H2O2 Is a Novel, Endogenous Modulator of Synaptic Dopamine Release

Billy T. Chen, Marat V. Avshalumov, and Margaret E. Rice

Departments of Physiology and Neuroscience and Neurosurgery, New York University School of Medicine, New York, New York 10016


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chen, Billy T., Marat V. Avshalumov, and Margaret E. Rice. H2O2 Is a Novel, Endogenous Modulator of Synaptic Dopamine Release. J. Neurophysiol. 85: 2468-2476, 2001. Recent evidence suggests that reactive oxygen species (ROS) might act as modulators of neuronal processes, including synaptic transmission. Here we report that synaptic dopamine (DA) release can be modulated by an endogenous ROS, H2O2. Electrically stimulated DA release was monitored in guinea pig striatal slices using carbon-fiber microelectrodes with fast-scan cyclic voltammetry. Exogenously applied H2O2 reversibly inhibited evoked release in the presence of 1.5 mM Ca2+. The effectiveness of exogenous H2O2, however, was abolished or decreased by conditions that enhance Ca2+ entry, including increased extracellular Ca2+ concentration ([Ca2+]o; to 2.4 mM), brief, high-frequency stimulation, and blockade of inhibitory D2 autoreceptors. To test whether DA release could be modulated by endogenous H2O2, release was evoked in the presence of the H2O2-scavenging enzyme, catalase. In the presence of catalase, evoked [DA]o was 60% higher than after catalase washout, demonstrating that endogenously generated H2O2 can also inhibit DA release. Importantly, the Ca2+ dependence of the catalase-mediated effect was opposite to that of H2O2: catalase had a greater enhancing effect in 2.4 mM Ca2+ than in 1.5 mM, consistent with enhanced H2O2 generation in higher [Ca2+]o. Together these data suggest that H2O2 production is Ca2+ dependent and that the inhibitory mechanism can be saturated, thus preventing further effects from exogenous H2O2. These findings show for the first time that endogenous H2O2 can modulate vesicular neurotransmitter release, thus revealing an important new signaling role for ROS in synaptic transmission.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Production of reactive oxygen species (ROS) is often considered to be a potentially harmful side effect of cellular respiration. In particular, superoxide and H2O2 have been linked causally to neurodegenerative states, including Parkinson's disease, which involves degeneration of dopaminergic pathways (Cohen et al. 1997; Ebadi et al. 1996; Olanow and Tatton 1999). Under normal physiological conditions, however, ROS are tightly regulated and can serve as cellular messengers (Topper et al. 1996; Wung et al. 1999). Indeed, ROS have recently been implicated in a variety of redox-based signaling mechanisms that can mediate changes in neural plasticity, including activation of oxidative stress-responsive transcription factors (Suzuki et al. 1997), modulation of LTP induction (Auerbach and Segal 1997; Klann et al. 1998), and mediation of nonsynaptic communication between neurons and glia (Atkins and Sweatt 1999). In addition, ROS have been implicated as modulators of synaptic transmission, following demonstration that H2O2 can reversibly depress evoked population spikes in slices of guinea pig hippocampus (Pellmar 1986, 1995).

The site of action of H2O2 on synaptic transmission in the hippocampus appears to be presynaptic because neither population spikes elicited by antidromic stimulation of CA1 pyramidal cells nor EPSPs evoked by exogenous glutamate application are altered by this ROS (Pellmar 1986, 1987). These observations led Pellmar (1987) to suggest that H2O2 might inhibit transmitter release, possibly by inactivation of presynaptic Ca2+ channels and thus decreased Ca2+ entry. Consistent with this hypothesis, previous studies have shown that preincubation with H2O2 can cause a decrease in glutamate release from synaptosomes (Gilman et al. 1992; Zoccarato et al. 1995) and dopamine (DA) release from striatal slices (Joseph et al. 1996) after H2O2 was washed out. In these experiments, however, release was not evaluated in the presence of H2O2 so that decreased release was presumably a consequence of irreversible oxidative damage during H2O2 exposure or washout. Reversible modulation of transmitter release by H2O2 has not been examined previously.

Although glutamate release cannot yet be monitored in real time with available microelectrode technology, electroactive neurotransmitters, including DA, can be detected directly using carbon-fiber microelectrodes with fast-scan cyclic voltammetry (FCV) (Bull et al. 1990; Rice et al. 1997) and other electrochemical techniques. In the present studies, we tested the hypothesis that H2O2 can inhibit transmitter release by determining the effect of this ROS on stimulated release of DA in slices of guinea pig striatum. The striatum is densely innervated with DA axon terminals arising from midbrain DA cell body regions (Smith and Bolam 1990); moreover Ca2+-dependent, vesicular release of DA from striatal synapses is well established (Dunlap et al. 1995; Moghaddam and Bunney 1989). Together, these characteristics make the striatal slice an ideal preparation in which to investigate modulation of synaptic transmitter release. Using an experimental protocol of H2O2 application similar to that used previously (e.g., Pellmar 1995), we first tested the hypothesis that exogenous H2O2 inhibits synaptic transmission by inhibiting transmitter release. Whether DA release was modulated by endogenous H2O2 was then addressed by examining the effect of the specific degradative enzyme for H2O2, catalase (EC 1.11.1.6). Because vesicular transmitter release (Dodge and Rahamimoff 1967; Llinás et al. 1982), as well as mitochondrial respiration and subsequent production of ROS (Dykens 1994; Jung et al. 2000; Kojima et al. 1994), are dependent on extracellular Ca2+ concentration ([Ca2+]o) and Ca2+ entry, we further examined the Ca2+ dependence of the effects of exogenous and endogenous H2O2 on DA release.


