse of the chick ciliary ganglion to test for synaptic cleft Ca(2+) depletion. The terminal was voltage clamped at a holding potential (V(H)) of -80 mV and a depolarizing pulse was applied to a range of potentials (-60 to +60 mV). The voltage pulse activated a sustained inward calcium current and was followed, on return of the membrane potential to V(H), by an inward calcium tail current. The amplitude of the tail current reflects both the number of open calcium channels at the end of the voltage pulse and the Ca(2+) electrochemical gradient. External barium was substituted for calcium as the charge-carrying ion because initial experiments demonstrated calcium-dependent inactivation of the presynaptic calcium channels. Tail current recruitment was compared in calyx nerve terminals that remained attached to the postsynaptic neuron and therefore retained a synaptic cleft, with terminals that had been fully isolated. In isolated terminals, the tail currents exhibited recruitment curves that could be fit by a Boltzmann distribution with a mean V(1/2) of 0.4 mV and a slope factor of 5.4. However, in attached calyces tail current recruitment was skewed to depolarized potentials with a mean V(1/2) of 11.9 mV and a slope factor of 12.0. The degree of skew of the recruitment curve in the attached calyces correlated with the amplitude of the inward current evoked by the step depolarization. The simplest interpretation of these findings is that during the depolarizing pulse Ba(2+) is removed from the synaptic cleft faster than it is replenished, thus reducing the tail current by reducing the driving force for ion entry. Ca(2+) depletion during presynaptic calcium channel activation is likely to be a general property of chemical transmission at fast synapses that sets a functional limit to the duration of sustained secretion. The synapse may have evolved to minimized cleft depletion by developing a calcium-efficient mechanism to gate transmitter release that requires the concurrent opening of only a few low conductance calcium channels.     

Prostaglandins induce calcium influx in human spermatozoa

Progesterone, prostaglandin and follicular fluid are reported to enhance the acrosome reaction through the influx of extracellular calcium into the cytoplasm of human spermatozoa. Prostaglandins are present within the male reproductive tract, and high concentrations of prostaglandins exist in seminal fluid. In order to investigate the mechanisms by which prostaglandins enhance the acrosome reaction through calcium influx, the intracellular calcium response induced by progesterone, prostaglandin E1 (PGE1), prostaglandin E2 (PGE2) and follicular fluid was measured using fura-2. PGE1 and PGE2 promoted calcium influx dose dependently through dihydropyridine insensitive calcium channels. Refractoriness of the elevation of intracellular Ca2+ concentration ([Ca2+]i) to a second stimulus occurred when 60 microg/ml PGE1 was administered 100 s after the prior administration of 60 microg/ml of PGE1, and similarly when 1 microg/ml of progesterone was administered 100 s after the prior administration of 1 microg/ml of progesterone. Refractoriness also occurred when 60 microg/ml PGE1 was administered after the prior addition of 60 microg/ml PGE2, but did not occur between PGE1 and progesterone. Pertussis toxin (PTX) did not modify the changes in [Ca2+]i after the addition of PGE1 or PGE2. In conclusion, PGE1 and PGE2 promoted calcium influx through PTX- insensitive calcium channels which appeared to be recognized by a common receptor different from that of progesterone.