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J Physiol (2003), 547.2, pp. 417-425
© Copyright 2003 The Physiological Society
DOI: 10.1113/jphysiol.2002.034561
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ABSTRACT |
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Striatal neurones are particularly vulnerable to hypoxia/ischaemia-induced damage, and free radicals are thought to be prime mediators of this neuronal destruction. It has been shown that hydrogen peroxide (H2O2), through the production of free radicals, induces rat insulinoma cell death by activation of a non-selective cation channel, which leads to irreversible cell depolarization and unregulated Ca2+ entry into the cell. In the study presented here, we demonstrate that a subpopulation of striatal neurones (medium spiny neurones) is depolarized by H2O2 through the production of free radicals. Cell-attached recordings from rat cultured striatal neurones demonstrate that exposure to H2O2 opens a large-conductance channel that is characterized by extremely long open times (seconds). Inside-out recordings show that cytoplasmically applied-nicotinamide adenine dinucleotide activates a channel with little voltage dependence, a linear current-voltage relationship and a single-channel conductance of between 70 and 90 pS. This channel is permeable to Na+, K+ and Ca2+ ions. Fura-2 imaging from cultured striatal neurones reveals that H2O2 exposure induces a biphasic intracellular Ca2+ increase in a subpopulation of neurones, the second, later phase resulting in Ca2+ overload. This later component of the Ca2+ response is dependent on the presence of extracellular Ca2+ and is independent of synaptic activity or voltage-gated Ca2+ channel opening. Consequently, this channel may be an important contributor to free radical-induced selective striatal neurone destruction. These results are remarkably similar to those observed for insulinoma cells and suggest that this family of non-selective cation channels has a widespread distribution in mammalian tissues.
(Received 22 October 2002; accepted after revision 12 December 2002; first published online 17 January 2003)
Corresponding author M. L. J. Ashford: Department of Pharmacology and Neuroscience, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, UK. Email: m.l.j.ashford{at}dundee.ac.uk
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INTRODUCTION |
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Oxygen-derived free radical formation is implicated in the aetiology of neurodegenerative disease (Simonian & Coyle, 1996) and in neuronal death after acute injury (Traystman et al. 1991). The superoxide anion can react with either nitric oxide to form peroxynitrite anions or with dismutate to form hydrogen peroxide (H2O2), a reaction that is catalysed by superoxide dismutase. H2O2 in turn exerts its toxic effects through either the ferrous ion-dependent (Fenton reaction) or superoxide-driven (Haber-Weiss reaction) formation of the highly reactive hydroxyl radical, which leads to alteration of lipids, proteins and DNA, and a change in the redox status of the cytosol (Halliwell, 1992).
The striatum is an area of the brain that is particularly vulnerable to hypoxia/ischaemia-induced damage, as evidenced by the large number of movement disorders that manifest themselves clinically following such insults (Hawker & Lang, 1990). This damage may arise from the generation of free radicals (Hyslop et al. 1995), as free radical scavengers and agents capable of lowering H2O2 are neuroprotective within the striatum (Desagher et al. 1997; Yu et al. 1998). In addition to their effects in acute injury, free radicals have also been implicated in the pathology of neurodegenerative diseases of the striatum such as Huntington's disease (Beal, 1995).
Substantial evidence exists to support the cytotoxic effect of H2O2 upon neurones, and many studies have been performed describing the chronic effects of free radical generation on cell processing. Less is known, however, about the acute effects of free radicals on cell physiology and the primary mechanism causing cell death. Recently, the cytotoxic effects of free radicals on the pancreatic cell have been ascribed to the activation of a novel non-selective cation channel, which results in membrane potential collapse, unregulated Ca2+ entry and cell death (Herson & Ashford, 1997; Herson et al. 1999). Thus, as striatal neurones are also extremely sensitive to free radical-induced damage, we have examined striatal slices and cultured neurones for evidence of this channel following H2O2 challenge.