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METHODS
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Slice preparation and solutions

Male Hartley guinea pigs (150-250 g) were deeply anesthetized with 40 mg/kg pentobarbital (ip) and decapitated. All animal handling procedures were in accordance with National Institutes of Health guidelines and were approved by the New York University School of Medicine Animal Care and Use Committee. Neostriatal slices, 400-µM thick, were prepared as previously described (Rice et al. 1997). In most experiments, slices were cut in ice-cold artificial cerebrospinal fluid (ACSF), which contained (in mM): 124 NaCl, 3.7 KCl, 26 NaHCO3, 1.5 CaCl2, 1.3 MgSO4, 1.3 KH2PO4, and 10 glucose saturated with 95% O2-5% CO2. Slices were allowed to recover in this normal ACSF for >= 1 h at room temperature before transfer to a submersion recording chamber (Warner Instrument, Hamden, CT). In some experiments, slices were cut and allowed to recover in HEPES-buffered ACSF, containing (in mM): 120 NaCl, 5 KCl, 20 NaHCO3, 6.7 HEPES acid, 3.3 HEPES salt, 2 CaCl2, 2 MgSO4, and 10 glucose saturated with 95% O2-5% CO2, which appeared to improve slice quality. Once in the recording chamber, slices were equilibrated with normal ACSF at 32°C for an additional 30 min before experimentation; superfusion media was ACSF with 1.5 or 2.4 mM Ca2+ as indicated, at a flow rate of 1.2 mL/min. To examine the effects of endogenous H2O2, slices were preincubated for 30 min with 250 IU/ml of catalase in normal ACSF (1.5 mM Ca2+) before transfer to the recording chamber; catalase was also included during the equilibration period in the chamber in 1.5 or 2.4 mM Ca2+ as indicated.

Microelectrodes and voltammetric instrumentation

Carbon-fiber electrodes were made from 7- to 8-µm carbon fibers (type HM, unsized, Courtaulds) that were spark-etched to a 2- to 4-µm tip (MPB Electrodes; Queen Mary and Westfield College, Miles End Road; London, UK). The voltammetric method used for all experiments was FCV. Electrodes were calibrated in the appropriate ACSF in the recording chamber at 32°C (Chen and Rice 1999; Kume-Kick and Rice 1998). Measurements of [DA]o in the presence of H2O2, sulpiride or catalase were calculated from calibration factors determined in the presence of these agents; only catalase consistently altered electrode sensitivity, with a decrease of <20%. Data were obtained using a Millar Voltammeter (PD Systems International, West Molesey, Surrey, UK). Data acquisition was controlled by Clampex 7.0 software (Axon Instruments, Foster City, CA), which imported voltammograms to a 333-MHz Pentium II PC via a DigiData 1200B D/A board (Axon Instruments). Scan rate for FCV was 800 V/s, with a sampling interval of 100 ms controlled by an external timing circuit. Scan rage was -0.7 to +1.3 V (vs. Ag/AgCl). Voltammograms were obtained in two-electrode mode, with an Ag/AgCl wire in the recording chamber as the reference electrode.

Electrical stimulation

Teflon-coated platinum wire bipolar stimulating electrodes (50-µm bare, 75-µm-coated diameter) with tip separation of 50 µm were placed on slice surface as previously described with the carbon-fiber microelectrode positioned between the electrical poles and inserted 50-100 µm below the slice surface (Rice et al. 1997). Two stimulation paradigms were used: normal stimulation with 50 pulses delivered at 10 Hz (Rice et al. 1997) and high-frequency stimulation with 10 pulses, delivered at 100 Hz (Cragg and Greenfield 1997; Singer 1988). In both paradigms, pulse duration was 100 µs with amplitudes of 0.4-0.8 mA.

Experimental design

Whether experiments were conducted using normal or high-frequency stimulation, the interval between stimulation trains was 10 min. Only slices with at least three consistent evoked increases in extracellular DA concentrations ([DA]o) under control conditions were tested further. For experiments with exogenous H2O2, after consistent DA release was confirmed, H2O2 was superfused for 15 min, followed by a 30-min washout period. Illustrated data were obtained 5 min before H2O2 application (control); after 15 min H2O2; and after 30 min washout of H2O2, unless otherwise indicated. To evaluate effects of endogenous H2O2 on evoked [DA]o, slices were preincubated with catalase (Auerbach and Segal 1997), then transferred to the recording chamber. Three consistent evoked [DA]o records were obtained in the presence of catalase, then the enzyme was washed out for 40 min. The three evoked [DA]o records obtained in catalase were averaged and compared with the average of three consistent records taken during washout.

