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ABSTRACT: The tetrodotoxin-resistant (TTX-R) voltage-gated Na(+) channels Na(v)1.8 and Na(v)1.9 are expressed by a subset of primary sensory neurons and have been implicated in various pain states. Although recent studies suggest involvement of TTX-R Na(+) channels in sensory synaptic transmission and spinal pain processing, it remains unknown whether TTX-R Na(+) channels are expressed and function presynaptically. We examined expression of TTX-R channels at sensory synapses formed between rat dorsal root ganglion (DRG) and spinal cord (SC) neurons in a DRG/SC co-culture system. Immunostaining showed extensive labeling of presynaptic axonal boutons with Na(v)1.8- and Na(v)1.9-specific antibodies. Measurements using the fluorescent Na(+) indicator SBFI demonstrated action potential-induced presynaptic Na(+) entry that was resistant to tetrodotoxin (TTX) but was blocked by lidocaine. Furthermore, presynaptic [Ca(2+)](i) elevation in response to a single action potential was not affected by TTX in TTX-resistant DRG neurons. Finally, glutamatergic synaptic transmission was not inhibited by TTX in more than 50% of synaptic pairs examined; subsequent treatment with lidocaine completely blocked these TTX-resistant excitatory postsynaptic currents. Taken together, these results provide evidence for presynaptic expression of functional TTX-R Na(+) channels that may be important for shaping presynaptic action potentials and regulating transmitter release at the first sensory synapse.
Neuroscience 01/2009; 159(2):559-69. · 3.38 Impact Factor
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ABSTRACT: In many cell types, cell death induced by a variety of insults is accompanied by an increase in intracellular calcium. The Ca(2+) homeostatic mechanisms affected by such insults, however, have not been fully determined. Recent evidence indicates that kainic acid-induced seizures alter plasma membrane calcium ATPase mRNA expression within vulnerable hippocampal cell populations before the onset of cell death. We examined the effects of altering plasma membrane calcium ATPase expression on cell vulnerability in rat pheochromocytoma 12 cells. Pheochromocytoma 12 cells are vulnerable to Ca(2+) overload induced by the Ca(2+) ionophore A23187. Reverse transcriptase-PCR and Western blot data indicated that plasma membrane calcium ATPase isoform 4b constitutes a major calcium pump isoform in the pheochromocytoma 12 cells. Therefore, permanently transfected pheochromocytoma 12-derived cell lines were established that either over-expressed plasma membrane calcium ATPase isoform 4b, or suppressed the expression of the endogenous plasma membrane calcium ATPase isoform 4. Over-expressing clones were less vulnerable to Ca(2+)-mediated cell death induced by A23187 whereas "antisense" clones were considerably more susceptible. These data indicate that regulation of plasma membrane calcium ATPase expression may be critical to cellular survival when cells are exposed to pathological increases in intracellular calcium.
Journal of Neuroscience Research 07/2001; 64(6):661-9. · 2.74 Impact Factor
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ABSTRACT: Precise regulation of intracellular Ca(2+) concentration ([Ca(2+)](i)) is achieved by the coordinated function of Ca(2+) channels and Ca(2+) buffers. Neuronal differentiation induces up-regulation of Ca(2+) channels. However, little is known about the effects of differentiation on the expression of the plasma membrane Ca(2+)-ATPase (PMCA), the principal Ca(2+) extrusion mechanism in neurons. In this study, we examined the regulation of PMCA expression during differentiation of the human neuroblastoma cell line IMR-32. [Ca(2+)](i) was monitored in single cells using indo-1 microfluorimetry. When the Ca(2+)-ATPase of the endoplasmic reticulum was blocked by cyclopiazonic acid, [Ca(2+)](i) recovery after small depolarization-induced Ca(2+) loads was governed primarily by PMCAs. [Ca(2+)](i) returned to baseline by a process described by a monoexponential function in undifferentiated cells (tau = 52 +/- 4 s; n = 25). After differentiation for 12-16 days, the [Ca(2+)](i) recovery rate increased by more than threefold (tau = 17 +/- 1 s; n = 31). Western blots showed a pronounced increase in expression of three major PMCA isoforms in IMR-32 cells during differentiation, including PMCA2, PMCA3 and PMCA4. These results demonstrate up-regulation of PMCAs on the functional and protein level during neuronal differentiation in vitro. Parallel amplification of Ca(2+) influx and efflux pathways may enable differentiated neurons to precisely localize Ca(2+) signals in time and space.