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METHODS |
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Preparation of rat striatal slices
All procedures used conformed to the UK Animals (Scientific Procedures) Act 1986. Sprague-Dawley rats (7-8 weeks old), weighing less than 500 g, were killed by cervical dislocation. Following decapitation, the brains were removed and 300 µm coronal slices containing the striatum were prepared as described previously (Lee et al. 1998).
Preparation of rat striatal cultures
Tissue culture plates (35 mm diameter; Life Technologies, Paisley, UK) were coated with 0.003 % (v/v) poly-L-ornithine (Sigma Chemical Company, Poole, UK) for 2 h and washed two times with sterile water, prior to the preparation of the striatal neurones. Pregnant Sprague Dawley rats were killed by CO2 inhalation on day 16 of gestation. The striatum was dissected from the embryos and incubated in 10 ml of Puck's saline (Life Technologies) containing 10 µg ml-1 penicillin/streptomycin, 250 µg ml-1 trypsin (type II-S) and 10 mM Hepes buffer at 37 °C for 20 min. Trypsin digestion was inhibited by application of 100 µg ml-1 trypsin inhibitor (type II-S) and the tissue was dissociated by gentle trituration with a glass Pasteur pipette. The cells were plated on the ornithine-coated culture plates at a density of 0.3 
106 cells ml-1 in Modified Eagle's Medium (Life Technologies) containing 5 % (v/v) fetal calf serum, 5 % (v/v) horse serum, 2 mM L-glutamine, 5 µg ml-1 insulin, 50 pg ml-1 fibroblast growth factor, 10 µg ml-1 penicillin/streptomycin and 5 mM Hepes buffer. Cells were grown in a humidified atmosphere in 5 % CO2 at 37 °C for 24 h prior to replacement of the medium with neurobasal medium supplemented with 1 % (v/v) N2-supplement, 2 mM L-glutamine and 10 µg ml-1 penicillin/streptomycin.
Electrophysiological recording and analysis
Recording electrodes were pulled from borosilicate glass capillaries and, when filled with electrolyte, had resistances of 5-10 M
for isolated patch experiments and 2-5 M for whole-cell current-clamp recordings. Electrophysiological signals were detected using an Axopatch 200A or List EPC-7 patch-clamp amplifier. Data were recorded onto digital audiotape for later analysis, and replayed for illustration onto a Gould TA 240 chart recorder. During current-clamp experiments, hyperpolarizing current pulses (50-100 pA and 300 ms duration) were applied every 10 s to monitor changes in input resistance. Single-channel data were analysed for current amplitude (I) and average channel activity (NfPo), where Nf is the number of functional channels and Po, the open state probability, as described previously (Lee et al. 1994). Striatal slices were maintained in physiological saline that contained (mM): 125 NaCl, 25 NaHCO3, 10 glucose, 2.5 KCl, 1.25 NaH2PO4, 2 CaCl2 and 1 MgCl2 and was bubbled with a 95 % O2/5 % CO2 gas mixture. For inside-out and cell-attached experiments, the pipette solution contained normal saline (mM): 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes, pH 7.4, whilst for whole-cell current-clamp recordings the pipette solution contained (mM): 120 potassium gluconate, 10.0 NaCl, 2.0 MgCl2, 0.5 K2EGTA, 10.0 Hepes, 4.0 Na2ATP, 0.1 Na2GTP, pH 7.4. The bath solution for whole-cell and cell-attached recordings from cultured neurones was normal saline, and for inside-out recordings was (mM): 140 NaCl, 0.6 MgCl2, 5.2 CaCl2, 5 EGTA, 10 Hepes, pH 7.2, (free Ca2+ of 200 µM). In experiments to determine cation selectivity, the electrode solution was replaced with (mM): 110 CaCl2 10 Tris-HCl, pH 7.2 to examine Ca2+ permeability, or 140 KCl, 0.6 MgCl2, 5.2 CaCl2, 5 EGTA, 10 Hepes, pH 7.2 to examine K+ permeability. To investigate internal Ca2+ dependence, the standard bath solution was replaced with (mM): 140 NaCl, 0.6 MgCl2, 5 EGTA, 10 Hepes, pH 7.2, (free Ca2+ < 50 nM). The potential across the membrane is described following the usual sign convention for membrane potential (i.e. inside negative). -Nicotinamide adenine dinucleotide (-NAD+) stocks were dissolved in 10 mM Hepes, pH 7.2 and stored at -20 °C; reduced glutathione (GSH) stocks were dissolved daily in the standard bath solution and stored at 4 °C. All reagents were purchased from Sigma (UK) and were added at the appropriate concentration to the bath solution by a gravity flow superfusion system, unless stated otherwise. All data in the text are presented as mean ± S.E.M.