HPLC analysis of DA content in neostriatal slices and in solution

In separate experiments, DA content in striatal tissue was analyzed following 32°C incubation of slices in normal ACSF or ACSF containing H2O2. Incubation chambers were bubbled continuously with 95% O2-5% CO2. Slices were prepared as usual, then incubated for 15 min at 32°C in ACSF or in ACSF plus H2O2. One hemisphere was used as control while the contralateral hemisphere was incubated with H2O2. Following incubation, excess ACSF was carefully removed from the slices, and a sample of striatal tissue (7-10 mg) was taken, weighted, frozen on dry ice, then stored at -80°C until HPLC analysis. On the day of analysis, samples were diluted 1:10 with ice-cold, deoxygenated mobile phase, sonicated and spun for 2 min at 14,000 g, and the supernatant was injected directly into the HPLC system. The analytical column was a 10-cm C18 reversed-phase catecholamine column (HRA-80, ESA, Wiggens, MA) preceded by a 7-µm guard column (1.5 cm, ODS, BAS, West Lafayette, IN); the detector was a glassy carbon electrode set at +0.7 V versus Ag/AgCl. The mobile phase was 50 mM NaH2PO4, with 3 mg/l sodium octylsulfate, 23.2 mg/L heptanesulfonic acid, 8 mg/L EDTA, and 10% methanol, pH 3 (Witkovsky et al. 1993). The mobile phase was deoxygenated with argon, filtered, then maintained under an argon atmosphere throughout the analysis. Flow rate was 1.2 mL/min. In separate experiments to test the stability of DA in ACSF in the presence of H2O2, nominally 1 µM DA was superfused through the recording chamber in 1.5 and 2.4 mM Ca2+-containing ACSF at 32°C, with and without H2O2. Samples of DA from the chamber were collected after 2 min of superfusion. Immediately after collection, samples were diluted 1:10 in ice-cold deoxygenated eluent and the concentration of DA determined using HPLC.

Lipid peroxidation assay

To address whether H2O2 might cause lipid peroxidation under the conditions tested, we determined total levels of the lipid peroxide byproducts, malonaldehyde (MDA) and 4-hydroxyalkenals (4-HNE), using a spectrophotometric assay (Bioxytech LPO-586 kit; Oxis International, Portland, OR). Slices were incubated for 30 min in normal ACSF (1.5 mM Ca2+), exposed to 1.5 mM H2O2 for 15 min, then H2O2 washed out for 30 min; control slices were maintained at 32°C for an equivalent incubation period of 75 min in normal ACSF. After incubation, excess solution was removed from each slice, three slices were pooled, weighed, and frozen on dry ice until analysis; for these pooled measurements, n was the number of samples rather than the number of slices.

Drugs and chemicals

ACS grade H2O2 (30% w/w), sulpiride, and all components of ACSF and HEPES-ASCF were obtained from Sigma (St. Louis, MO). At 30%, H2O2 is 8.8 M; the concentration of H2O2 used in all experiments was 1.5 mM (0.005%). The stability of H2O2 solutions in some experiments was determined spectrophotometrically using a commercially available kit (Oxis International); after 15 min superfusion, H2O2 concentration in the recording chamber remained within 5% of the initial concentration. Catalase (from bovine liver) was obtained from Calbiochem (San Diego, CA), a preparation that has been shown to have pure catalase activity (Esch et al. 1998); enzyme concentration was 250 IU/mL, which was mid-range for effective levels reported previously (Auerbach and Segal 1997; Desagher et al. 1997). All solutions were freshly made immediately before use.

Statistical analysis

All data are expressed as means ± SE, where n is the number of slices unless otherwise indicated. The controls in each experiment with H2O2 were the stimulated [DA]o increases obtained before H2O2 exposure; the maximum evoked [DA]o under these control condition was considered to be 100%. Two different statistical analyses were used to assess the effects of H2O2. First, the average maximal evoked [DA]o during stimulation was compared using paired Student's t-test, then [DA]o throughout the stimulus was compared using one-way ANOVA followed by Tukey post hoc analysis. The same statistical assessments were used to evaluate the effect of catalase except that instead of comparing single stimulation records, as was necessary for the H2O2 experiments, three catalase and three washout records were averaged from each slice. Significance of differences in striatal DA content, media DA concentrations, or tissue lipid peroxidation products between control and H2O2-exposed samples were determined using unpaired t-tests.


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INTRODUCTION
METHODS
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REFERENCES

Inhibition of evoked DA release by exogenous H2O2

Reproducible, TTX-sensitive DA release in striatal slices could be elicited using a 10-Hz, 50-pulse stimulation train at 10-min intervals for at least 2 h under control conditions. As described in METHODS, two different aspects of the effect of H2O2 on stimulated DA release were examined: maximum evoked [DA]o during stimulation and [DA]o throughout the pulse train.

In normal ACSF containing 1.5 mM Ca2+, the average maximum evoked [DA]o was 0.66 ± 0.07 µM (n = 10; Fig. 1A). Maximum evoked [DA]o was decreased by 56% to 0.29 ± 0.05 µM after 15-min exposure to H2O2 (P < 0.001, n = 10). Moreover, throughout the stimulus, evoked increases in [DA]o were depressed in the presence of H2O2, when compared with control stimulation (P < 0.001). Both peak [DA]o (0.58 ± 0.08 µM, n = 11) and DA release throughout the stimulus recovered to control levels after 30-min washout of H2O2 (P > 0.05; Fig. 1B).