Journal of Neurochemistry 04/2001; 76(6):1756-65. · 4.06 Impact Factor
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ABSTRACT: Gene transfer into neuronal cells provides an important approach to study their function. Particle-mediated gene delivery was used to transfect rat dorsal root ganglion (DRG) and hippocampal neurons in primary culture with the genes for the enhanced blue and green fluorescent proteins (EBFP and EGFP) under control of the cytomegalovirus promoter. Quantitative analysis of marker protein fluorescence detected expression at 3 h that continued to increase for 48 h. For DRG neurons the optimal expression efficiency of 8+/-2% was obtained 24 h following transfection. In contrast, approximately 2+/-1% of hippocampal neurons in culture expressed EGFP at 3 h which subsequently declined. Co-transfection of DRG cultures with two plasmids produced reliable expression of both genes. Transfected DRG neurons exhibited normal electrophysiological properties, and resting and stimulated intracellular Ca2+ concentrations were unchanged. After transfection, 44% of hippocampal neurons remained in functional synaptic networks as indicated by glutamatergic Ca2+ spiking activity. Particle-mediated gene delivery provided a straightforward, reproducible and efficient method for transfection of neurons in primary culture. Transfected cells were easily identified by EGFP fluorescence, enabling subsequent physiological analysis. Biolistic particle bombardment was well tolerated by peripheral neurons, although caution was required when this method was applied to CNS cultures.
Pflügers Archiv - European Journal of Physiology 05/2000; 439(6):730-8. · 4.46 Impact Factor
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ABSTRACT: Gene transfer into neuronal cells provides an important approach to study their function. Particle-mediated gene delivery was used to transfect rat dorsal root ganglion (DRG) and hippocampal neurons in primary culture with the genes for the enhanced blue and green fluorescent proteins (EBFP and EGFP) under control of the cytomegalovirus promoter. Quantitative analysis of marker protein fluorescence detected expression at 3 h that continued to increase for 48 h. For DRG neurons the optimal expression efficiency of 8DŽ% was obtained 24 h following transfection. In contrast, approximately 2ǃ% of hippocampal neurons in culture expressed EGFP at 3 h which subsequently declined. Co-transfection of DRG cultures with two plasmids produced reliable expression of both genes. Transfected DRG neurons exhibited normal electrophysiological properties, and resting and stimulated intracellular Ca2+ concentrations were unchanged. After transfection, 44% of hippocampal neurons remained in functional synaptic networks as indicated by glutamatergic Ca2+ spiking activity. Particle-mediated gene delivery provided a straightforward, reproducible and efficient method for transfection of neurons in primary culture. Transfected cells were easily identified by EGFP fluorescence, enabling subsequent physiological analysis. Biolistic particle bombardment was well tolerated by peripheral neurons, although caution was required when this method was applied to CNS cultures.
Pflügers Archiv - European Journal of Physiology 02/2000; 439(6):730-738. · 4.46 Impact Factor
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ABSTRACT: Changes in the intracellular calcium concentration ([Ca2+]i) convey signals that are essential to the life and death of neurons. Ca(2+)-induced Ca(2+)-release (CICR), a process in which a modest elevation in [Ca2+]i is amplified by a secondary release of Ca2+ from stores within the cell, plays a prominent role in shaping neuronal [Ca2+]i signals. When CICR becomes regenerative, an explosive increase in [Ca2+]i generates a Ca2+ wave that spreads throughout the cell. A discrete threshold controls activation of this all-or-none behavior and cellular context adjusts the threshold. Thus, the store acts as a switch that determines whether a given pattern of electrical activity will produce a local or global Ca2+ signal. This gatekeeper function seems to control some forms of Ca(2+)-triggered plasticity in neurons.