Imaging [Ca2+]i of single striatal neurones
Ca2+ imaging experiments were performed on rat cultured striatal neurones that had been in culture for between 5 and 14 days. After washing with normal saline supplemented with 10 mM glucose, the striatal cultures were incubated with 2 µM Fura-2/AM for 45-120 min at room temperature (22-25 °C). Measurements of [Ca2+]i in individual neurones were made from the fluorescence ratio (excitation at 340/380 nm and emission > 510 nm for Fura-2) using a filter wheel assembly, incorporating a CCD camera and a photomultiplier. The MAGICAL (Applied Imaging, Sunderland, UK) suite of software was used to sample emissions following excitation at 340 and 380 nm wavelengths at 15 s intervals, and the [Ca2+]i calculated as described previously (Herson et al. 1999). All intact cell imaging was performed on at least three separate cultures and numbers quoted (n) represent the number of individual cells analysed.
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RESULTS |
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Whole-cell recordings
Recordings were made predominantly from a population of striatal slice neurones, which, in the presence of 10 mM glucose, were silent at rest and had a mean resting membrane potential and input resistance of -72.4 ± 4.3 mV and 87.8 ± 6.5 M (n = 16), respectively. These parameters were stable for as long as recordings were maintained under control conditions (Fig. 1A(i)). These properties are consistent with those reported for medium-sized spiny neurones (Calabresi et al. 1995, 1997). High, non-physiological concentrations of H2O2 were used to challenge neurones. This was necessary in order to shorten the time for the terminal pathological events to occur (Herson & Ashford, 1997) and thus allow electrophysiological examination of the acute consequences of the oxidative stress. At a concentration of 10 mM, H2O2 (in the presence of 1 mM sodium azide (NaN3) to prevent H2O2 breakdown by endogenous catalase) caused, after a short period (5-30 min), a complete and irreversible collapse of the membrane potential (n = 5/8), which was associated with an increase in cellular conductance (Fig. 1A(ii)). This depolarization was unaffected by the addition of a cocktail of agents (1 µM TTX, 50 µM DL-2-amino-5-phosphonovalerate (D-APV), 5 µM 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX), 10 µM bicuculline) designed to prevent excitatory and inhibitory synaptic transmission (n = 4). Concomitant with the irreversible depolarization of these neurones were distinct morphological changes, which embodied membrane shrivelling and blebbing, consistent with cellular destruction (data not shown). These effects were not due to the presence of NaN3, as this compound induced no depolarization per se (n = 3; data not shown). The free radical scavenger, dimethylthiourea (DMTU), has been demonstrated previously to protect neurones from the effects of forebrain ischaemia (Pahlmark et al. 1993). Striatal neurone slices pretreated with 1 mM DMTU did not display the H2O2-induced irreversible depolarization or increased conductance described above (n = 4; Fig. 1A(iii)) in recordings for up to 1 h following the challenge with H2O2. Similar results were obtained in recordings from cultured striatal neurones (data not shown). These data mirror the effects of H2O2 on cell membrane potential and conductance reported for the insulin-secreting cell line, CRI-G1 (Herson & Ashford, 1997).