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Fig. 1. Reversible inhibition of evoked [DA]o by H2O2 in the presence of 1.5 mM [Ca2+]o. A: average maximum evoked [DA]o and stimulated [DA]o throughout the 10-Hz, 50-pulse stimulation in normal artificial cerebrospinal fluid (ACSF) were decreased significantly (P < 0.001) by 15-min exposure to H2O2. B: evoked peak [DA]o and DA release throughout the stimulus reversibly returned to control levels (P > 0.05) after 30-min washout of H2O2. Data are means ± SE (n = 10). Solid bar indicates stimulation period (5 s).

Because decreased DA release in the presence of H2O2 might reflect oxidative loss of DA during H2O2 exposure, we determined the DA content of striatal tissue immediately after incubation with H2O2. Striatal DA content was not altered significantly (P > 0.05) by H2O2 exposure, with 62 ± 5 nmol/g tissue wet weight (n = 24) in control slices and 56 ± 5 nmol/g (n = 24) in slices incubated for 15 min with H2O2. Similarly, media concentrations of DA were relatively stable in the presence of H2O2 after superfusion through the recording chamber for 2 min at 32°C; this was substantially longer than the 5-s period of [DA]o elevation in the extracellular space during electrical stimulation. In ACSF with 1.5 mM Ca2+, the measured concentration of a nominally 1 µM DA was 0.98 ± 0.02 µM (n = 6) and 0.93 ± 0.02 µM (n = 6) in the same medium plus H2O2. In ACSF with 2.4 mM Ca2+, DA concentration was 0.97 ± 0.02 µM (n = 6) and 0.91 ± 0.01 µM (n = 6) with H2O2. This 5-6% decrease in DA concentration was statistically significant in 2.4 mM Ca2+ (P < 0.05) but not in 1.5 mM Ca2+. In addition, total MDA + 4-HNE levels, reflecting lipid peroxide formation, were indistinguishable between control and H2O2-exposed slices, with 1.6 ± 0.2 nmol/g tissue wet weight (n = 6) in control slices and 1.4 ± 0.2 nmol/g (n = 8) in slices sampled after 30-min washout of H2O2. Levels of MDA + 4-HNE were not determined in slices sampled during H2O2 exposure because of the potential for interference of this oxidant under assay conditions.

Ca2+ dependence of inhibition of DA release by exogenous H2O2

To investigate the potential involvement of Ca2+ entry on the effect of H2O2 on DA release, the Ca2+ dependence of H2O2 inhibition was examined in ACSF containing normal (1.5 mM) [Ca2+]o or 2.4 mM [Ca2+]o. As expected, evoked [DA]o was enhanced in 2.4 mM [Ca2+]o compared with that in 1.5 mM (2.16 ± 0.38 µM, n = 8). Under these conditions, however, H2O2 had no significant effect on either peak [DA]o or overall release levels (P > 0.05). Moreover, concentrations of exogenous H2O2 of up to 10 mM did not alter evoked [DA]o when [Ca2+]o was 2.4 mM (data not illustrated).

To determine whether the inhibition of DA release seen in 1.5 mM Ca2+ could be reversed as well as prevented by raising [Ca2+]o to 2.4 mM, we first determined the effect of H2O2 on stimulated DA release in 1.5 mM [Ca2+]o, then switched to 2.4 mM [Ca2+]o in the continued presence of H2O2 (Fig. 2). In 1.5 mM [Ca2+]o, maximum evoked [DA]o decreased from 0.69 ± 0.16 to 0.37 ± 0.10 µM (P < 0.001, n = 5) in the presence of H2O2 (Fig. 2A), with lower release levels throughout stimulation (P < 0.001). When [Ca2+]o was increased to 2.4 mM in H2O2 for an additional 10 min, evoked [DA]o increased significantly, with a peak level of 2.27 ± 0.63 µM (n = 5), which was 320% of control in 1.5 mM [Ca2+]o in the absence of H2O2 (P < 0.001; Fig. 2B). Evoked DA release throughout the stimulus was also elevated (P < 0.001). Maximum (2.26 ± 0.65 µM, n = 5) and overall evoked [DA]o in 2.4 mM [Ca2+]o were the same as release levels in the presence of H2O2 (P > 0.05) or after H2O2 washout (Fig. 2B).



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Fig. 2. Reversal of H2O2-induced inhibition of dopamine (DA) release in 2.4 mM [Ca2+]o. A: average evoked [DA]o during 10-Hz, 50-pulse stimulation under control conditions (1.5 mM [Ca2+]o) and after H2O2. B: average evoked [DA]o after 10 min further H2O2 exposure but with [Ca2+]o increased to 2.4 mM, followed by washout of H2O2 in the continued presence of 2.4 mM [Ca2+]o. Control DA release levels (average peak and DA release throughout stimulation) differed significantly between 1.5 mM [Ca2+]o and 2.4 mM [Ca2+]o (P < 0.001). There was no difference, however, between average maximum [DA]o, and overall evoked [DA]o during stimulation in 2.4 mM [Ca2+]o, and that in 2.4 mM [Ca2+]o + H2O2. Data are means ± SE (n = 5). Solid bar indicates stimulation period (5 s).