BioEssays 10/1999; 21(9):743-50. · 4.95 Impact Factor
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ABSTRACT: 1. Store-operated, voltage-independent Ca2+ channels are activated by depletion of intracellular Ca2+ stores and mediate Ca2+ influx into non-excitable cells at resting membrane potential. We used microfluorimetry, patch-clamp and Mn2+-quench techniques to explore the possibility that a similar mechanism exists in rat dorsal root ganglion (DRG) neurones in primary culture. 2. Following caffeine-induced depletion, ryanodine-sensitive Ca2+ stores refilled with Ca2+ at resting membrane potential. The refilling process required extracellular Ca2+, was blocked by 2 mM Ni2+, and was facilitated by membrane hyperpolarization from -55 to -80 mV, indicating a key role for Ca2+ influx. This influx of Ca2+ was not affected by the voltage-operated Ca2+ channel (VOCC) antagonists nicardipine (10 microM), nimodipine (10 microM) or omega-grammotoxin SIA (1 microM). 3. When ryanodine-sensitive Ca2+ stores were depleted in Ca2+-free media, a return to 2 mM external Ca2+ resulted in a pronounced [Ca2+]i overshoot, indicating an increased permeability to Ca2+. Depletion of Ca2+ stores also produced a 2-fold increase in the rate of Mn2+ influx. The [Ca2+]i overshoot and Mn2+ entry were both inhibited by Ni2+, but not by VOCC antagonists. 4. Caffeine induced periodic Ca2+ release from, and reuptake into, ryanodine-sensitive stores. The [Ca2+]i oscillations were arrested by removal of extracellular Ca2+ or by addition of Ni2+, but they were not affected by VOCC antagonists. Hyperpolarization increased the frequency of this rhythmic activity. 5. These data suggest the presence of a Ca2+ entry pathway in mammalian sensory neurones that is distinct from VOCCs and is regulated by ryanodine-sensitive Ca2+ stores. This pathway participates in refilling intracellular Ca2+ stores and maintaining [Ca2+]i oscillations and thus controls the balance between intra- and extracellular Ca2+ reservoirs in resting DRG neurones.
The Journal of Physiology 09/1999; 519 Pt 1:115-30. · 4.72 Impact Factor
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ABSTRACT: Ca2+-induced Ca2+ release (CICR) from intracellular stores amplifies the Ca2+ signal that results from depolarization. In neurons, the amplification has been described as a graded process. Here we show that regenerative CICR develops as an all-or-none event in cultured rat dorsal root ganglion neurons in which ryanodine receptors have been sensitized to Ca2+ by caffeine. We used indo-1-based microfluorimetry in combination with whole-cell patch-clamp recording to characterize the relationship between Ca2+ influx and Ca2+ release. Regenerative release of Ca2+ was triggered when action potential-induced Ca2+ influx increased the intracellular Ca2+ concentration ([Ca2+]i) above threshold. The threshold was modulated by caffeine and intraluminal Ca2+. A relative refractory period followed CICR. The pharmacological profile of the response was consistent with Ca2+ influx through voltage-gated Ca2+ channels triggering release from ryanodine-sensitive stores. The activation of a suprathreshold response increased more than fivefold the amplitude and duration of the [Ca2+]i transient. The switch to a suprathreshold response was regulated very precisely in that addition of a single action potential to the stimulus train was sufficient for this transformation. Confocal imaging experiments showed that CICR facilitated propagation of the Ca2+ signal from the plasmalemma to the nucleus. This all-or-none reaction may serve as a switch that determines whether a given electrical signal will be transduced into a local or widespread increase in [Ca2+]i.