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Figure 1. The effect of hydrogen peroxide (H2O2) on the resting electrical properties of striatal neurones A(i), recording illustrating that the resting membrane properties of visually identified medium-sized spiny neurones, recorded from striatal slices, are stable. A(ii), current-clamp recording illustrating that application of 10 mM H2O2 (vertical arrow) caused decreased input resistance and slow depolarization followed by sudden and irreversible depolarization. A(iii), pre-application of the free-radical scavenger dimethylthiourea (DMTU, 1 mM) prevented the H2O2-induced depolarization. B, induction of single-channel currents by H2O2. Representative continuous cell-attached recording (no applied pipette potential) illustrating the spontaneous, sudden appearance of an inward current, approximately 30 min following exposure of the neurone to 45 mM H2O2 + 5 mM sodium azide. The current appears abruptly and expansion of the initial part of the current trace illustrates the rapid recruitment of six channels, giving rise to a sustained inward current. Note the large amplitude of these channel events and the slow kinetics, exemplified by the long, stable open durations. | ||
Cell-attached recordings
In order to identify, unequivocally, the nature of the ionic conductance underlying the H2O2-induced depolarization, cell-attached single-channel recordings from cultured striatal neurones were performed. Recordings from multipolar striatal neurones, demonstrated a sudden, spontaneous induction of inward current (11.8 ± 6.8 pA, n = 4) following ~ 30 min of exposure to H2O2 (Fig. 1B), which was associated with complete destruction of the neurones soon afterwards. Expansion of the time base demonstrates discrete step-like currents, indicating rapid recruitment of individual channels (mean amplitude, 1.8 ± 0.3 pA) characterized by prolonged openings. Cell-attached recordings from bipolar neurones displayed no spontaneous inward current (not shown) or obvious morphological changes associated with cellular destruction, at a time when the multipolar neurones had clearly succumbed to the H2O2-induced oxidative stress. These morphological characteristics are consistent with spiny striatal and large aspiny cholinergic interneurones, respectively (Calabresi et al. 1997).
As the appearance of this inward current appeared to be directly associated with the destruction of the neurone, we surmised that it also mediated the spontaneous depolarization described above. In insulin-secreting cells, H2O2 or alloxan, through the generation of free radicals, causes irreversible depolarization and consequent cell destruction by opening of a novel channel (Herson & Ashford, 1997). This channel (termed NSNAD) is non-selective for cations and in isolated patches is activated by -NAD+ applied to the intracellular membrane aspect (Reale et al. 1994; Herson et al. 1997). Therefore we examined cultured striatal neurones for evidence of this unique channel.
Inside-out recordings
Inside-out patches were excised from cultured striatal neurones into normal saline (i.e. symmetrical NaCl) containing 0.5 mM NAD+. Approximately 20 % of patches excised from multipolar striatal neurones contained channels that were activated by bath-applied 0.5 mM NAD+. Inside-out patches from bipolar neurones did not exhibit this channel. The -NAD+-activated channel was characterized by extremely long open times (seconds), a single-channel conductance of 73.3 ± 1.6 pS (n = 5), a reversal potential of -0.2 ± 0.3 mV (n = 5) and a linear current-voltage relationship, with no obvious voltage dependence of channel activity (Fig. 2A). The NSNAD channel in CRI-G1 insulin-secreting cells does not discriminate between Na+ and K+ ions and demonstrates a significant permeability to Ca2+ ions (Reale et al. 1994: Herson et al. 1997). Consequently, we examined the ion selectivity of the NAD+-activated channel in inside-out patches from cultured striatal neurones by replacement of the electrode solution (140 mM NaCl) with one containing 140 mM KCl or 110 mM CaCl2, maintaining 140 mM NaCl in the bathing solution. In experiments using KCl (Fig. 2B), the current-voltage relationship remained linear, reversed at -0.1 ± 1.2 mV (n = 7), and exhibited a single-channel conductance that was significantly (P < 0.001) increased to 90.3 ± 1.7 pS (n = 7). Using 110 mM CaCl2 as the electrode solution (Fig. 2C), the currents had characteristic slow kinetics, a linear current-voltage relationship, reversal at -2.3 ± 1.3 mV (n = 4) and a conductance of 35.8 ± 4.0 pS (n = 4).