Loss of exogenous H2O2-mediated inhibition under conditions of enhanced excitation

The dependence of the effect of H2O2 on [Ca2+]o suggested that other conditions that increase Ca2+ entry and subsequent DA release might also decrease the effectiveness of exogenous H2O2 on inhibiting release. We tested this hypothesis using two additional experimental paradigms, both conducted in 1.5 mM [Ca2+]o. First, we assessed the effect of enhanced stimulus frequency by using brief, high-frequency stimulation (10 pulses at 100 Hz) to evoke DA release. Because of the short duration of this stimulus paradigm, only mean peak evoked [DA]o was compared statistically in these experiments. With this protocol, maximum evoked [DA]o was significantly higher than with 10-Hz stimulation (167% of control, n = 5; P < 0.05). Moreover, DA release evoked by high-frequency stimulation in 1.5 mM [Ca2+]o was unaffected by H2O2 (P > 0.05 for both H2O2 and washout compared with control; Fig. 3A). Representative voltammograms of the maximum [DA]o obtained under each conditions are also illustrated (Fig. 3B).



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Fig. 3. Loss of effect of H2O2 on evoked [DA]o during high-frequency stimulation. A: average evoked [DA]o during brief, high-frequency stimulation (100 Hz, 10 pulses). DA release under these conditions was unaffected by H2O2 (P > 0.05). Data are means ± SE (n = 3-5). Black bar indicates time of stimulation (0.1 s). B: representative voltammograms of DA obtained either during calibration with 1 µM DA or at the maximum [DA]o during high-frequency stimulation under different conditions in the same striatal slice.

Second, we assessed the effect of blocking D2 autoreceptors (Fig. 4). Under normal conditions, D2 receptor activation should hyperpolarize presynaptic DA terminals (Lacey et al. 1987, 1988; Liu et al. 1994). In addition, D2 receptor activation can have a direct effect on Ca2+ entry, and hence DA release, by inhibiting voltage-dependent N- and P/Q-type Ca2+ channels (Kuzhikandthil and Oxford 1999; Yan et al. 1997). Consistent with these actions, a supramaximal concentration of the D2-receptor antagonist sulpiride (1 µM) (Cragg and Greenfield 1997; Lacey et al. 1987) caused a significant increase in peak evoked [DA]o during 10-Hz, 50-pulse stimulation, compared with evoked levels in the absence of D2-receptor blockade (P < 0.01) with an average peak [DA]o of 1.76 ± 0.40 µM (n = 5), which was 275% of control. In the presence of sulpiride, exogenous H2O2 again caused a significant decrease in peak evoked [DA]o (to 1.36 ± 0.36 µM, n = 5, P < 0.01) and a decrease in overall release (P < 0.01; Fig. 4). However, the decrease was attenuated from that in control conditions, with an inhibition of only 20%, compared with the roughly 60% decrease in the absence of D2-receptor blockade (compare Figs. 1A and 4). Unlike control conditions, maximum evoked [DA]o did not return to pre-H2O2 levels after H2O2 was washed out in the presence of sulpiride (not shown). By contrast, maximum evoked [DA]o during high-frequency stimulation (10 pulses at 100 Hz) was unaffected by sulpiride (P > 0.05, n = 5), as shown previously (Cragg and Greenfield 1997). Neither exposure to H2O2 nor subsequent washout in the continued presence of sulpiride altered [DA]o evoked by high-frequency stimulation (data not illustrated).



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Fig. 4. Decreased H2O2-mediated inhibition of DA release after blockade of D2 autoreceptors. Average maximum evoked [DA]o during 10-Hz, 50-pulse stimulation (in 1.5 mM [Ca2+]o) in the presence of the D2-receptor antagonist, sulpiride (1 µM) was decreased significantly (P < 0.01) after 15 min in H2O2, although to a lesser extent than in the absence of sulpiride (compare with Fig. 1A). In the presence of sulpiride, the decrease in overall DA release by H2O2 was also significant (P < 0.01). Data are means ± SE (n = 5). Solid bar indicates stimulation period (5 s).

Catalase increased evoked [DA]o

To determine whether DA release was modulated by endogenously produced H2O2 during stimulation, we tested the effect of catalase on evoked [DA]o during 10-Hz, 50-pulse trains and compared this to evoked [DA]o recorded after catalase was washed out (Fig. 5). Following three consistent recordings in the presence of catalase, washout was initiated; evoked [DA]o typically began to decrease after washout for 10 min. A new, lower level was seen by 20-min washout, which remained stable until the end of recording (up to 1 h of washout). In the presence of catalase, average peak [DA]o in 1.5 mM Ca2+ was significantly higher than in paired control stimulations after catalase washout (165% of control, n = 5; P < 0.01) as was release throughout the stimulus (P < 0.001 vs. washout; Fig. 5A). In contrast to exogenously applied H2O2, catalase was not only effective in the presence of 2.4 mM [Ca2+]o, but its effect was enhanced compared with that in 1.5 mM [Ca2+]o (Fig. 5B). Peak [DA]o in catalase was 181% higher than after washout in 2.4 mM [Ca2+]o (n = 7; P < 0.001 for peak [DA]o and for [DA]o throughout the train vs. washout). The greater effect of catalase in higher [Ca2+]o can also be seen in the normalized DA voltammograms shown in Fig. 5. To ascertain that the enzymatic action of catalase was responsible for these effects, catalase was inactivated in some experiments by boiling; when active catalase was washed out with inactivated enzyme (in either 1.5 or 2.4 mM [Ca2+]o), a decrease in evoked [DA]o was observed that was indistinguishable from that seen with ACSF washout alone.