Journal of Neuroscience 11/1997; 17(19):7404-14. · 7.11 Impact Factor
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ABSTRACT: The free intracellular Ca2+ concentration ([Ca2+]i) is governed by the balance between the activation of Ca2+ channels and buffering and efflux processes. We tested the hypothesis that Ca2+ efflux pathways are susceptible to modulation. The whole-cell patch-clamp technique was used in combination with Indo-1-based microfluorometry to record Ca2+ current and [Ca2+]i simultaneously from single rat dorsal root ganglion (DRG) neurons grown in culture. Depolarizing test pulses (-80 to 0 mV, 100-300 msec) elicited [Ca2+]i transients that recovered to basal levels by a process best-fit with a single exponential (tau = 5.1 +/- 0.4 sec; n = 14) and were independent of Ca2+ load (40-500 pC) over this range of test pulses. [Ca2+]i transients recorded in whole-cell configuration were similar to those elicited by a brief train of action potentials in unclamped neurons. Inhibition of Ca2+ sequestration into intracellular stores with thapsigargin had no effect on the kinetics of recovery. Inhibition of plasma membrane Ca2+ ATPase (PMCA) function by including a peptide inhibitor (C28R2) in the patch pipette significantly slowed recovery to basal [Ca2+]i (tau = 9.9 +/- 0.8 sec; n = 4). Preincubation with calmidazolium, a calmodulin antagonist, produced modest slowing of Ca2+ efflux. Phorbol dibutyrate, an activator of protein kinase C (PKC), accelerated Ca2+ efflux only when the PMCA had been inhibited by C28R2. We conclude that in DRG neurons PMCAs are responsible for lowering [Ca2+]i after small Ca2+ loads and that PMCA-mediated Ca2+ efflux is modulated by calmodulin- and PKC-signaling pathways.
Journal of Neuroscience 03/1996; 16(3):1008-15. · 7.11 Impact Factor
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ABSTRACT: 1. Optical fibres were used to excite and record fluorescence from the lumenal face of rat aorta or tail artery loaded with fura-2. 2. Acetylcholine (ACh) evoked an endothelium-dependent rise in the fura-2 340/380 nm excitation ratio in both vessels. High [K+] or phenylephrine evoked an endothelium-independent rise in ratio in tail artery but failed to increase the ratio in aorta. These observations indicate that fura-2 fluorescence and therefore cytosolic calcium concentration ([Ca2+]i) may be selectively recorded from the endothelium of intact rat aorta. 3. In aortic endothelium, resting [Ca2+]i was 95 +/- 8 nM (n = 44). ACh evoked a monophasic rise in [Ca2+]i which was temporally coincident with a membrane hyperpolarization. 4. ATP in most (22/35) preparations evoked a rise in [Ca2+]i which declined towards resting and was followed by a secondary rise. The biphasic [Ca2+]i responses were accompanied by biphasic electrical responses of initial hyperpolarization followed by depolarization above the resting potential and subsequent restoration towards rest. In the presence of high [K+] or the K+ ionophore valinomycin, ATP did not evoke changes in membrane potential and only monophasic rises in [Ca2+]i were observed. In some (7/35) preparations, ATP evoked oscillations in [Ca2+]i, with membrane potential oscillating in antiphase. 5. These data suggest interplay between [Ca2+]i and membrane potential in the generation of agonist-evoked responses in native endothelium in situ. The observed oscillations in [Ca2+]i imply spatio-temporal synchronization of Ca2+ signalling in large groups of endothelial cells in intact rat aorta.
The Journal of Physiology 01/1996; 489 ( Pt 2):309-17. · 4.72 Impact Factor
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ABSTRACT: The tetrodotoxin-resistant (TTX-R) voltage-gated Na+ channels Nav1.8 and Nav1.9 are expressed by a subset of primary sensory neurons and have been implicated in various pain states. Although recent studies suggest involvement of TTX-R Na+ channels in sensory synaptic transmission and spinal pain processing, it remains unknown whether TTX-R Na+ channels are expressed and function presynaptically. We examined expression of TTX-R channels at sensory synapses formed between rat dorsal root ganglion (DRG) and spinal cord (SC) neurons in a DRG/SC co-culture system. Immunostaining showed extensive labeling of presynaptic axonal boutons with Nav1.8- and Nav1.9-specific antibodies. Measurements using the fluorescent Na+ indicator SBFI demonstrated action potential–induced presynaptic Na+ entry that was resistant to tetrodotoxin (TTX) but was blocked by lidocaine. Furthermore, presynaptic [Ca2+]i elevation in response to a single action potential was not affected by TTX in TTX-resistant DRG neurons. Finally, glutamatergic synaptic transmission was not inhibited by TTX in more than 50% of synaptic pairs examined; subsequent treatment with lidocaine completely blocked these TTX-resistant excitatory postsynaptic currents. Taken together, these results provide evidence for presynaptic expression of functional TTX-R Na+ channels that may be important for shaping presynaptic action potentials and regulating transmitter release at the first sensory synapse.
Neuroscience.