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Figure 2. Single-channel characteristics of the NSNAD channel Recordings from inside-out patches at different membrane potentials (given at the side of the traces) in symmetrical 140 mM NaCl (A), a bath solution of 140 mM NaCl and electrode solution containing 140 mM KCl (B) and a bath solution of 140 mM NaCl with an electrode solution containing 110 mM CaCl2 (C). On the right of each set of traces are the corresponding single-channel current (I)-voltage (V) relationships. The straight lines show the best fit, with slope conductances of 74.9 pS (A), 89.2 pS (B) and 38.4 pS (C), and no obvious rectification. The currents were recorded with 0.2 mM free internal Ca2+ and 0.5 mM | ||
The activity of the NSNAD channel in CRI-G1 insulin-secreting cells depends on the presence of both - NAD+ and Ca2+ at the cytoplasmic side of the membrane (Herson et al. 1997). This is also true for the non-selective cation channel present in striatal neurones. In the presence of intracellular Ca2+, channel activity is not sustained on removal of -NAD+, but is recovered on re-application (Fig. 3A). The mean channel activity (NfPo) in 0.5 mM -NAD+ and 0.2 mM Ca2+ was 6.11 ± 2.26, which decreased to 0.002 ± 0.0001 and returned to 2.68 ± 2.08 (n = 5) on removal and re-addition of the -NAD+, respectively. Similarly, buffering Ca2+ to nanomolar levels in the presence of 0.5 mM -NAD+, caused complete and reversible cessation of channel activity (Fig. 3B). The corresponding values of NfPo were 10.73 ± 2.24, 0.003 ± 0.0001 and 2.00 ± 0.45 (n = 5) for the presence, absence and re-application of Ca2+, respectively. The decline in the mean current is probably due to channel rundown, invariably observed for the NSNAD channel in CRI-G1 cells following formation of the isolated patch configuration (Herson et al. 1997).
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Figure 3. Dependence of the NSNAD channel on Ca2+ and Removal of | ||
These data demonstrate that the striatal neurone cation channel has the essential characteristics of its counterpart in insulin-secreting cells (i.e. equal permeability to the main monovalent physiological cations, a higher conductance in K+ ions and significant Ca2+ permeability; Herson et al. 1997). The NSNAD channel of CRI-G1 cells (Herson & Ashford, 1999) is completely inhibited by 10 mM reduced GSH. The striatal neurone cation channel, however, is insensitive to GSH, with mean channel activity (in 0.5 mM -NAD+) of 2.76 ± 1.88, 1.73 ± 0.08 and 1.62 ± 0.63 (n = 3) in control conditions, and in the presence and after washout of 10 mM GSH (P > 0.4), respectively (data not shown).
Ca2+ imaging of neurones
The destruction of CRI-G1 cells induced by H2O2 through the production of free radicals, is correlated with changes in [Ca2+]i levels, leading to Ca2+ overload (Herson et al. 1999). Consequently, we examined the effects of H2O2 on [Ca2+]i in cultured striatal neurones. H2O2 caused a biphasic rise in [Ca2+]i; within 3 min the Fura-2 ratio (340/380) increased from 0.45 ± 0.01 (~ 59 nM [Ca2+]i) to 0.91 ± 0.02 (184 nM [Ca2+]i; n = 42) and 20-45 min later the Fura-2 ratio increased to near dye saturation (> 1 µM; Fig. 4A). In the absence of extracellular Ca2+, H2O2 caused an initial rise in [Ca2+]i within 3 min, the Fura-2 ratio increasing from 0.46 ± 0.02 (61 nM [Ca2+]i) to 0.87 ± 0.01 (171 nM [Ca2+]i; n = 54). This increased level of [Ca2+]i was maintained for as long as the extracellular Ca2+ was absent with no second rise in [Ca2+]i observed. Re-addition of extracellular Ca2+, however, resulted in an immediate and synchronous increased [Ca2+]i (Fig. 4B).