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Fig. 5. Enhanced DA release in the presence of catalase. A: average maximum evoked [DA]o and stimulated [DA]o throughout the 10-Hz, 50-pulse stimulation in 1.5 mM [Ca2+]o were significantly enhanced in the presence of catalase (P < 0.01 for averaged peak [DA]o and P < 0.001 for overall DA release throughout the stimulus; n = 5). B: average maximum evoked [DA]o and stimulated [DA]o throughout the 10-Hz, 50-pulse stimulation in 2.4 mM [Ca2+]o was further enhanced in the presence of catalase (P < 0.001 for both averaged peak [DA]o and stimulated [DA]o throughout the stimulus; n = 7). Data are means ± SE. Solid bars indicate stimulation period (5 s). Insets: representative averaged voltammograms of peak evoked [DA]o under the conditions indicated.

In studies with active catalase, the shape of evoked [DA]o responses differed somewhat from that in control slices never exposed to catalase (compare pre- or post H2O2 records in Figs. 1 and 2 with catalase washout records in Fig. 5). During stimulation under control conditions, evoked [DA]o reached a maximum after 200-400 ms, then declined to plateau levels that were roughly 40-50% of the peak concentration (Figs. 1A and 2B). By contrast, after catalase incubation and washout, [DA]o fell to 10% of peak concentration by the end of stimulation in both 1.5 and 2.4 mM [Ca2+]o.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The data presented here demonstrate for the first time that neurotransmitter release can be modulated by H2O2. In these experiments, both endogenous and exogenous H2O2 were shown to inhibit DA release in striatum. These findings, together with previous reports that exogenous H2O2 can inhibit glutamatergic transmission in the hippocampus (Avshalumov et al. 2000; Pellmar 1986, 1987, 1995), suggest that inhibition of transmitter release by H2O2 might represent a general synaptic response to ROS.

Ca2+ dependence of H2O2-mediated inhibition of DA release

In the present studies, exogenous H2O2 had no effect on evoked DA release in the presence of 2.4 mM [Ca2+]o or during high-frequency stimulation. Both of these conditions would increase presynaptic Ca2+ entry. At face value, this result suggests that increased intracellular Ca2+ might act competitively with H2O2. However, even when H2O2 concentration was increased to 10 mM, there was still no effect on evoked release in 2.4 mM [Ca2+]o, as noted in RESULTS, indicating that direct competition between Ca2+ and H2O2 was unlikely. Furthermore unaltered evoked [DA]o in the presence of exogenous H2O2 under these conditions argued against a direct chemical interaction between H2O2 and DA to decrease [DA]o. This was verified by our studies of DA stability in H2O2-containing ACSF.

In contrast to the lack of effect of exogenous H2O2 in 2.4 mM [Ca2+]o, the effect of catalase was somewhat greater in 2.4 than in 1.5 mM [Ca2+]o. This suggests that a Ca2+-dependent increase in endogenous H2O2 levels generated during stimulation caused a maximal inhibitory effect that occluded any further response to exogenous H2O2. In support of this hypothesis, previous studies have shown that mitochondrial respiration (the primary source of endogenous H2O2, see following text) and consequent ROS production are Ca2+ dependent. Increases in intracellular Ca2+ concentration ([Ca2+]i) are accompanied by enhanced mitochondrial O2 consumption (Jung et al. 2000; Kojuma et al. 1994). In addition, in presynaptic terminals, elevated [Ca2+]i serves as a trigger for mitochondrial uptake of Ca2+ (Giovannucci et al. 1999) and for active clearance of [Ca2+]i by plasma membrane transporters (Zenisek and Matthews 2000). Both of these processes are ATP-dependent processes and thus require increased oxidative metabolism. Moreover, Dykens (1994) has shown directly that increases in [Ca2+] cause an increase in ROS production by isolated brain mitochondria.

The effect of H2O2 on DA release was also minimal or absent when release was elicited in 1.5 mM [Ca2+]o under conditions that enhance Ca2+ entry. The first paradigm tested was brief, high-frequency stimulation (Fig. 3). Physiologically, both terminal excitability and DA release are dependent on stimulation frequency, in part reflecting frequency-dependent Ca2+ entry (Chergui et al. 1994; Garris et al. 1997). In vivo, DA cells exhibit two main activity patterns: single spike firing at 0.5-8 Hz and burst firing of three to eight spikes at frequencies of 6-15 Hz (Grace and Bunney 1984), with the ability to follow stimulation frequencies up to 100 Hz (Tepper et al. 1991). Increasing stimulus frequency from 10 to 100 Hz in the present experiments caused an increase in evoked [DA]o as expected. Consistent with a concomitant increase in H2O2 generation under these conditions, exogenous H2O2 had no effect on evoked [DA]o during high-frequency stimulation, again suggesting that the inhibitory mechanism was already fully activated by endogenous H2O2.