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Figure 4. H2O2 causes a biphasic rise in [Ca2+]i A, representative imaging experiment, from three neurones (also B and C), illustrating the biphasic [Ca2+]i response caused by 10 mM H2O2. The initial rise occurred within 3 min, and a second, larger rise (> 1 µM) after 20-45 min. B, a separate experiment where addition of 10 mM H2O2 in the absence of extracellular Ca2+ caused increased resting [Ca2+]i, which was sustained until re-addition of extracellular Ca2+, which resulted in an immediate [Ca2+]i increase (> 1 µM). Note that the time of exposure to H2O2 in the absence of extracellular Ca2+ was longer than the time taken to reach a near-maximal response in A. C, identical experiment to B, except that prior to re-application of extracellular Ca2+, a cocktail of blockers (1 µM TTX, 10 µM NBQX, 50 µM D-APV and 100 µM Cd2+), was applied; this did not prevent Ca2+ entry on addition of extracellular Ca2+. | ||
In separate experiments, H2O2 was applied in the absence of external Ca2+ and the Fura-2 ratio increased from 0.42 ± 0.01 (52 nM [Ca2+]i) to 0.84 ± 0.02 (161 nM [Ca2+]i; n = 32) within 3 min (Fig. 4C). After 90 min, during which [Ca2+]i was unchanged, a cocktail of blockers (1 µM TTX, 10 µM NBQX, 50 µM D-APV and 100 µM Cd2+) designed to inhibit other Ca2+ permeation pathways was applied. This did not affect [Ca2+]i (the Fura-2 ratio was 0.84 ± 0.02). The subsequent addition of extracellular Ca2+ induced a rapid rise in the Fura-2 ratio (Fig. 4C). These results indicate that the H2O2-induced early increase in [Ca2+]i is caused by Ca2+ mobilization from an intracellular source and that the second, large rise in [Ca2+]i is due to extracellular Ca2+ influx. These data are in remarkable consonance with those of insulin-secreting cells and lead us to conclude that the most likely explanation for the second phase of increased Ca2+ is NSNAD channel opening.
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DISCUSSION |
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Hypoxia/ischaemia-induced neuronal cell death is prevalent in the striatum and is particularly associated with neuropathology of various movement disorders (Hawker & Lang, 1990). There is evidence that this neuronal damage arises from the production of free radicals (Hyslop et al. 1995; Lipton, 1999), most notably following the generation of H2O2 (Desagher et al. 1998; Yu et al. 1998). A currently favoured mechanism for neuronal cell death during oxidative stress is through the excessive release of glutamate and prolonged glutamate receptor stimulation producing postsynaptic [Ca2+]i overload (Choi & Rothman, 1990; Lipton, 1999). In the study reported here, we have demonstrated an alternative mechanism, also involving an increased cellular conductance and increased [Ca2+]i, which acts independently of changes in synaptic transmission and release of transmitter.
Under conditions where synaptic transmission was attenuated pharmacologically, in a subpopulation of neurones, application of H2O2 to striatal slices induced an irreversible depolarization, concomitant with an increased input conductance. These events were also associated with membrane blebbing and shrivelling of the recorded neurone, consistent with cell necrosis. Complete inhibition of these H2O2-driven events by the presence of DMTU, a free radical scavenger, attests to their crucial role. Consonant with the brain slice data, extracellular application of H2O2 rapidly depolarized a subpopulation of cultured striatal neurones over a similar time course. A more detailed analysis of the actions of H2O2 was performed on cultured neurones as this also allowed us to examine the changes in Ca2+ homeostasis associated with oxidative stress and free radical induction. Cell-attached recordings from cultured neurones demonstrated that H2O2, albeit at a very high concentration, induced the appearance of a spontaneous inward current after a short time delay, consistent with the increased conductance associated with irreversible depolarization. The appearance of this current coincided with cellular destruction, as evidenced by membrane blebbing. Resolution of the single-channel events associated with this inward current showed that it consisted of the stepwise recruitment of individual channel openings of prolonged open duration (seconds). As observed in the slice experiments, only a proportion of cultured striatal neurones was susceptible to the effects of H2O2. The morphology (approximately 20 µm in diameter with extensive dendritic tree) and electrophysiological characteristics of the affected neurones (high membrane potential, low input resistance and lack of firing at rest) are consistent with them being medium-sized spiny neurones (Calabresi et al. 1997; Lee et al. 1998).
At the concentrations of H2O2 used in this study, the electrical events described above and the morphological changes occurred rapidly, usually 
30 min following the initiation of the oxidative stress. These concentrations are approximately 100 times greater than that reported to be produced in the striatum during forebrain ischaemia (Hyslop et al. 1995). At lower, more physiological concentrations of H2O2 ( 100 µM), these events were not observed during the time frame of the electrophysiological recordings. In parallel studies using the insulin-secreting cell line, CRI-G1, lower concentrations of H2O2 (100-200 µM) were shown to induce the effects described herein, but over a more prolonged time (> 2 h). However, application of 100 µM FeCl2, concomitant with 100 µM H2O2, induced irreversible depolarization and increased conductance of CRI-G1 cells within 20 min (unpublished observations). Thus, it is probable that the activity of free radicals (determined by the levels of free ion or other catalytically active transition metal ion) governs the time course of the response rather than the absolute concentration of H2O2 (Emerit et al. 2001).
Studies on the CRI-G1 insulin-secreting cell line have shown that a non-selective cation channel underlies free radical-induced depolarization and consequent cellular destruction (Herson & Ashford, 1997). This channel (NSNAD) has a unique biophysical profile, with large single-channel conductance (
70 pS) for monovalent cations, significant divalent cation permeability, extremely slow kinetics (in the order of seconds), and a requirement for the presence of -NAD+ at the cytoplasmic aspect for activity in isolated patches (Herson et al. 1997). Inside-out patch studies conducted on cultured striatal spiny neurones have demonstrated the presence of a channel with almost identical characteristics. The neuronal channel displays essentially identical conductance values and relationships for Na+/Na+ (73 vs. 74 pS) and K+/Na+ (90 vs. 88 pS), and similar permeability to Ca2+ in comparison to CRI-G1 cells. In addition, channel activity in inside-out patches requires the presence of internal -NAD+ and micromolar concentrations of Ca2+; removal of -NAD+ in the continued presence of 0.2 mM Ca2+, or reduction of Ca2+ to < 50 nM in the presence of 0.5 mM -NAD+, could not sustain activity. The neuronal channel also displays the most striking feature of NSNAD, namely the slow kinetic behaviour that is characterized by extremely long and stable open durations. These features suggest strongly that the striatal channel is a member of the NSNAD channel family. However, one notable difference between the neuronal channel and CRI-G1 cells is the lack of inhibition of channel activity by internally applied GSH. At present, it is unclear why this should be the case. Possible reasons are: variability in molecular composition or protein partners, differential post-translational modification or different second-messenger systems may alter channel sensitivity to intracellular factors such as GSH. Whatever the mechanism responsible for this change, it may have the effect of making this channel more susceptible to oxidative stress and hence more prone to cell death under ischaemic conditions (Wallin et al. 2000).