The second approach was to manipulate DA terminal excitability using an antagonist of D2 autoreceptors, which should also increase Ca2+ entry. Normally, D2-receptor activation hyperpolarizes the presynaptic membrane of DA terminals, which decreases excitability and thus decreases DA release (Cragg and Greenfield 1997; Lacey et al. 1987, 1988). In addition, D2-receptor activation can decrease Ca2+ currents through direct links to N- and P/Q-type Ca2+ channels (Kuzhikandathil and Oxford 1999; Yan et al. 1997), which are required for synaptic release of DA (Turner et al. 1993). Consistent with these actions, the presence of a D2-receptor antagonist markedly enhances evoked [DA]o (Cragg and Greenfield 1997) (Fig. 4). In the present studies, the D2-receptor blockade with sulpiride also decreased the effect of exogenous H2O2 on DA release (Fig. 4).

Sources of endogenous H2O2

Oxidative metabolism in brain tissue occurs in the mitochondria as in all cells. During the process of oxidative phosphorylation, a significant amount of O2 consumed is diverted to form superoxide (O<UP><SUB>2</SUB><SUP>•−</SUP></UP>), which is the stoichiometric precursor of H2O2 (Chance et al. 1979). The amount of H2O2 produced by brain mitochondria is up to 5% of the amount of O2 consumed (Arnaiz et al. 1999). Given that the rate of O2 consumption in gray matter is 2-5 µmol/g tissue wet weight per minute (Hagerdal et al. 1975; McIlwain and Bachelard 1985), or 2-5 mM (assuming 1 g = 1 mL), this would mean that concentrations of up to 250 µM H2O2 could be generated every minute within brain neuropil. Because the rate of O2 consumption is roughly 10-fold higher in neurons than in glia (Siesjö 1980), however, this H2O2 would be produced predominantly in the neuronal compartment, which could lead to higher, intra-neuronal concentrations. Moreover, the presence of mitochondria within 250 nm of the synapse in DA terminals (Delle Donne et al. 1997; Nirenberg et al. 1997) suggests that even higher levels could be reached in the restricted, intracellular compartment of a synaptic terminal. It is relevant to note, however, that such local increases are likely to be transient because H2O2 is membrane permeable and thus can readily leave the compartment in which it is produced.

In addition to mitochondrial sources, H2O2 will also be produced in DA terminals by monoamine oxidase (MAO), which is a metabolizing enzyme for DA (Cohen et al. 1997; Sandri et al. 1990). Importantly, MAO is localized on the outer membrane of mitochondria, which would further enhance H2O2 concentrations near DA synapses. Given that increased DA metabolism accompanies enhanced DA release, generated H2O2 might provide additional feedback inhibition to decrease transmitter release and thus augment the effect of D2-autoreceptor-mediated inhibition.

Actual concentration of H2O2 at a given location will depend not only on the activity of sources of H2O2 and the size of the compartment it enters, but also on the activity of the local antioxidant network. In brain tissue, H2O2 levels are regulated largely by the intracellular enzyme, glutathione (GSH) peroxidase, and by endogenous catalase in peroxisomes (Cohen 1994). Importantly, the present results with exogenous catalase indicate that cellular antioxidant regulation does not completely remove endogenously generated H2O2. Rather these processes appear to permit levels of H2O2 that are sufficient to exert modulatory actions. The lack of effect of up to 10 mM exogenous H2O2 in elevated [Ca2+]o, however, indicates that H2O2-mediated effects are saturable; this is also consistent with the calculation that competing levels of endogenous H2O2 might be millimolar. Indeed, the concentration of exogenous H2O2 used in the present studies (1.5 mM) is within the range (1-3 mM) found to have biological effects in many other studies in the literature (e.g., Frantseva et al. 1998; Krippeit-Drews et al. 1999; Pellmar 1987, 1995; Seutin et al. 1995). One caveat in these arguments is that the activity of antioxidant enzymes and other cellular antioxidants could result in levels of exogenously applied and endogenously generated H2O2 that are much lower than this. Available analytical methods cannot yet address this question quantitatively. Regardless of absolute concentrations, however, the fact that catalase enhanced evoked [DA]o by roughly the same percentage that exogenous H2O2 decreased peak [DA]o suggests that similar effective levels were achieved with both sources.

Possible mechanisms of H2O2-mediated modulation of DA release

It has been previously suggested that inhibition of synaptic transmission in the hippocampus by H2O2 might be the result of decreased presynaptic Ca2+ entry and consequent loss of transmitter release (Pellmar 1987). The present results confirm that H2O2 can indeed inhibit transmitter release. In addition, in separate studies in hippocampal slices, we have monitored presynaptic Ca2+ entry indirectly using ion-selective microelectrodes to monitor decreases in [Ca2+]o during pulse-train stimulation (Avshalumov et al. 2000). Evoked [Ca2+]o decreases were unaffected by H2O2, suggesting that its action was downstream from Ca2+ entry. Inhibition of evoked population spikes in the hippocampus by H2O2 in those studies was further shown to be mediated by hydroxyl radical (·OH) formation, which can be catalyzed by metal ions (Avshalumov et al. 2000). It is likely that ·OH is also involved in modulating DA release; however, we were not able to test this in the present studies because the metal ion chelator typically used to prevent ·OH generation, deferoxamine, is electroactive and thus interfered with voltammetric detection of DA.