Numerous studies have indicated that ischaemia-induced neuronal cell death is associated with episodes of [Ca2+]i overload (Choi, 1995; Kristián & Siesjö, 1998). In addition, CNS neurones that are subjected to ischaemia/hypoxia have demonstrated a causal relationship between free radical production and Ca2+-mediated neuronal death (Kristián & Siesjö, 1998; Lipton, 1999). A crucial question relates to the sources of this Ca2+. Undoubtedly, a significant contribution is derived from internal stores, but much of the ultimate insult associated with neuronal necrosis results from a massive and relatively sudden influx of Ca2+ (Kristián & Siesjö, 1998). It has been argued that such Ca2+ entry during the final phase of oxidative stress in insulin-secreting cells arises from Ca2+ conducted through activated NSNAD channels (Herson et al. 1999). Fura-2 monitoring of [Ca2+]i in cultured striatal neurones exposed to 10 mM H2O2 has revealed a temporal profile of increased [Ca2+]i remarkably similar to that reported for CRI-G1 insulin-secreting cells (Herson et al. 1999). In the presence of external Ca2+, H2O2 induced a biphasic increase in [Ca2+]i. However, in the absence of external Ca2+, H2O2 induced only the first-phase [Ca2+]i increase, with no second phase until external Ca2+ was re-applied. These data indicate that striatal neurones, like CRI-G1 cells, respond to an acute exposure to H2O2 by a rapid and sustained mobilization of internal Ca2+, followed by a later influx of external Ca2+, the latter occurring concomitantly with morphological indications of neuronal cell death. The presence of pharmacological agents designed to inhibit synaptic transmission did not prevent either phase of the H2O2-induced increase in [Ca2+]i, suggesting little or no involvement of oxidative-stress-induced glutamate release. The lack of effect of 100 µM Cd2+ on these [Ca2+]i changes also indicates little contribution from voltage-gated Ca2+ channels. Consequently, we suggest that the later influx of Ca2+ that is associated with cellular destruction occurs through free radical-induced opening of the striatal equivalent of the NSNAD channel. It is plausible that such a Ca2+ entry pathway may also contribute to the delayed neuronal death that is associated with transient exposure to NMDA (Chen et al. 1997).
Striatal spiny neurones are selectively vulnerable to Huntington's disease and hypoxia/ischaemia, whereas the larger aspiny neurones are spared from these pathological conditions (Mitchell et al. 1999; Sieradzan & Mann, 2001). This differential neuronal vulnerability is associated with membrane depolarization, increased [Ca2+]i and increased [Na+]i (Calabresi et al. 1997, 1999; Pisani et al. 1997). Electrophysiological analyses of these neurones demonstrate that the irreversible depolarization associated with ischaemia (> 10 min), is due to the appearance of an inward current that is carried by Na+ and Ca2+ and is resistant to the presence of TTX and glutamate receptor antagonists (Calabresi et al. 1999). Thus it is feasible that the selective vulnerability of spiny striatal neurones to ischaemia is due to the opening of the NSNAD channel described above.
Recently, members of a class of proteins, termed the transient receptor potential (TRP) family of channels have been shown to function as Ca2+ influx channels (Vennekens et al. 2002). One member of the TRP melastatin group, TRPM2 (also known as LTRPC2) is a likely candidate for the NSNAD channel. Following heterologous expression in human embryonic kidney 293 cells, electrophysiological studies have demonstrated that this channel has many of the attributes of the NSNAD channel described above and in insulin-secreting cells (Herson & Ashford, 1997; Herson et al. 1997, 1999). The TRPM2 channel functions as a Ca2+-permeable non-selective cation channel that is activated by intracellular -NAD+, and which endows intact cells with sensitivity to H2O2, resulting in Ca2+ and Na+ entry (Sano et al. 2001; Hara et al. 2002; Wehage et al. 2002). TRPM2 has also recently been reported to be expressed in human islets (Qian et al. 2002). In summary, this striatal neurone non-selective cation channel, which is activated by the overproduction of free radicals, may be an important contributor to neuronal cell death and may represent a novel target for strategies that prevent oxidant stress-induced neuronal destruction.
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REFERENCES |
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Acknowledgements
This work was supported by the Wellcome Trust (Grant no. 042726). M.A.S. was a Wellcome Trust Prize Student.
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