A number of presynaptic mechanisms that are downstream from Ca2+ entry could be involved in mediating the actions of H2O2 on DA release. For example, it was recently shown that H2O2 caused a decrease in ATP production, which interfered with stimulus secretion in pancreatic cells (Krippeit-Drews et al. 1999). Another possible mechanism might be through oxidative modification of one or more intracellular proteins involved in vesicular release, including those involved in Ca2+-dependent SNARE binding interactions (Bezprozvanny et al. 1995; Rettig et al. 1997; Sheng et al. 1996; Südhof 1997). Such a process would involve oxidation of redox-sensitive amino acids like cysteine. Alternatively, because ROS shift the balance between phosphatases and kinases (Klann and Thiels 1999; Klann et al. 1998), the effects of H2O2 could involve increased phosphorylation of Ca2+ binding proteins or other synaptic proteins. Tying these latter two mechanisms together is the fact that cysteine residues on protein tyrosine phosphatases have been shown to be selective targets of H2O2-induced oxidation (Denu and Tanner 1998).

In addition to these possible mechanisms, there is evidence in the literature to suggest that H2O2 application can cause membrane hyperpolarization mediated by an increased K+ in some cell types, including CA1 pyramidal neurons (Krippeit-Drews et al. 1999; Seutin et al. 1995), which could lead to a decrease in transmitter release. This is not seen in all cells, however. For example, although H2O2 was also found to cause changes in membrane properties of some cells in thalamocortical slices, the net effect of H2O2 application was excitatory, apparently by selective loss of inhibitory transmission (Frantseva et al. 1998). How changes in membrane properties might contribute to inhibition of release of DA from synaptic terminals in striatum has not been examined.

The slightly altered shape of evoked [DA]o records after prolonged catalase incubation suggests that H2O2 might contribute to DA regulation by mechanisms in addition to inhibition of release. The shape and amplitude of [DA]o records during normal stimulation is a consequence of many factors, including specific inhibitory effects of D2 autoreceptors and DA uptake via the dopamine transporter (DAT) as well as general slice quality. The influence of D2 receptors can be seen in Fig. 4, in which the increase in [DA]o was enhanced and prolonged when inhibitory autoreceptors were blocked by sulpiride compared with control records (e.g., Fig. 1). Whether sulpiride was present or not, however, DA release curves typically fell to 40-50% of peak value by the end of the pulse train. After incubation in catalase, however, the fall in evoked [DA]o was exaggerated, with a decay to only 10% of peak by the end of the pulse train (compare Figs. 1, 2, and Fig. 5). One possible explanation is that prolonged depletion of tonic levels of H2O2 could decrease protein kinase C-dependent phosphorylation of the DAT, which would increase DA uptake activity (Bauman et al. 2000; Vaughan et al. 1997). In addition, there is evidence in the literature to suggest that decreased phosphorylation of D2 receptors might increase agonist sensitivity (Elazar and Fuchs 1991). Either effect could contribute to the faster return of evoked [DA]o to baseline after catalase incubation. On the other hand, direct oxidation of the DAT under conditions of oxidative stress can inhibit DAT activity (Berman et al. 1996) such that added catalase might protect the DAT by decreasing oxidative stress. It should be noted, however, that possible oxidative inhibition of the DAT by H2O2 during exogenous application would increase rather than decrease evoked [DA]o. Moreover, the similarity of evoked [DA]o in the presence and absence of H2O2 in 2.4 mM [Ca2+]o (e.g., Fig. 2B) suggests that DAT activity is not altered during 15-min H2O2 application.

Independent of the possible mechanism(s) by which H2O2 may act, the finding that H2O2 can inhibit transmitter release reveals a novel process by which synaptic transmission might be modulated physiologically. In addition, these data point to a normal process that could lead to neuropathology if regulation were disrupted. As noted earlier, Parkinson's disease, which involves degeneration of DA cells and terminals, has been linked to oxidative stress (Cohen et al. 1997; Ebadi et al. 1996; Olanow and Tatton 1999). An imbalance in ROS production and regulation could contribute to this process. Moreover, excess H2O2 can damage mitochondria (Hoyt et al. 1997), such that disregulation could initiate a cycle of increased [Ca2+]i, increased ROS production, cytotoxicity in striatum (Brouillet et al. 1995), and degeneration of DA neurons in midbrain (Sonsalla et al. 1997). The present findings, therefore not only provide a new perspective on modulation of synaptic transmission by ROS but also suggest normal regulatory processes that could, over time, contribute to the pathology of neurodegenerative disorders.


    ACKNOWLEDGMENTS

We are grateful to M. Chesler and S. J. Cragg for insightful comments on the manuscript, to M. L. Chao for HPLC analysis of dopamine, and to K. A. Helmin for lipid peroxide determination.

These studies were supported by National Institute of Neurological Disorders and Stroke Grant NS-36362.


    FOOTNOTES

Address for reprint requests: M. E. Rice, Dept. of Physiology and Neuroscience, NYU School of Medicine, 550 First Ave., New York, NY 10016 (E-mail: margaret.rice{at}nyu.edu).

Received 31 August 2000; accepted in final form 17 January 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society